FORMULATION OF

MICROBIAL
BIOPESTICIDES
FORMULATION OF
MICROBIAL
BIOPESTICIDES
Beneficial microorganisms,
nematodes and seed treatments

Edited by

H.D. Burges

SPRINGER SCIENCE+BUSINESS MEDIA, B.V.

Library of Congress Cataloging in Publication Card Number: 98-70274

ISBN 978-94-010-6066-0 ISBN 978-94-011-4926-6 (eBook)

DOI 10.1007/978-94-011-4926-6

02-0601-100 ts

Al l Rights Reserved
1998 Springer Science+Busines s Media Dordrecht
Originall y published by Kluwer Academic Publishers in 1998
Softcover reprint of the hardcover 1st edition 1998
No part of the material protected by thi s copyright notice may be reproduced or
utilize d in any form or by any means, electronic or mechanical, includin g
photocopying, recording, or by any information storage and retrieval system,
withou t prior permission from the copyright owner.
CONTENTS

List of contributors vii

Preface IX

Abbreviations xi

1 Introduction 1
H. Denis Burges and Keith A. Jones

PART ONE PRINCIPLES OF FORMULATION 5

2 Technology of formulation and application 7
Keith A. Jones and H. Denis Burges

PART TWO ORGANISMS WITH A PERORAL MODE OF ACTION 31

3 Formulation of bacteria, viruses and Protozoa to control insects 33
H. Denis Burges and Keith A. Jones

PART THREE ORGANISMS WITH A CONTACT MODE OF ACTION 129

4 Formulation of mycoinsecticides 131
H. Denis Burges
5 Formulation of microorganisms to control plant diseases 187
Deborah R. Fravel, William J. Connick, Jr and Jack A. Lewis
6 Formulation of microbial herbicides 203
Michael P. Greaves, Peter J. Holloway and Bruce A. Auld
7 Formulation of beneficial organisms applied to soil 235
Alan S. Paau
8 Application of microorganisms to seeds 255
Mark P. McQuilken, Peter Halmer and David J. Rhodes

PART FOUR ORGANISMS WITH A POWER OF SEARCH 287

9 Formulation of entomopathogenic nematodes 289
Rayman Georgis and Harry K. Kaya
vi Contents
PART FIVE THE FUTURE 309

10 Trends in formulation of microorganisms and future research requirements 311

H. Denis Burges and Keith A. Jones

APPENDICES 333

A catalogue of formulation additives: function, nomenclature, properties

and suppliers 333
Konrad Bernhard, Peter J. Holloway and H. Denis Burges
II Spray application criteria 367
Keith A. Jones
III Glossary (including list of product and additive types) 377
H. Denis Burges

Index 383
CONTRIBUTORS
BRUCE A. AULD
Orange Agricultural Institute,
Forest Road, Orange, NSW 2800, Australia
E-mail bruce.auld@agric.nsw.gov.au
KONRAD BERNHARD
Ettlinger Strasse 33/3, 0-76307 Karlsbad
Germany
E-mail Konrad.Bernhard@t-online.de
H. DENIS BURGES
21 Withdean Avenue, Goring-by-Sea,
Worthing, West Sussex, BN12 4XD, UK
E-mail denis@hdburges,freeserve.co.uk
WILLIAM J. CONNICK JR
Commodity Utilisation Research Unit,
USDA-ARS,
Southern Regional Research Center,
1100 Robert E. Lee Boulevard, PO Box 19687,
New Orleans, LA 70124-0687, USA
E-mail wconnick@nola.srrc.usda.gov
DEBORAH R. FRAVEL
Room 275, Building OlIA, BARC-West,
Biocontrol of Plant Disease Laboratory,
Plant Sciences Institute, USDA-ARS,
Beltsville, MD 20705-2350, USA
E-mail dfravel@asrr.arsusda.gov
RAMON GEORGIS
ThermoTrilogy Corp., 7500 Grace Drive,
Columbia, MD 21044, USA
E-mail RGeorgis@ThermoTrilogy.com
MICHAEL P. GREAVES
IACR-Long Ashton Research Station,
Department of Agricultural Sciences,
University of Bristol, Weston Road,
Long Ashton, Bristol, Avon, BS18 9AF, UK
E-mail Mike.Greaves@BBSRC.ac.uk
PETER HALMER
Germain's (UK) Ltd, Hansa Road,
Hardwick Industrial Estate, King's Lynn,
Norfolk, PE30 4LG, UK
E-mail phalmer@germains.com
PETERJ. HOLLOWAY
IACR-Long Ashton Research Station,
Department of Agricultural Sciences,
University of Bristol, Weston Road,
Long Ashton, Bristol, Avon, BS18 9AF, UK
E-mail peterj_holloway@hotmail.com
KEITH A. JONES
Natural Resources Institute,
University of Greenwich, Central Avenue,
Chatham Maritime, Kent, ME4 4TB, UK
E-mail keith.jones@nri.org
HARRY K. KAYA
One Shields Avenue,
Department of Nematology,
University of California, Davis,
CA 95616, USA
E-mail hkkaya@ucdavis.edu
JACK LEWIS
Room 275, Building OlIA, BARC-West,
Biocontrol of Plant Disease Laboratory,
Plant Sciences Institute, USDA-ARS,
Beltsville, MD 20705-2350 USA
E-mail dfravel@asrr. arsusda.gov
viii Contributors
MARK P. MCQUILKEN San Diego, 9500 Gilmen Drive, USA
Unit of Plant Protection, E-mail apaau@ucsd.edu
Department of Plant Biology,
The Scottish Agricultural College, DAVID RHODES
Auchincruive, Ayr, KA6 5HW, Scotland, UK Zeneca Agrochemicals,
E-mail m.mcquilken@au.sac.ac.uk Fernhurst, Haslemere, Surrey, GU27 3JE, UK
E-mail DavidD.J.Rhodes@
ALAN S. PAAU AGUK.ZENECA.COM
University of California,
Technology Transfer & Intellectual Property
PREFACE
Formulation vies with genetic engineering as
one of the two most important recent areas of
progress in developing microorganisms for
use in agriculture and forestry. The subject
has not previously been comprehensively
covered at book length. Early formulation,
stylized from that of chemicals, led to many
initial failures. This book goes back to basics,
i.e. ecological and biological knowledge, to
analyse the special requirements when formu-
lating microorganisms and to build up a
detailed account of modern formulation
technology. The function of the organisms in
nature is examined with a view to bettering
natural performance by mass producing their
survival stages. These stages are optimized
for storage and effectiveness, then mixed
with carriers, supplemented by a wide range
of additives that further improve efficiency
and survival during harvest, storage and
application, as well as protect and nurture
the organisms afterwards while they lie in
wait to take effect. There are 15 authors, all
widely experienced in their own fields.
The scope of the book is broad, spanning 10
chapters. The scene is set by a description of
application technology and machines, which
depend heavily on formulation to improve
efficiency. Bacterial and viral insect pathogens
attack perorally and must be eaten to take
effect. These organisms are used to control
mainly chewing, foliar pests, posing the de-
manding task of creating an even, palatable
cover over the foliage, or they are used to
control larvae of vectors of human disease in
water bodies. This task is particularly challen-
ging for sprays because of the particulate, live
and / or proteinous nature of the organisms.
The ultimate in efficacy - systemic formula-
tion - is obtained by the formation of the
toxins of Bacillus thuringiensis in the tissues
of transgenic plants. While the application
target of these peroral pathogens is the in-
sects' food, sprays of entomopathogenic fungi
target the insects themselves because the
fungi attack through the insect cuticle, so can
control sucking as well as chewing insects.
Although more susceptible than bacterial
spores to the environment, dry fungal conidia
formulated in oil provide a breakthrough, en-
abling their use in arid climates. The insect
pathogens have led progress in formulation
for sprays, but the fungi are also formulated
for use in the moist environment of soil. How-
ever, formulations for soil have mainly been
developed for three other types of organisms:
those used to control plant diseases, to control
weeds, and to improve plant growth, largely
with nitrifying organisms. Seeds can be used
as vehicles, taking formulation into another
industry, that of seed treatment. Entomo-
pathogenic nematodes set the formulator the
most demanding task, to preserve mobility
and the power of search. Each chapter consid-
ers research needs and probes the future, both
assessed overall in the final chapter. Because
these varied areas progressed largely inde-
pendently, intensive cross-referencing be-
tween chapters has been inserted to cross-
fertilize information between them.
Momentum for progress comes mainly
from research interest and from design of
products for sale. In this book, great attention
has been paid to the needs of cost effective-
ness and user acceptance. The book is de-
signed for a wide readership. Thus readers
x Preface
new to the field are served by many practical
illustrations; for experienced workers there
are in-depth analyses of available data and a
bewildering array of additives (including fail-
ures), shown in Tables and Appendices. These
analyses and models have enabled the best
additives to be assessed in a concise text.
Great effort has been devoted to making the
book reader-friendly.
This book will interest readers from univer-
sities, government research laboratories and
industry, as well as operatives of environ-
mentally friendly pest and disease control,
whether they are engaged in research, devel-
opment, commerce, teaching or practical use.
The disciplines involved are entomology,
botany, microbiology, virology, nematology,
chemistry and equipment design. Information
is gleaned from a huge range of journals and
patents; it outweighs confidential information
locked up in industry.
I wish to thank my team of authors, numer-
ous peer reviewers - particularly Peter J.
Holloway, who assiduously applied his spe-
cialist chemical knowledge - and all those
who so generously answered my appeal for
pre-publication material to push the book as
far ahead as possible; also my daughter Stella,
son-in-law Michael, grandson Robert Irons
and my wife Sheila, for all their help and
support.
H. Denis Burges
February 1998
ABBREVIATIONS

AAU Aedes aegypti unit of mosquitocidal activity

AMEA acetamide-(3-mercaptoethylamine, humectant
aw water activity
Bt Bacillus thuringiensis
CDA controlled-droplet application
c.f.u. colony-forming unit
CIAT Centro Internacional de Agricultura Tropical, Colombia
CMA corn meal agar
CMC carboxymethykellulose
CPV cytoplasmic polyhedrosis virus
DOT dissolved O 2 tension
EDTA ethylenediaminetetra-acetic acid
GCPF Global Crop Protection Federation (formerly GIFAP)
HLB hydrophile-lipophile balance of a surfactant
HV high-volume spray
IPA iso-propanol
IPM integrated pest management
IU,ITU international units of bacterial insecticidal activity
l.a.i. ratio of leaf surface area of a plant to the area of ground occupied by the plant
LAMEA lactamide-fJ-mercaptoethylamine, humectant
LD so lethal dose to kill half the test insects
LSTB lauryl sulphate tryptose broth
LTso lethal time to kill half the test insects
LV low-volume spray
mil 0.001 inch
MNPV multiple-embedded NPV
NMD number median diameter
NPV nuclear polyhedrosis (nucleopolyhedrosis) virus
OAR original activity ratio
OB see PIB
PEG polyethylene glycol
PGPR plant growth-promoting rhizobacteria
PIB,OB (polyhedral) occlusion body of an NPV
POE [a]-(p-nonylphenyl)-w-hydroxypoly (oxyethylene)
PVP polyvinylpyrrolidone
RH relative humidity
SMP solid matrix priming
ULV ultra low-volume spray
xii Abbreviations
USDA-ARS US Department of Agriculture, Agricultural Research Service
UV, UVA, UVB, uve ultraviolet radiation
VAM vesicular-arbuscular endomycorrhiza
VLV very low-volume spray
VMD volume median diameter
WDG water-dispersible granule
WHO World Health Organisation
INTRODUCTION
H. Denis Burges and Keith A. Jones
Some pesticidal and other beneficial organ-
isms have been very effective in the laboratory
only to fail at some stage in the field, even
after development of a product for marketing.
Common causes of this demise are poor stab-
ility of the product during storage prior to
application, too little active material actually
reaching the field target, and rapid degrada-
tion of the active material on the target.
Formulation plays a vital role in helping to
solve these problems and in making an organ-
ism effective in practice. However, this must
be achieved in a cost-effective manner if the
final product is to survive commercially.
What is formulation? Defined collectively,
formulation comprises aids to preserving
organisms, to delivering them to their targets
and - once there - to improving their activ-
ities. A technical concentrate of an organism
that has been formulated is termed a formula-
tion, or a product, which may be stored and
put on sale commercially. A product often
does not fully serve all the requirements of
use on all crops. More additives may be
needed to achieve optimum application on
some crops. These are normally added just
before application and the final formulation
applied is termed a tank mix.
This book deals specifically with formula-
tion of beneficial microorganisms and nem-
atodes. These are divided into six groups: (1)
microbial insecticides; (2) microbes that
destroy, inactivate or compete with plant
Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0412625202.
1
pathogens; (3) microbial herbicides; (4) bene-
ficial organisms that improve plant nutrition;
(5) microbes applied to seeds; and (6) entomo-
philic nematodes. Formulation has tended to
be considered and research has tended to
proceed independently in these six groups.
However, they have common strengths and
technological weaknesses, as well as unique
features, so would benefit from cross-fertiliza-
tion and assimilation of ideas and data. The
various groups of microorganisms are sum-
marized and compared in Table 1.1.
General principles of formulation are estab-
lished in Part 1 of this book. Formulation is
determined not only by scientific require-
ments, e.g. uniform spray cover on foliage,
but also by commercial requirements, e.g.
user friendliness. Products must be easy to
store, easy to use and compatible with users'
equipment.
Critical formulation requirements are
determined by features of the organisms
themselves and of their environments. An
overriding feature is mode of action: it dict-
ates the formulator's ultimate objectives and,
therefore, is used as the basis for three sepa-
rate parts in the book. Some organisms, such
as insect pathogenic bacteria and viruses, act
through the gut and must be eaten to take
effect; these are dealt with in Part 2. Part 3
covers others - such as insect pathogenic
fungi, bacteria and fungi that control plant
pathogens and weeds, Rhizobium inoculants
2 Introduction
Table 1.1 Main types of formulated organism and environments to which they are applied

Organism Distributed life stage Mode of action Main environment

Spore-forming bacterial Crystal toxin, durable Stomach poison, Plant surfaces,

insecticide spore infection water, soil
Protozoan insecticide Durable spore Infects via gut Plant surfaces
Insect viruses Durable inclusion body Infects via gut Plant surfaces
Mycoinsecticide Relatively delicate or Infects on contact Soil, plant surfaces,
durable spore water, insect cuticle
Mycoherbicide Relatively delicate Infects on contact Plant surfaces, soil
spore
Bacterial herbicide Delicate bacterial cell, Infects on contact Plant surfaces, soil
tough spore
Fungi, bacteria Durable or delicate Infects or inhibits on Plant surfaces, soil
combating plant spore, bacterial or contact
pathogens actinomycete cell
Bacterial, fungal Bacterium, delicate Infects on contact Soil
symbionts
Entomophilic Infective stage (and Infects after search Soil, water
nematodes associated bacteria)
delicate

and avirulent strains of plant pathogens -
which act, infect or colonize after contact
with external surfaces of the pest or plant.
Part 4 deals with entomophilic nematodes
which have a power of search before infecting
(Table 1.1). Thus the formulator must concent-
rate on encouraging the pest to eat the organ-
isms, or on facilitating contact between the
organism and the external surfaces of pest
and plant, or on preserving search mobility,
as well as providing a food base for prolifera-
tion of the organisms in their new surrounds.
Contrasting problems and challenges are
often presented to the formulator by these
requirements, together with the needs of
the environments to which the organisms are
applied and the purposes for which they
are used. The organisms are applied to target
insects, or plants or their substrates, which
include foliage, soil, water and commodities
in food stores.
Various life stages and toxins of the organ-
isms are applied and these vary greatly in
their robustness. Their formulation require-
ments are more stringent than those for
chemical products. The active ingredient is
often a living organism, which must be kept
alive and in good health in order to achieve
the desired effect. It should not, therefore, be
subjected to harsh chemical or physical treat-
ment during the formulation process. There
may also be critical nutritional or physical
requirements for organism maintenance.
Organisms are particulate, which further
complicates formulation. Since beneficial
organisms are regarded as environmentally
friendly, it is desirable that any formulation
additive also should be environmentally
benign in order to retain this advantage.
On the positive side, many organisms
multiply and spread from the site of applica-
tion. Also, through genetic engineering,
improvements can be made in activity, stabi-
lity or distribution of an organism (or its
active ingredient). Any of these features can
reduce the formulation requirements for
effective activity. The insertion of genetic fac-
tors into the plant genome to produce a trans-
genic plant is regarded for the purposes of
this book as the ultimate formulation, analo-
gous to a systemic insecticide but superior as
it lasts the life of a plant and is transmitted in

the seed. This method made a big impact in
1996, its first year of wide commercial use,
and should encourage the use of other specific
control measures, including microbial insecti-
cides, in integrated pest management (IPM)
systems. However, together with recent new
chemical insecticides, it will cause severe
competition with Bacillus thuringiensis, the
leading conventional microbial. Just when
the biopesticide manufacturers are learning
how to compete with chemical pesticides,
these two innovations lend a note of urgency
to further improve the effectiveness of con-
ventional microbial products by better formu-
lation.
Despite the central role that effective formu-
lation plays in the practical and economic
success of conventional microbial products,
the formulation of organisms is a neglected
area when compared to fundamental aspects
of research. Although this may be due partly
to a lack of published information resulting
from commercial secrecy (usually the most
effective way of 'protecting' formulation
information), it is due also to a lack of
acknowledgement of the importance of the
subject. This is compounded by some
reported field trials which apparently indic-
ated that the formulation did not drastically
improve field activity (Bull, 1978; Couch and
Ignoffo, 1981; Payne, 1986). However, such
trials are subject to the normal extensive var-
iations of field plots which often mask the
desired effects, a handicap to field experi-
ments that is countered in the following chap-
ters by assembling and assessing as much
data as possible.
Review literature on formulation require-
ments of organisms has not kept pace with
the development of the subject. Formulation
of microbial pesticides has been reviewed by
Most and Quinlan (1986) and Devisetty
(1988), and for a wider range of organisms
by Rhodes (1993), amongst others. These
reviews, however, have concentrated on gen-
eral principles and have not presented data
about formulation processes, additives or
Introduction 3
effects. Some earlier reviews giving limited
data have been confined to products aimed
at specific narrow target ranges, and even
these lack practical information on specific
formulation ingredients and methods (e.g.
Angus and Luthy, 1971; Ignoffo and Falcon,
1978; Couch and Ignoffo, 1981; Soper and
Ward, 1981; Young and Yearian, 1986; Con-
nick et ai., 1990; Diagle and Connick, 1990;
McIntyre and Press, 1991).
The present volume endeavours to remedy
these shortcomings in depth, but in a reader-
friendly fashion. Here are some suggestions
about how best to use it. Part 1 describes types
of application machinery common for many
groups of organisms and for chemicals too,
explaining the types of formulation needed
for efficient use of products. In subsequent
Parts, the arrangement of organisms in groups
around mode of action facilitates penetrative
assessment of the basic requirements of for-
mulation in relation to target organism and
environment, by explaining in an analytical
way why each group needs somewhat differ-
ent formulation. The book brings together
information in a comparative and critical
manner so that each group of organisms can
benefit from ideas, technology and progress
in the other groups, within the framework of a
major reference work. Much effort has been
made to insert cross-references between chap-
ters to speed comparisons; the text has been
designed to facilitate navigation and locating
figures and tables quoted in cross-references,
particularly by giving the section in which
each occurs. Readers with varied interests
are serviced, ranging from those probing
theory and science (developed in the text of
chapters), to those desiring practical detail on
individual additives. Detail is partitioned into
tables and appendices. Attention is directed to
a series of recipe-type tables which explain
how to make specimen formulations. Within
the chapters comparable data are assembled,
assessed and compared as far as possible. In
contrast, in Appendix I additives - which are
the basic tools of formulation - are listed
4 Introduction
alphabetically for easy reference. Since there
is a bewildering array of most sorts of addi-
tives, only those actually tried with organisms
are included, not only those that have been
successful, but also those that have failed,
together with information on harmful effects
or unexpected beneficial effects. More data on
each additive can be sought by reference to
the index, which has purposely been made
comprehensive. Inevitably, formulation
terminology relating to microbials has become
somewhat specialized. The glossary in
Appendix III defines relevant additives and
terms, plus a variety of additional useful
words. Obviously a global system is desirable
for terminology of formulation/product types
for microbials and it seems reasonable to
follow the international system recommended
for chemical pesticides, so these terms are
listed in the glossary. Research needs and
the future are assessed at the end of each
chapter, then reviewed, compared and dis-
cussed in Part 5; this is possibly the most
important aspect of the book.
REFERENCES
Angus, T. A. and Luthy, P. (1971) Formulation of
microbial insecticides, in Microbial Control of
Insects and Mites (eds H. D. Burges and N. W.
Hussey), Academic Press, London, pp. 623-38.
Bull, D. L. (1978) Formulation of microbial insect-
icides: microencapsulation and adjuvants. Mise.
Pub/. Ent. Soc. Am. 10, 11-20.
Connick, W. J. Jr, Lewis, J. A. and Quimby, P. C. Jr.
(1990) Formulation ofbiocontrol agents for use in
plant pathology, in New Directions in Biological
Control (eds R. R. Baker and P. E. Dunn), Alan
Liss, New York, pp. 345-72.
Couch, T. L. and Ignoffo, C. M. (1981) Formulation
of insect pathogens, in Microbial Control of Pests
and Plant Diseases (ed. H. D. Burges), Academic
Press, London, pp. 621-35.
Devisetty, B. N. (1988) Microbial formulations -
opportunities and challenges, in Pesticide Formu-
lations and Application Systems, Vol 8 (eds D. A.
Hovde and G. E. Beestman), American Society
for Testing Materials (ASTM STP 980), Philadel-
phia, pp. 46-64.
Diagle, D. J. and Connick, W. J. Jr. (1990) Formula-
tion and application technology for microbial
weed control, in Microbes and Microbial Products
as Herbicides (ed. R. E. Hoagland), ACS Sympo-
sium Series No. 439, American Chemical Society,
Washington DC, pp. 288-304.
Ignoffo, C. M. and Falcon, L. A. (eds) (1978) For-
mulation and application of microbial insect-
icides. Mise. Pub/., Ent. Soe. America 10, 1-80.
McIntyre, J. L. and Press, L. S. (1991) Formulation,
delivery systems and marketing of biocontrol
agents and plant growth promoting rhizobac-
teria (PGPR), in The Rlzizosphere and Plant Growth
(eds D. L. Keister and P. B. Gregan), Kluwer, The
Netherlands, pp. 289-95.
Most, B. H. and Quinlan, R. J. (1986) Formulation of
biological pesticides, in Fundamental and Applied
Aspects of Invertebrate Pathology (eds R. A. Sam-
son, J. M. Vlak and D. Peters), in Proceedings of
the IV International Colloquium on Invertebrate
Pathology, Wageningen, Society for Invertebrate
Pathology, pp. 624-7.
Payne, C. C. (1986) Insect pathogenic viruses and
pest control agents, in Biological Plant and Health
Protection - Biological Control of Plant Pests and of
Vectors of Human and Animal Disease, Progress in
Zoology Vol. 32 (ed. J. M. Franz), Gustav Fisher,
Stuttgart, pp. 183-200.
Rhodes, D. J. (1993) Formulation of biological con-
trol agents, in Exploitation of Microorganisms (ed.
D. G. Jones), Chapman & Hall, London, pp.
411-39.
Soper, R. S. and Ward, M. G. (1981) Production,
formulation and application of fungi for insect
control, in Biological Control in Crop Protection,
BARC Symposium No.5 (ed. G. C. Papavizas),
Allanheld, Ottawa, pp. 161-80.
Young, S. Y. and Yearian, W. C. (1986) Formulation
and application of baculoviruses, in The Biology of
Baculoviruses, Vol. 2 (eds R. R. Granados and B.
A. Federici), CRC Press, Boca Raton, pp. 151-79.
PART ONE

PRINCIPLES OF FORMULATION

Formulated organisms are suspended in a suitable carrier, which is supple-

mented by additives to maximize survival in store, optimize application to the
target and protect the organisms after application. In contrast to chemical active
ingredients, they are particulate and live or proteinous in nature, making them
relatively sensitive to storage conditions and the environment.
TECHNOLOGY OF FORMULATION AND 2
APPLICATION
Keith A. Jones and H. Denis Burges

CONTENTS
2.1 Introduction 7
2.2 Functions of formulation 9
2.2.1 Stabilization 9
2.2.2 Handling and application 10
2.2.3 Environmental persistence 19
2.2.4 Improvement of action 21
2.3 Types of formulation 21
2.3.1 Dry products 22
2.3.2 Liquid suspensions 24
2.4 Conclusions 27
References 27

2.1 INTRODUCTION target in the most appropriate manner and

form;
Formulation technology must be considered
to protect the agent from harmful environ-
at all stages from production of an organism
mental factors at the target site, thereby
to its eventual action on the target. The
increasing persistence;
method of production often dictates sub-
to enhance activity of the organism at the
sequent formulation activities, which in turn
target site by increasing its activity, repro-
may lead to alterations being made in the
duction, contact and interaction with the
production process. The range of organisms
target pest or disease organism.
is given in Table 1.1, section 1.1.
There are four basic functions of formula-
A wide variety of approaches are available to
tion. These are:
the formulator to achieve these basic func-
to stabilize the organism during production, tions, ranging from production of liquid sus-
distribution and storage; pensions to solid briquettes, and even
to aid handling and application of the incorporation of the agent in a living organ-
product so that it is easily delivered to the ism. The final product developed depends on

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
8 Technology offormulation and application
Table 2.1 Problems faced by a formulator

Stage Function
Harvest Reduction of material bulk.
Division into particles able to pass spray nozzles
Stabilization Prevent growth of agent and contaminant microorganisms.
Prevent proteases denaturing active agent
Storage Avoid powders caking due to moisture uptake.
Control viscosity of liquids to keep particles in suspension without aggregation so
that they flow.
Retain viability
Application Ensure good performance in applicators for dusts/powders/granules.
Maintain appropriate viscosity of liquids to form spray droplets.
Ensure good performance of sprayers without making foam
Post-application Ensure good coverage of target and good product retention.
Reduce physical loss by rain or other means.
Protect agents from inactivating factors, e.g. sun.
Ensure deposit is palatable and preferably attractive to target pest

several factors, such as type and location of
target, availability of formulation materials
and application equipment, as well as user
preference. A formulator is, therefore, faced
with several problems and challenges, sum-
marized in Table 2.1. The aim of a formulation
process is to address these problems.
Optimization of formulation to address one
problem may result in an adverse effect on
another aspect of the product's function.
Formulation therefore normally represents a
compromise between these opposing effects.
Due to the diverse nature of climatic situa-
tions, targets and user preferences, a single
organism will often be formulated in several
different forms, each aimed at a particular
market. Broadly formulations of organisms
can be split into two groups, dry solid and
liquid. All formulations, however, must still
be practical and economically viable. For
example, it is not practical to develop a prod-
uct that includes a protective dye that also
stains the skin of the operator. It is not eco-
nomically viable to add expensive sunscreens
to a product that will be applied at a high
volume per unit area unless they are formu-
lated to stay in close juxtaposition to the
organism to minimize the quantity required
(Appendix II, Fig. II.6). Further challenges are
presented by a product that must be eaten by
a target pest. It must be distributed evenly in
the area where a pest feeds, or - to attract pest
to active organism - the product must be pre-
ferentially palatable to the feeding stage of a
pest. In contrast, an organism acting by con-
tact can attack all stages of a pest and may be
picked up as the target pest moves about, so
distribution is less critical and palatability is
not a concern as long as the product is not
repellent.
This chapter elaborates the four basic func-
tions of a formulation and how they affect
formulation approach: it also reviews the
main formulation types suitable for use
with organisms. Both function and type
are interrelated, each influencing the other.
However, other considerations must be
taken into account. General principles and
approaches are discussed, and descriptions
of particular products used for different
organisms are covered in ensuing chapters.
Formulation types for application to soil are
reviewed in section 7.5. Technology for seed
treatment is described in Chapter 8. Appendix
III lists and defines types of formulation and
additive. Appendix I describes individual
additives known to have been tried with
microbials.

2.2 FUNCTIONS OF FORMULATION
2.2.1 STABILIZATION
The longest period of time in the life of a
product elapses during storage, i.e. the time
between manufacture and eventual use in the
field. This period can range from several
weeks to years. Organisms are required to
remain viable during storage, with minimum
loss of potency/activity and without loss
or breakdown of the desired formulation
properties.
Couch and Ignoffo (1981) state that a shelf-
life of 18 months is the minimum practical for
microbial pesticides. However, most currently
available commercial chemical pesticides
require a minimum shelf-life of 2 years, and
Rhodes (1993) indicates that up to 4 years is
desirable. This should be achievable at tem-
peratures at which the product may be stored.
In the tropics it is not unusual for tempera-
tures in some pesticide stores to reach 40 C or
more for extended periods. For chemicals, this
target cannot be achieved with many unstable
active ingredients without appropriate addi-
tives to prevent breakdown, e.g. antioxidants.
Biopesticides and beneficial microorganisms
(Table 1.1 in section 1.1) are usually live, albeit
often in a dormant stage, so are generally less
stable than chemicals and cannot easily be
altered chemically to improve stability (sec-
tion 10.2). Improved stability can, however,
be achieved by treatment before formula-
tion, for example by appropriate growth
conditions during production (e.g. Whipps
and McQuilken, 1993; sections 4.7.3, 9.2), by
appropriate storage prior to formulation (e.g.
Georgis and Hague, 1991) or by appropriate
processing after production, such as drying
(e.g. sections 3.2.1, 3.2.2, 4.7.3; Jones, 1994).
In addition, or alternatively, additives are
included to improve stability.
Depending on the organisms involved, dif-
ferent problems must be addressed to
improve stability. Some organisms, such as
nematodes, may require air or free oxygen
2.2.1 Stabilization 9
(section 9.3.1) to remain viable, as well as
requiring appropriate energy stores. Deple-
tion of energy stores in this case may be lim-
ited through restricting movement during
storage (section 9.3.2). Others, such as fungi,
may be stored as a resting stage, and additives
that prevent premature growth may be
needed or those with a nutritive value
avoided (sections 4.6.4, 4.6.6); also moisture
may be a critical factor (section 4.6.3). Some
additives that inhibit germination of the
organisms after application must be avoided
(sections 4.3.7, 6.5; Roberts and Yendol,
1971).
With dry products, deterioration acceler-
ates if the product moisture content is allowed
to increase progressively above 5% (e.g.
Couch and Ignoffo, 1981). When exposed
freely to ambient air, the moisture content of
a material always approaches an equilibrium
with the air humidity. Equilibrium moisture
content increases as the air humidity increases
and may be excessive for hygroscopic mater-
ials. Even those not normally regarded as
hygroscopic may moisten enough to cause
particles to adhere together, sometimes indu-
cing caking and sometimes deterioration, par-
ticularly at high temperatures. Similarly,
moisture above 5% can impair products with
oil as carrier, as has been shown with insect
pathogenic fungi (section 4.6.5). It is therefore
important to store products in water vapour-
proof containers, sometimes with a hygro-
scopic additive such as silica gel (sections
3.3.6, 4.6.3). Removing water from, or drying,
oil carriers by heating prior to incorporation
of the microorganism into the formulation
may be necessary.
The pH of a product must be stabilized
within certain ranges. Very high and low pH
conditions will normally inactivate agents
(e.g. Griffiths, 1982; Salama and Morris, 1993;
sections 3.3.6, 4.6.6b). A buffer may therefore
be required, and certain additives with
extreme pH values must be avoided. Mainten-
ance of optimal pH may improve shelf-life
(Date, 1970).
10 Technology offormulation and application
Products based on water, such as some
baculovirus formulations, may support
growth of contaminant microorganisms,
most with alkaline or near neutral optima.
Contaminants may include antagonists of the
formulated organism; they may cause pH
changes due to accumulation of waste
products; or they may produce enzymes
harmful to the organism. Gases from micro-
bial activity can cause explosive release of
the product prior to, or on, opening. Initial
levels of contaminants and the range of
species present may be limited by appropri-
ate conditions or treatment during produc-
tion and timing, as well as handling, of
harvest (McKinley et al., 1989; Lisansky et al.,
1993); the problem may be minimized by
ensuring that inoculants or substrates used
in the production are, as far as possible, free
from contaminants, e.g. use of food-grade
ingredients (Sherman, 1985). A useful method
of minimizing growth of contaminants in
a product is to maintain the pH of the
suspension at a value outside the optima
for growth of contaminants, but not inhibitory
of infection, growth or replication of the bio-
control organism after application (section
3.3.6; Jones and Burges, 1997). In addition,
or alternatively, it may also be necessary to
include an additive that restricts the growth
of contaminants. Caution is required, how-
ever, in the use of medically important
antibiotics which by wide-scale application
in the environment might cause resistance
to develop in microorganisms pathogenic
to humans and other vertebrates. Growth of
contaminants can be avoided by drying the
product.
Many commercial products currently
available avoid part of the problem by recom-
mending immediate use or storage in con-
trolled conditions, e.g. refrigeration (Rhodes,
1993; Jones and Burges, 1997). However, this
may not be practical or possible in many
situations, such as in developing countries.
Moreover, it increases costs, thereby reducing
competitiveness with chemical alternatives.
Rhodes (1993) points to the commercial
success of some organisms despite the need
for restricted storage requirements, but their
total market value, now and in the future, is
limited. Special storage conditions should not
be regarded as an alternative to appropriate
formulation.
Stabilizing agents may also be needed to
protect the agent from the harsh physical or
chemical treatment sometimes required in the
formulation process, for example to reduce
shear effects in grinding or mixing, or to pro-
tect the organism from heat inactivation dur-
ing spray drying (section 4.6.2).
The product itself may require stabilization.
For example, granules and briquettes may
need binders to maintain their integrity over
a period of time. Liquid emulsions require
stabilizers to maintain the emulsion.
2.2.2 HANDLING AND APPLICAnON
Additives ensure that the product is easy to
handle and apply. For example, in suspen-
sions thickeners or suspenders help maintain
even distribution of the organism in the car-
rier, prevent clumping of the organism and
ensure its ready resuspension after prolonged
storage. Dusts and wettable powders contain
additives to prevent clumping and caking
(sections 2.3.1a, 2.3.1c). Additive types are
listed and defined in Appendix III and addit-
ives tried with microbials are described in
Appendix 1.
Effective and economic use of a product
requires the active ingredient to reach the
target: no matter how good the product, if it
does not reach the target it will not perform
the required function. With some chemical
insecticides this problem was partly solved
through the development of active ingredi-
ents or formulations able to move within the
plant - translaminar or systemic action.
Nematodes apart, movement of microbial
agents after application is not possible using
conventional technology, emphasizing the
importance of effective application. Genetic

engineering has developed transgenic plants
and microorganisms able to express microbial
products such as the toxins of Bacillus thurin-
giensis. These can be considered as highly spe-
cialized formulations, equivalent to systemic
chemical insecticides. However, beneficial
organisms have to be applied in some way,
usually - but not always - requiring the use of
application equipment.
Effective application is influenced by two
main parameters: appropriate equipment
and formulation. These two parameters are
interrelated; equipment will not perform at
an optimum if the characteristics of a product
are not suitable, and vice versa (e.g. Appendix
Tables 11.4, 11.7; Figs 11.4, 11.5). The formulator
aims to provide the product in the most suit-
able form for optimal performance of the
application equipment.
By far the most research and information on
application technology concerns chemical
pesticides. There are comprehensive general
reviews (e.g. Southcombe, 1985; Matthews,
1992), reviews on specific application techni-
ques (e.g. Maas, 1971; Reardon, 1991), and
others on application of specific groups of
pesticides (e.g. Ross and Lembi, 1985). Some
reviews concentrate on formulation principles
and the physics of pesticide application
(Hartley and Graham-Bryce, 1980; Barlow,
1985). However, many of the principles can
be extrapolated to microorganisms. Some crit-
icism has been made that application of
microbial pesticides has relied too heavily on
technology developed for chemical pesticides,
and more research should be devoted to novel
methods for the biologicals. Even so, in most
situations a farmer (particularly on a small
scale) is unlikely to buy special equipment
for microbials, whereas equipment used for
chemicals, mostly hydraulic nozzle systems
used at low to high volumes (Table 2.3
below), is already likely to be on site, and
compatibility of microbes with such systems
is more likely to lead to their use. Application
of microorganisms has been discussed by
Smith and Bouse (1981); Diagle and Connick
2.2.2 Handling and application 11
(1990); Reardon (1991); Jones (1993); Bateman
(1994).
A pesticide is broadcast in the region where
the target is located: some will impact on or
near the target; much will miss completely.
For example, Himel et al. (1990) estimate that
as little as 5% of the total active ingredient
applied reaches the target site, and Graham-
Bryce (1977) estimates that >97% of applied
pesticides are lost to the general environment.
Much can be lost through being blown or
drifting off target (Wodageneh and Matthews,
1981; Appendix Table 11.3), and a significant
amount of product can bounce or be reflected
from a target. With microorganisms there is
also some suggestion that particles are 'lost'
between the spray tank and target, as estim-
ates for the volume of suspension recovered
and number of microorganisms do not tally
(Smith and Bouse, 1981; Richards, 1984).
Application equipment is designed to max-
imize the effectiveness of a product through
accurate and safe delivery within the con-
straints of practicality and efficiency. Equip-
ment for solid and liquid formulations ranges
from small hand-held apparatus to large
machinery mounted on tractors and aircraft.
Types of equipment suitable for use with
microorganisms are summarized in Table
2.2. Types of formulation are discussed in
section 2.3.
2.2.2a Application of solid formulations These
consist of dusts, granules and briquettes; their
properties are described in section 2.3.1.
Walker (1976) summarized the requirements
of a good granule applicator as being able to
deliver accurately calibrated amounts of
product and to spread them evenly, without
damaging products by grinding or impaction.
The machine should be robust and easy to
handle, calibrate and repair, as well as in-
expensive. These criteria can be applied not
only to granule applicators, but also to all
types of applicator in general.
Dry products, particularly granules and bri-
quettes, have the advantage that they can be
 ......
Table 2.2 Equipment suitable for application of beneficial microorganisms' N

TlfPe Prillciple Uses Adz>allfl1ges Disl1dvl1l1fl1gcs

A LIQUID FORMULATraNS
Hand operated
Hose-end sprayer Container feeds concentrated product into water Gardens, Convenient, low cost Needs pressurized water
from hose and expels it via high-volume nozzle landscaped areas for small areas supply. Much product
runs-off target
Trigger pump Small pump, trigger activated, expels spray via Small, confined Low cost, easy use Low capacity, ideal for
sprayers nozzle areas liquid/soluble
formulations
Syringe/piston/ Spray drawn into cylinder of pump and forced Small areas, walls, Strong, robust, low Tiring to use. Variable
stirrup pump through hose and nozzle surface water cost coverage. Stirrup pump
needs two operators
Compressed air Hand pump or CO2 pressurizes air to force spray Indoor /outdoor, Convenient for many Maintenance/cleaning
sprayers through hose and nozzle small areas types of application essential
Knapsack sprayer Hand-operated hydraulic pump forces spray via Small areas, Durable, easy to Heavy when full
hose and nozzle(s) indoors and maintain, convenient
outdoor for many types of
application
Motorized, hand-held sprayers
Powered knapsack Small engine drives pump to force the spray Small/medium Larger areas covered Can be heavy for long
through nozzles(s). Engine also drives airblowers areas, normally LV by small volume. Air periods of use.
to help propel spray droplets stream improves Maintenance frequent
coverage of target
Controlled droplet For ULV /LV. Product gravity fed to spinning Medium/large Good target coverage Equipment may be
applicators disc/serrated edged cup/spinning cage, areas using spray under optimal delicate. Good coverage
distributing spray in small droplets of narrow drift to aid droplet conditions. Light and needs correct
size spectrum. Can have a motorized fan to help distribution. Fan- easy to use environmental conditions
break up and distribute droplets. Drops may be driven models can
electrostatically charged to attract them to target be used in
surfaces, in some cases to avoid the need of a enclosed areas e.g.
spinning disc. CDAs also vehicle or aircraft greenhouses
mounted
Vehicle-mounted power sprayers
Low-pressure boom Product pumped via boom and nozzles, normally Large area Price relatively low. Poor penetration of dense
sprayer on tractor/truck/trailer. Handguns can be fitted Can cover large areas canopy. Servicing
for remote/spot treatment rapidly frequent
High-pressure HV dilute product pumped via nozzle(s). Often Dense vegetation/ Good canopy Heavy, costly. Servicing
hydraulic sprayer on boom specially designed to improve coverage trees/buildings/ penetration. Durable frequent. Large water
livestock volumes as carrier
Air-blast sprayers Product pumped via a series of nozzles into Dense foliage, Good canopy High-powered motor
airstream from motorized fan trees penetration and normally required.
coverage at LV /HV Servicing frequen t
ULV sprayers Product pumped to LV nozzles or ULV CDA Large areas Smaller and lighter Accurate calibration
applicators as above. Airstream from fan propels covered without than HV sprayers. essential. Servicing
drops to target large volumes of Little water needed frequent. Good coverage
water needs optimal conditions
Aerosol generators Very small droplets formed by heat, rapidly Confined spaces, Fog reaches Particulate material
and foggers whirling discs/airblast/fine nozzles. Droplets e.g. houses/ inaccessible and difficult to use without
remain suspended as a fog greenhouses/ cryptic areas, e.g. blocking nozzles, if
stores small cracks present. Droplets can
drift far from target
Injection pumps Product pumped directly into carrier, e.g. water, Other application Product accurately Needs careful calibration
prior to distribution. Can be used in combination machinery can be metered. One system and valves to stop
with an irrigation pump used in various suitable for several product backflow,
situations products especially if combined
with irrigation systems
Aerial applicators
Fixed-wing aircraft Product pumped from hoppers via boom to La rge / very la rge Can trea t areas Expensive. High capital
hydraulic nozzles or CDA spinning cages. areas trea ted inaccessible to ground cost. Limited to
Droplets formed and distributed by airspeed. rapidly sprayers. No meteorological conditions
Aircraft single or multi-engine mechanical damage to that avoid excessive
crop. Rapid spray drift. Use difficult
in small areas and in
areas with many tall
obstructions
Helicopters Product pumped from hoppers via boom to Large areas, water As above. Higher initial cost and
hydraulic nozzles, or spinning disc/cage CDA cou rses trea ted Manoeuvrability and maintenance, thus
nozzles. Spray unit can be suspended by a cargo rapidly slower speed allow relatively high unit area
hook under helicopter more accurate cost
placement. No
runway
Radio-controlled Product pumped from small reservoir to CDA Medium-sized Can treat inaccessible Difficult to control.
and microlight nozzles areas trea ted areas rapidly. Low Delicate
aircraft rapidly capital cost. Take- off /
landing area small

B DUST AND GRANULE FORMULATIONS

Hand-operated applicators
'Pepper-pot' shaker Simple, hand-held container with holes through Small areas/ Very low cost, can be Amount applied can be
which product is poured individual plant home-made highly variable
parts
......
W
 ......
Table 2.2 (Contd.)
"'"
Type Principle Uses Advantages Disadvantages
Bulb/plunger Small hand-held unit with a piston/pump used Small areas, Simple, easy to use Amount applied can be
duster to expel product via tube/orifice especially forcing highly variable
dust into cracks
Mechanical duster/ Lever-operated bellows or crank-operated fan Small areas Light-weight, easy to Amount applied can be
granule applicator propels product via hose. In granule applicator, use. Useful for spot highly variable
product gravity-fed down a tube, can be fed treatment
below soil surface by spike attachment
Power dust/granule Knapsack-type. Product fed into air-stream from Small/medium Can be metered to Heavy for long periods of
applicator motorized fan and propelled via discharge nozzle areas and provide even use. Maintenance
buildings application frequent
Compressed air High velocity air picks up product from airtight Confined spaces Application into wall Amount applied variable
duster container and expels it via nozzle voids and similar
situations
Mechanically driven Metered product, fed from small, hand-pushed Small areas with Low cost, easy to use Amount applied can be
granule applicator hopper on wheels, falls to ground even surface, e.g. variable
lawns
Vehicle-mounted applicators
Tractor-mounted Hopper with ground-wheel-driven meter down Large areas Accurate, even Servicing frequent
granule applicator which product falls by gravity, normally via a distribution and
tube; often several fixed to tractor tool bar. Or placement below
hopper with a chain/auger at bottom, feeding ground. For treatment
onto a spinning disc - dispersion, can be of leaf whorls on corn,
directional. Airflow can be used to aid plant is bent as
distribution. Can be combined with ploughs to hopper passes over it
incorporate product in soil
Aerial applicators
Fixed wing Gravity / revolving agitator, feeds product from Large/very large Able to treat areas Application can be
hopper into tube with guide vanes hence to areas inaccessible to ground uneven/variable.
propeller slipstream. Or product is fed onto sprayers. No Expensive equipment
hydraulic/electrically driven spinning plate, or a mechanical crop and maintenance
rotary-cylinder damage. Rapid
Rotary wing Air from engine-driven fan forced along ducts on Large areas/water As above. Slower Product rate can be
side-mounted hoppers, product driven out via courses speed and uneven/variable. High
short booms; or single hopper suspended on manoeuvrability give initial cost and
cable under aircraft uses same principle. May use more accurate maintenance so unit area
spinning plates placement. No cost relatively high
runway needed
Miscellaneous
Soil injectors Liquid/ granular product pumped/ fed via Medium/large Precise application Easily blocked. Need
tube into soil. Normally vehicle mounted, areas and penetration regular maintenance
often used with chisel cultivators/shanks. into soil
Product can be fed 30 cm or more deep.
Hand injectors available consisting of point
injector and piston pump
Tree injectors Product forced via tube into holes drilled in Individual trees Accurate placement Slow, labour intensive,
tree trunks, can be a simple syringe treated often costly
Seed-coating Product sprayed (liquid) or fed (dusts) into Seed treatment Placement direct on Product may separate
machinery a mixing chamber and mixed with seed target during seed storage.
Treatment may not be
needed
Traps Pest attracted (often by feeding attractant/ Traps put in Avoids need for Traps can be
pheromone/kairomone) to open product houses/stores/ complete coverage removed/destroyed.
container where it is contaminated, then greenhouses or of infested area. May attract only a few
normally allowed to escape open-field Useful in pests or a non-pest
situations inaccessible stores stage. Good control
may rely on contagion
between individuals
* Adapted from Matthews (1992); Marer et al. (1988); Bohmont (1990).
LV, low-volume spray; CDA, controlled-droplet applicator; ULV, ultra low-volume spray; HV, high-volume spray

.....
U1
16 Technology offormulation and application
easily distributed by hand. Specialized equip-
ment ranges from simple hand-held devices -
such as a 'pepper-pot shaker', which is par-
ticularly suited to small-plot, low-input
agriculture and generally allows the product
to be placed accurately on the target - to large
tractor-borne equipment and aircraft suited to
application over large areas (Table 2.2). Avail-
able machinery is described in more detail by
Matthews (1992).
2.2.2b Application of liquid formulations In
these the organism is carried in a liquid,
normally oil or water. Addition of surfactant
(section 2.3.2) or oil and emulsifier to water, or
use of pure oil, forms drops of more even size
(Appendix II, Fig. ILl) than those of water
alone, with consequently better controlled
spray. Oil is preferred for ultra low-volume
(ULV) sprays, water is normally used as the
diluent at higher volumes. Table 2.3 classifies
volume application rates. Low volumes are
preferred in areas where water is scarce,
because they reduce the volume and weight
to be transported and the time needed for
application. Higher volumes are aimed at pro-
viding complete wetting of a target surface,
although this also causes high run-off of spray
from the target. The general trend has there-
fore been toward reduction of volume. With
chemicals, control has been best with low
volumes (Matthews, 1992). With microbials,
results have varied (e.g. Smith and Bouse,
1981; Topper et al., 1984; Jones, 1994, Jones et
al., 1997), but this may be due to a lack of
attention to efficient application.
Table 2.3 Volume application rates of liquid formulations (l/ha)
Designation Field crops'
High volume (HV) >600
Medium volume (MV) 200-600
Low volume (LV) 50-200
Very low volume (VLV) 5-50
Ultra low volume (ULV) <5
* After Matthews (1992).
Reduction of volume increases the need to
optimize spray droplet size to maximize
coverage of the target. Coverage is influenced
by droplet viscosity, impaction and retention,
and depends on several factors. Impaction is
influenced by complex interactions between
droplet size and velocity, as well as obstacles
in its path (Johnstone et al., 1977; Reardon,
1991; Matthews, 1992). Collection efficiency
of a target in an airstream is defined as the
ratio of droplets striking the object to the
number that would strike it if the air was not
deflected by the object. Collection increases
with droplet size and velocity, and decreases
with increase in target size. Himel et al.
(1990) divided drop sizes into two main
classes: small drops 100-150 j.lm diameter)
and large drops (>150 j.lm). The small
drops are primarily transported on turbulent
eddies and therefore penetrate through
foliage canopies, whereas the large drops are
primarily affected by gravity and impinge
on the first substrate in their path. A spray
cloud normally contains a range of droplet
sizes (e.g. AppendiX II, Fig. ILl). The
droplet spectrum is measured in terms of
volume median diameter (VMD) or number
median diameter (NMD). VMD represents
the droplet diameter at which half the
liquid volume is contained in smaller drops.
NMD is the diameter below which half the
number of drops is smaller. VMD is more
commonly used. Spray uniformity (even-
ness of drop size) can be measured as the
ratio VMD /NMD; a ratio of ~ 2 represents
controlled-droplet application spray. The
Bushes and trees' Forest (by air)
>1000
500-1000
200-500
50-200 3-5
<50 0.5-3

statistics for relating measured droplet
spectra are discussed in more detail by Bate-
man (1993).
A droplet impacting on a surface can be
retained, or lost through bouncing or reflec-
tion (discussed in detail by Spillman, 1984).
Retention is greatly influenced by the ability
of the droplet to wet the surface, largely deter-
mined by formulation (Appendix II, Fig. 11.6).
For example, the spread of a droplet on a leaf
is increased 8-16 times, and bounce off the
leaf on impaction is prevented or reduced
with formulation in oil or as an oil emulsion
(E. M. Chadd, Imperial College, Silwood Park,
Ascot, personal communication).
It is critical to ensure that the correct
amount of active ingredient reaches the target;
this is mainly influenced by the number of
drops available for impaction (Appendix
Tables 11.5, 11.6, 11.7, Figs 11.2, 11.7; Graham-
Bryce, 1977). Low volumes require the genera-
tion of smaller droplets, limited by the
increasing importance of effects such as eva-
poration of droplets and reduced collection
efficiency such that an optimum is reached
(Johnstone, 1985). Table 2.4 summarizes
optimum droplet size ranges for selected
targets. With microbial pesticides the particu-
late nature of the active ingredient may influ-
ence this optimum. Primarily an appropriate
number of active particles must be contained
within a droplet (Appendix Tables 11.5, 11.6).
For example, a low - i.e. sublethal- number of
B. thuringiensis spores/toxin crystals in a
small droplet may stop insect feeding before
lethal poisoning, and the insect may recover
and survive. It may be physically impossible
to obtain enough active particles in very small
droplets. In contrast, with a very active prod-
uct, used at low concentration, it is likely that
a significant number of smaller droplets
would contain no particles at all (Appendix
Table 11.6). In this case the distribution of part-
icles between droplets should follow a Pois-
son distribution (Amesellum et al., 1990). The
relationship between amount of active ingre-
dient and droplet size is discussed further,
2.2.2 Handling and application 17
Table 2.4 Droplet size ranges for optimum appli-
cation to selected targets'
Target Droplet size (j.Lm)
Flying insects 10-80
Insects on foliage 30-80
Foliage 40-100
Soil 250-500
After Matthews (1992); Bateman (1991).
with examples, in Appendix II and section
3.3.3a.
Effectiveness of additives in a formulation
may also be influenced by droplet size. For
example, the degree of filtration by a sun-
screen will depend both on its concentration
and on droplet size, which determine the
amount of screen covering the organisms
(Appendix II, Fig. II.6). Similarly the influence
of a feeding attractant may vary according to
concentration and number of point sources
(drops).
The production of optimum sized droplets
from a sprayer is greatly influenced by formu-
lation. Droplet formation and size are influ-
enced by viscosity (Appendix Table II.4, Fig.
II.4), volatility and to a lesser extent surface
tension of a liquid suspension (Sundaram,
1988). The influence of organisms on viscosity
depends on their concentration. Normal con-
centrations of Metarhizium flavoviride conidia
had negligible effect, but high concentration
(50 g spores/I) increased viscosity of a ULV
spray in oil from 5-8centipoises (c.p.) (Bate-
man, in press). Because of the pseudoplastic
behaviour of suspension concentrates, simple
cup viscometers may give a more reliable pre-
diction of flow in spinning disc sprayers than
more sophisticated viscometers (Bateman,
1996).
Particular sprayer types may need spray
liquid with physical properties confined
within certain limits. For example, a ULV
sprayer requires a solution/suspension
within a predefined viscosity range (Appen-
dix II, Figs 11.3, II.4, II.5). This is achieved by
formulation in an appropriate carrier,
18 Technology offormulation and application
normally a mixture of vegetable and mineral
oils (Appendix Table II.4), although water-
based ULV sprays (very low-volume, VLV)
are also possible. Many other sprayer types
are available (Table 2.2), requiring formula-
tions with different physical properties. Sev-
eral types, including knapsack and tractor-
mounted systems, commonly have hydraulic
nozzles of various types, all relying on pres-
sure to force liquid through a narrow orifice
to form droplets. Some microbial products
may contain large particles, e.g. insect debris
in viral insecticides. Large particles can block
nozzles causing incomplete or poor applica-
tion, avoidable by grinding dry products, wet
milling or filtration. In other sprayers droplets
are formed by a spinning cage inside which
formulation must prevent particles becoming
'caked'. Sprayer types are described in Table
2.2, and in more detail by Matthews (1992);
Reardon (1991) gives a comprehensive review
of aerial application, including the application
of B. thuringiensis.
The final size of a droplet reaching the
target depends also on the amount of
evaporation. The general characteristics of
evaporation from water-based droplets have
been fairly well described by a theoretical
model; evaporation rates from B. thuringiensis
formulations were essentially the same as
those from water droplets until the droplets
crystallized (Luo et al., 1994). Oil evaporates
much less than water as a result of different
vapour pressure and viscosity. Also, evapora-
tion rate increases as droplet size decreases
due to increasing surface to volume ratios.
For these reasons oil carriers are used most
often for ULV sprays. If water is used, espe-
cially with small droplets, an anti-evaporant
or humectant is added. Anti-evaporants may
form a film at the droplet surface to reduce
evaporation: often these are water-soluble
polymers (Appendix I). Humectants, such as
glycerol, glycols or molasses, increase hygro-
scopicity to reduce evaporation.
Suspended particles, such as microbes, also
alter physical aspects of drops. Their influ-
ence, particularly on spray disintegration, is
reduced if they are wetted (Smith and Bouse,
1981). Microorganisms can be hydrophobic or
hydrophilic, which will affect both choice of
carrier and wetter (section 2.3.2a; Appendix
Tables I.1-1.3, 1.5.).
Fogs or aerosols are the equivalent of the
lowest volume ULV spray with the finest dro-
plets. There are two types of fogging machine,
cold and thermal. Carriers such as cottonseed
oil or water can be used with cold foggers.
Thermal foggers generally use specialized
chemical carriers; it must be determined that
neither carrier nor the process of fogging
inactivates the organism. One carrier (VK2)
containing methanol and ethoxyethanol
added to water is limited in volume per acre
by phytotoxicity.
Fogs are particularly suited for use in
greenhouses because gentle wind does not
carry the fog away from the target area, and
high in-house humidity delays evaporation of
water from droplets. However, fogs should
not be applied in windy weather because
strong wind blows fog through spaces
between sheets of glass in modern metal-
framed houses, reducing deposit of droplets
on foliage to windward. This does not occur
in plastic-sheeted houses.
Performance of the product following
application is improved by several different
types of additive; these include sunscreens
and stickers (section 2.3; Appendix Table 1.6).
However, additives also influence features
such as viscosity and the wetting ability of
the suspension. Ideally, therefore, choice of
thickeners, humectants and wetting agents
should be made in the context of the formula-
tion as a whole.
Additives may be incorporated into formu-
lated products or added later to spray tank
mixes. For ease of use, and to ensure that all
necessary ingredients are present in the cor-
rect proportions, the former is preferable,
designed for the most amenable pest/
plant combination, but with the intention
that further additions should be made to

tank-mix formulations for more intractable
combinations.
Dyes are added experimentally in trials to
visualize deposits on foliage or target cards
sited in target areas (Appendix Table 1.9).
Although not a normal part of a formulation,
their presence may affect the performance of a
product, e.g. by additional protection from
UV light. This should be taken into account
when assessing the results of trials using
these dyes. Water- or oil-sensitive paper is
an alternative.
2.2.2c Application to water Initially a product
must readily penetrate through the water sur-
face. This has tended to lead to the use of
larger droplet sizes with sprays. Further
requirements depend on the type of water
body and the habits of the target within. For
example, target larvae of mosquitoes and
simuliid blackflies feed by filtering particulate
food from the water (Table 3.19 in section
3.6.). Blackfly larvae attach themselves by
silken threads to a substrate in rapidly
flowing water and trap particles moving
past. In contrast, mosquito larvae are
mobile, living mainly in static or slow flowing
waters. These range from large rivers to
small streams and from swamps to small
bodies like water butts, car tyres and leaf
axils. Logistics range from application across
large areas with or without dense foliage, to
searching for a host of small and varied
containers. The same target species may
occupy different habitats. Some species are
surface feeders, others bottom feeders (Table
3.19 in section 3.6). Appropriate formulations
must therefore be designed to deliver the
correct dose of the organism to the zone
harbouring target species, often over a period
of time. Thus slow release formulations, such
as briquettes, may be designed to disintegrate
into some particles that remain at, or just
below, the water surface and others that
slowly sink to the bottom. Slow release formu-
lations are not generally suited to fast flowing
streams.
2.2.3 Environmental persistence 19
2.2.3 ENVIRONMENTAL PERSISTENCE
Normally an organism must remain active for
a time after application, ideally throughout
the period that a pest is likely to attack the
crop, or in soil throughout the crop cycle.
Microbes are inactivated by several environ-
mental factors, including sun, high tempera-
ture, humidity, leaf surface exudates and
competitors (sections 3.4.1 to 3.4.4c, 4.3.3,
4.3.4, 7.4). Also they may be lost physically
from the target location by the action of
wind, rain or leaching (Jones and McKinley,
1987; sections 3.4.1, 3.4.2, 4.3.3 to 4.3.6, 4.4.2).
The relative importance of each of these
factors depends on why and where a product
is used. For example, inactivation by sunlight
is the most important factor reducing persist-
ence of microbes applied to foliage, whereas
field temperatures and humidity have rela-
tively little effect except on fungi and nema-
todes (Jaques, 1985; Jones and McKinley,
1987). In contrast, sunlight is not important
when microbial insecticides are applied to
pests in stores or for control of soil dwelling
pests; here temperature, humidity and soil
biota are more likely to affect persistence.
Formulations normally contain additives to
protect agents from ravages of the environ-
ment. The main effects are discussed below.
2.2.3a Inactivation by sunlight The most
harmful wavelengths reaching the Earth's
surface are between 280 and 320 nm (UVB);
320-400nm (UVA) are less damaging, but x8
greater in quantity. However, there may be
sensitivities of some organisms to wave-
lengths outside this range (e.g. Harm, 1980).
The power of light to penetrate into solids
increases with increasing wavelength,
UVC(<280 nm) ~ UVB ~ UVA ~ visible light ~
infra red rays (Shaath, 1990a, b). Greenhouse
glass transmits no DVB and some UVA ran-
ging from nearly zero at 320 nm to 90% at
400 nm (Pilkington Bros Ltd, St Helens, UK,
personal communication). Similarly some
plastics do not transmit UVB (K. Jones,
20 Technology offormulation and application
unpublished results). Penetration of UYB in
water is also limited.
To counter harmful effects, sunscreens are
often added to a formulation (sections 3.4.4e,
4.3.3; reviewed by Shaath, 1990a, b). Sun-
screens act by physically reflecting and scat-
tering, or by selectively absorbing radiation,
converting short wavelengths to harmless
longer ones. Reflectors include zinc oxide,
titanium oxide, silicate and talcum. Absorb-
ents comprise most of the materials tested
with microorganisms (Table 3.16 in section
3.4.4; Table 4.4 in section 4.3) including
specialized dyes and chemicals, such as
Congo Red and stilbene derivatives, which
absorb specific wavelengths given in Appen-
dix Tables I.9-I.12, as well as cheap and
readily available additives that absorb over a
wider range, e.g. molasses.
2.2.3b Effect of temperature Temperature is
important for the shelf-life of microbial prod-
ucts (section 4.6.1) and it can affect their activ-
ity once applied. Temperature optima and
limits vary with the microorganism (Table
4.5 in section 4.6). For example, insect patho-
genic viruses are unaffected by 1-10 C and
<OC has little effect (Tanada, 1953; David
and Gardiner, 1967; David et a!., 1971). At
the other end of the scale, exposure to 60 cC
for more than 10 min inactivates these viruses
(Entwistle and Evans, 1985). Infection pro-
ceeds at field temperatures in the plant grow-
ing season, but slows at lower temperatures.
Insect pathogenic fungi can be frozen; sur-
vival is improved by additives (section 4.6.1).
Although inactivated by high temperatures
(Table 4.5 in section 4.6) fungi, if protected
by additives, can survive spray drying
(section 4.6.2). Strains used in pest control
normally will not grow at 37C, a feature of
their safety to mammals. Rhizobia inoculants
have well defined optima for growth, and are
sensitive to high temperatures, which may
limit their use in the tropics (Sbmasegaran et
a!., 1984), although this may also be an effect
of desiccation at high temperatures.
Generally, most prolonged temperature
effects cannot be countered through formula-
tion. A more promising approach is selection
of strains of microorganisms active over a
wider range of temperatures.
2.2.3c Effect of humidity/water availability As
well as the effect of moisture content on
storage stability (sections 4.6.3, 4.6.5), some
organisms may also have certain moisture
needs for activity. Thus, nematodes often
need free water to survive on a target surface
and to move and locate their hosts. Antago-
nistic bacteria used against plant pathogens
need the plant surface to be wet in order to
establish themselves (section 5.3.5). Fungal
spores normally need high humidity to
germinate. (section 4.3.4). These needs may
be overcome by appropriate formulation in
oil or oil emulsion, or by the addition of a
humectant to the product (sections 4.3.4,
4.6.5). In general there is little direct effect of
relative humidity on the activity of viruses
and spore forming bacteria (Tanada, 1973;
Jaques, 1977); probably, however, humidity
does influence the rate or extent to which
these organisms are inactivated by other
factors, such as heat, leaf-surface chemicals
(next section) and sun (Jones, 1988).
2.2.3d Effect of chemicals at the target substrate
Chemicals on or in the target substrate may
influence organisms in various ways (section
3.4.5). These include chemicals present in the
soil, as well as plant secretions and chemicals
on the phylloplane. For example, on cotton
and many other xerophytic plants, high levels
of magnesium, calcium and manganese as
carbonates and bicarbonates are exuded onto
the leaf surface. This can raise the pH of the
leaf surface to values as high as 10 or 11 (Han
et a!., 1980; Jones and McKinley, 1987; Jones,
1988). The presence of these ions or the asso-
ciated pH value, when wetted by dew or
water spray, can inhibit or inactivate some
organisms (Andrews and Sikorowski, 1973;
Elleman and Entwistle, 1985). Thus a product
 2.2.4 Improvement of action 21
~ay be fo~mulated in microcapsules to pro- 2.2.4 IMPROVEMENT OF ACTION
vide phySIcal protection from the chemical
With organisms that act after ingestion, pha-
effect.
gostimulants encourage pests to eat more of
Plant extracts can also harm organisms.
the organism before it deteriorates on foliage
Some extracts depress the growth of B. thur-
or before a microbial toxin arrests feeding
ingiensis (Morris and Moore, 1975). However,
(section 3.4.3). Insect larvae are encouraged
since B. ~huringiensis acts more by poisoning
to eat more rapidly and may be attracted to
larvae WIth the crystal toxin than by infection,
the formulated products, particularly baits:
the bactericidal effect of leaves probably has a
neonate larvae may find a soft, nutritious
relatively minor effect on pest control. Plant
product easier to browse on than the hard
~xtra~ts have also directly or indirectly
surface of a leaf. Bait is generally a more effi-
mactlvated baculoviruses, or at least affected
cient method of dosage transfer than blanket
activity (Uchida et al., 1984; Richter et al.,
application and random transfer (Caudwell,
1987; Santiago-Alverez and Ortez-Garcia,
1993), although it is inferior to dust on ants
1992). Similarly, chemicals on the insect cuti-
(section 4.4.3). Phagostimulants can be added
cle may suppress the growth of fungal spores
to a spray tank mix or incorporated into a
(St Leger, 1993). Thus additives designed to
solid bait. Plant extracts and liquid or ground
overcome antimicrobial activity may be
plant products are often used. However, a
needed to improve the activity of products
stimulant must be selected with care as its
on these target substrates (see sections 3.4.3,
effect may not be the same with all target
3.4.5, 3.4.6).
pests Oones, 1990). Common solid baits
include bran and calcium alginate/wheat
2.2.3e Physical loss Microorganisms can be
mixtures for grasshopper control (section
lost from target areas through action of wind
4.4.3).
and rain, physical abrasion or flowing water.
Some chemicals synergize the action of an
Loss varies with type and properties of the
organism, e.g. sodium tetraborate increases
formulation. The effects of drop size and
the potency of some insect viruses (section
liquid properties on spray retention are dis-
~.4.6). Similarly, certain mixtures of organ-
cussed in section 2.2.2. Similarly, particle size
Isms or mixtures with chemical pesticides
in dry solid formulations also influences
cause potentiation. However, caution is
retention (sections 2.3.1a, 2.3.1b below). Fine
~eeded as some pesticides antagonize organ-
dusts readily adhere to leaf surfaces. Larger
Isms, and the effect can vary with pesticide
and heavy solid formulations will not easily
dose (sections 3.4.6, 6.5). Moreover, the
be washed away in moving water.
formulator is adding toxic chemicals to a pro-
Stickers (Appendix Table 1.6) improve
duct, which may negate the environmental
adherence of organisms to foliage and persist-
benefits of using safe microorganisms.
ence during wind and rain. Effectiveness var-
ies, from the delaying action of water-soluble
materials such as molasses, to the fastness of
2.3 TYPES OF FORMULAnON
materials such as resins, which dry to become
insoluble. Stickers (Appendix Table 1.6) may There are a wide variety of formulation types,
double up as thickeners (Appendix Table 1.4), both liquid and solid (GIFAP, 1989; Appendix
i.e. additives such as gums and molasses to ~II). The main types currently used for organ-
increase spray viscosity and reduce evapora- Isms have been classified by Rhodes (1993)
tion from drops, or they may double up as into dry products (dusts, granules and bri-
phagostimulants such as molasses (Appendix quettes) and suspensions (oil- or water-based
Table 1.7). and emulsions). A wider range of formulation
22 Technology offormulation and application
types, together with additive types, are listed
and defined in Appendix III using nomencla-
ture recommended for adoption worldwide.
Additional specialized methods may be used,
such as sponges for nematodes. Chapter 7
describes formulation types from the view-
point of application to soil.
2.3.1 DRY PRODUCTS
These comprise dusts, granules and bri-
quettes, a classification based on particle or
aggregate size (Appendix III). Also included
in this group are wettable powders, which are
formulated as dry powder designed to be
added to a liquid carrier, normally water,
just before application.
2.3.1a Dusts Based on inert diluents or
carriers, normally of low absorbent capacity,
these have particle sizes ranging from
5-20 mm. Particles of <10 mm are abrasive
and insecticidal, also an inhalation hazard,
but the smaller particles adhere best.
Minerals such as clays are often the first
choice of diluent, but silica minerals are also
used (Appendix Table 1.2), varying the pro-
portions to obtain the desired bulk density
(Matthews, 1992). Diluents with high surface
acidity or alkalinity are usually avoided as
they tend to form an unstable product
(Polon, 1973). Inert fillers (Appendix Table
1.2) used with insect pathogens are listed by
Angus and Luthy (1971), also Couch and
Ignoffo (1981); longer lists of available fillers
are given by Polon (1973) and Becher (1973).
Dusts typically contain <10% of an organ-
ism by weight. They are normally prepared
by feeding the organism into an airstream for
mixing with the mineral diluent in a ribbon
blender or Muson mixer (e.g. Table 3.5 in sec-
tion 3.3.1). Particle size, bulk density and
flowability are extremely important. The pro-
portion of components is varied to form a
free-flowing, fluffy powder which does not
stick to machinery (section 4.4.3) or allow
separation of the organisms from the diluent
during transport, storage and application.
Separation occurs if diluent particles are not
within 10 mm of the size of the organism par-
ticles. Bulk density should be 320-800 kg/m 3
(Polon, 1973).
At application, dust particles are carried on
air currents to penetrate plant canopies.
Smaller particles collect on target surfaces,
larger ones fall through. Usually only 10% of
the particles adhere to the surface. Stickers
can be included, such as milk powder
(which is hygroscopic so the air must be dry
during mixing), or a desiccant such as sodium
sulphate (added to prevent caking).
2.3.1b Granules, pellets, capsules and briquettes
Granules are discrete masses 5-10 mm 3 in
size, pellets are >10 mm 3, and briquettes are
large blocks up to several cubic centimetres;
the sizes are defined in Appendix III. Like
dusts, these products contain an inert
carrier holding the organisms. Carriers
include clay minerals, starch polymers, dry
fertilizers and ground plant residues (Ross
and Lembi, 1985; section 7.6.1). Choice of car-
rier depends on sorption (more important for
formulating slurries of organisms), hardness,
bulk density and product disintegration rate
in water (Polon, 1973). Soft carriers, e.g. ben-
tonite, disintegrate quickly to release the
organism. The product can be coated with
various materials to slow and control the
rate of release, which also depends on unit
size.
Typically the concentration of organisms
is 5-20%, usually <15%. There are three
types of granules: (1) the organisms are
attached to the outer surface of a granular
carrier in a rotating drum by a sticker (Table
3.20 in section 3.6.3); (2) the organisms are
sprayed onto a rotating granular carrier with-
out a sticker (Table 3.21 in section 3.6.4); (3)
the organisms are incorporated into a carrier
paste or powder which sets as a matrix (Table
6.8 in section 6.8; Table 7.6 in section 7.5.3),
size being controlled by passing the product
through a sieve. Type 3 is the most common

with nitrifying microorganisms. When the
carrier forms a protective coat around a core
aggregate of organisms, the unit is termed a
capsule. A code of nomenclature for granules,
matrices and capsules is described in section
3.3.1.
These products present no inhalation
hazard, do not readily drift in the wind and
can be measured easily or weighed out, in
contrast to dusts. Although the smaller prod-
ucts can be applied to surfaces such as foliage
and leafaxils, much material falls to the
ground. The larger products are applied
mainly to water bodies, having the advantage
of penetrating a foliage canopy. Briquettes,
used for mosquito control, can be placed
individually in water containers.
2.3.1c Wettable powders These predominated
among early commercial products and
comprise technical powders (e.g. Table 3.2 in
section 3.2.1; Table 3.3 in section 3.2.2)
blended with additives to make them stable
during storage on the shelf and readily
miscible with water, to which the powder is
added shortly before spraying (Table 3.11 in
section 3.3.4). As with other formulations
using water as carrier, chlorinated water
must be avoided (or the water allowed to
stand to evaporate the chlorine) for fear of
damaging the organisms.
Most wettable powders contain 50-80%
technical powder, 15-45% filler, 1-10%
dispersant and 3-5% surfactant by weight
(wetter; section 2.3.2a; Table 3.11 in section
3.3.4). The filler should be inert and hydro-
philic to mix well with water. Normally a
mineral such as silica is added to prevent
clumping and fusing during grinding (grind-
ing may not be possible with some organ-
isms) and aids flowability by minimizing
caking during storage. Caking reduces wet-
tability and clogs spray nozzles. However,
the silica in a product should be kept to a
minimum as silica is abrasive and wears
formulation equipment, as well as spray noz-
zles. Other materials such as clays or lactose
2.3.1 Dry products 23
(Appendix Table 1.2) may also be used as
fillers, with or without silica (e.g. McKinley
et al., 1989).
The dispersant (Appendix Table 1.4) neutral-
izes attractive interactions of like particles and
ensures that particles of the technical product
do not attract each other, but suspend uni-
formly in a water column without settling.
The World Health Organisation (WHO)
recommends that a product suspended in a
graduated cylinder for 30 min should have a
concentration of at least 50% of the original
concentration at a point halfway up the
cylinder. Polon (1973) lists dispersants suita-
ble for use with chemical pesticides. Excessive
amounts of soap or salts (sodium, calcium)
must be avoided with lignosulphate dispers-
ants to avoid killing or flocculating spores
(Darvan, 1978).
A dry powder, put into a liquid, must pene-
trate the surface and overcome surface tension
at the liquid-solid interface. The surfactant
helps to reduce this tension and allows liquid
to displace air around particles. Too much
surfactant, however, can cause excessive
foaming, preventable to some degree by sili-
cone antifoams and by using low-foam surfac-
tants. As a general rule no more than 10 ml of
foam should remain in a 100 ml cylinder 5 min
after mixing. Foam should be avoided not
only because it is a nuisance, but also because
some organisms, such as spores, separate dif-
ferentially into foam. When rain re-wets
sprays dried on foliage, surfactants also act
as detergents, increasing the vulnerability of
organisms to wash-off. Often one additive
acts as both dispersant and surfactant.
Usually, wettable powders tend to mix
slowly into water and separate mixers may
be needed before filling spray tanks, since
tank agitators are often not forceful enough.
Powders form unwetted balls, a few of which
may persist even after protracted mixing. A
filter, preferably of nylon mesh, is essential in
the spray line to prevent nozzle blocking.
Mixing problems can largely be solved by
dry blending a powder with a binder and
24 Technology offormulation and application
forming the mix into water-dispersible
granules. These break surface tension more
easily than powders. They allow high concen-
trations of organisms, flowing freely with lit-
tle dust and can be accurately measured by
volume like a liquid. However, production
costs are high, more agitation is needed for
dispersion in cold water in the spray tank,
and small particle sizes may be difficult to
achieve. These granules are gaining popular-
ity: a new range of 'Altox' wetting and disper-
sing agents has been developed for their use
(Appendix Table 1.5).
When a wettable powder or water-dispers-
ible granule is mixed into water to form a
spray, solutes are dissolved. These include
enzymes, bacterial nutrients and additives,
such as surfactants and sugars, which may
stimulate germination of some microbes. The
spray must not be allowed to stand, as deter-
ioration may be significant in 1-2 days and
could be accelerated by some surfactants
harmful to organisms.
Particles in these products settle rapidly in
spray tanks, so tanks fitted with agitators may
be essential. Settling can be reduced by min-
imizing particle size and adding thickeners to
increase viscosity of the spray tank mix.
2.3.2 LIQUID SUSPENSIONS
This range of formulations uses a liquid as
carrier, usually water or oil, but solvents are
also possible. The commonest are suspension
concentrates and emulsions, although there
are also specialized types such as microcap-
sules (Appendix III).
2.3.2a Suspension (flowable) concentrates These
are essentially suspensions of particulate
organisms in liquids. Particles account for
10-40%, suspender ingredient 1-3%, dispers-
ant 1-5%, surfactant 3-8% and carrier liquid
(oil or water) 35-65% by weight (e.g. Tables
3.9 and 3.10 in section 3.3.3). Viscosity should
roughly equal the settling rate of the particles.
This is achieved by the use of colloidal clays,
polysaccharide gums, cellulose or synthetic
polymers (Appendix Table 1.4; Battista, 1975;
Theng, 1979). A product should resist a small
force such as particle settling during storage,
but should flow freely under a greater force.
Dispersants reduce the rate of sedimentation
of solids, which is increased through reversi-
ble clumping (flocculation). Surfactants act as
emulsifiers as well as wetters and spreaders;
they are described by Anderson (1983) and
Smith (1993) among other additives, with
lists of available products. The oldest types
of surfactant are soaps, normally sodium or
potassium salts of weak fatty acids, e.g.
sodium stearate, which have a polar hydro-
philic (water-soluble) and a non-polar lipo-
philc (oil-soluble) end of the molecule.
Although soaps are effective anionic surfact-
ants, they precipitate as an insoluble scum in
hard water or acidic solutions and surfactant
properties are lost.
Synthetic surfactants are split into anionic,
cationic and non-ionic types (Appendix Table
1.5). They are similar to soap in possessing a
non-polar, lipophilic group (long 12-18
hydrocarbon chain), and a polar hydrophilic
group. The hydrophilic group differs from
that in soap. Anionic surfactants are very
good wetters and detergents; they include
alkylarylsulphonates, fatty alcohol sulphates
and alkylsulphonates. Cationic surfactants
are derived from ammonia and are generally
phytotoxic and effective bactericides; they
precipitate in hard water and are poor deterg-
ents, generally unsuitable for use with most,
if not all, microorganisms. A third group,
amphoteric surfactants, act as either anionic
or cationic depending on the acidity.
Non-ionic surfactants do not ionize in solu-
tion and so there is no detrimental effect of
hard water and highly acid solutions. They
are good dispersing agents and detergents
and can form stable emulsions. Wetting
can be improved by addition of anionic
surfactants. Non-ionic surfactants are mainly
derivatives of polyoxyetheylene and polyoxy-
propylene. They have a lipophilic hydrocar-

bon chain, e.g. fatty acid, and corresponding
alcohol attached to a second, hydrophilic,
oxyethylene chain. The length of hydrophilic
chain and the nature or length of hydrophobic
chain determine the surfactant properties; the
relationship between the lengths of these
chains is the hydrophile-lipophile balance or
HLB scale. The HLB scale is arbitrary, ranging
from 1 (lipophilic) to 20 (hydrophilic) (Becher,
1973). Table 2.5 summarizes properties of sur-
factants on the scale. Non-ionic surfactants are
the most useful type for use in formulation of
microorganisms.
Suspension of particles in the carrier
requires a surfactant (Appendix Table 1.5).
Depending on the reactions of the organism
and carrier used, the surfactant needs to be
hydrophilic, lipophilic or mid-way between
these. For example, lipophilic fungal spores
can be suspended in oil using a lipophilic
surfactant; highly purified baculoviruses
easily suspend in water with hydrophilic wet-
ters, but for suspension of unpurified insect
viruses the surfactant needs both hydrophilic
and lipophilic properties because they contain
much fatty insect material. Similarly, forma-
tion of a stable emulsion (see below) requires
a surfactant which is partly hydrophilic and
partly lipophilic (Table 2.5).
As well as their problems of foaming and
wash-off (section 2.3.1c), some surfactants
harm or inhibit microorganisms. Although
Table 2.5 Hydrophilic-lipophilic balance (HLB)
range and properties of non-ionic surfactants
(ranges shown in bold are not suitable for
formulation of beneficial microorganisms)*
HLB range Property
3-6 Invert (water-in-oil emulsion)
7-9 Wetting agent
8-15 Normal (oil-in-water) emulsion. Good
for wetting organisms with
hydrophilic and lipophilic properties
13-15 Detergent
15-18 Solubilizer
* From Anderson (1983).
2.3.2 Liquid suspensions 25
there is little experimental work designed to
critically test surfactants, repeated good
bioassay and/or field results with individual
wetters accumulate to allow significant con-
clusions to be drawn. However, laboratory
results should be extrapolated to the field
with caution because field conditions differ
with, for example, variable exposure times,
biodegradation, concentration of surfactants
by drying of drops and their periodic remo-
istening to make active solutions. Non-ionic
surfactants are less likely than ionic surfact-
ants to damage microorganisms. Their selec-
tion is discussed in sections 3.4.1, 4.3.6 and
4.6.6, and by Ward (1984). Surfactant manu-
facturers can provide HLB values as well as
advice on suitable uses and compatibilities of
their products. Surfactants added for storage
stability should be minimized because, due to
long exposure, they are more likely to harm
organisms than those put in the spray tank,
which can be varied according to the wettabil-
ities of different leaf surfaces.
Formation of a concentrate by suspending
finely ground dry technical powder in oil
increases sedimentation compared with sus-
pending in water, because of relatively larger
particle size and low density of the oil carrier.
Of oils used with microbial pesticides
(Appendix Table 1.1; Couch and Ignoffo,
1981), vegetable oils are preferable to mineral
oils to minimize phytotoxicity if rancidity and
solidifying during storage are avoided. Liquid
('wet') milling of a product in oil results in
smaller particles that stay in suspension
longer and are easily resuspended. Weinber-
ger and Greenhalgh (1984) give content ana-
lyses of oils from the ecological viewpoint.
2.3.2b Emulsions Emulsification reduces
sedimentation of particles during storage
and in the spray tank, because buoyancy of
the oil counteracts high relative densities of
the particles.
Appropriate surfactants (emulsifiers) are
required for the formation of a stable emul-
sion. Their molecules contain structurally dis-
26 Technology offormulation and application
similar groups - with opposing tendencies -
that enable them, in dilute solution, to adsorb
at the interface of the solution and a phase in
contact with it (section 2.3.2a). They modify
properties of the interfaces to allow formation
of emulsions and to enable the solvent, e.g.
water, more effectively to wet both particle
and target surfaces, as well as to spread on
those surfaces.
Once formed an emulsion needs to be
maintained. Separation and creaming can
occur at temperature extremes, and emulsions
are also affected by the hardness of water and
pH.
With normal emulsions the oil is the dis-
persed phase and the liquid in which it is
suspended, water, the continuous phase; vice
versa for invert emulsions. Invert emulsions
have high viscosity, so are less likely to separ-
ate. They produce larger drops with most
sprayers and, becZluse the external phase is
oil, evaporation from drops is minimal, both
factors combining to present less drift hazard.
Invert emulsions can be made by mixing two
phases at the spray nozzle, or are pre-mixed
with the addition of fatty amine salts. The
stability and possible phytotoxicity of these
formulations need to be carefully assessed.
2.3.2c Encapsulated formulations Microcap-
sules contain the organism and are compared
with granules in section 3.3.1. Sher (1977) lists
22 commonly used encapsulating materials,
including gelatin, starch, cellulose and several
types of polymers. Capsules give good protec-
tion from environmental factors, such as sun-
light and leaf-surface chemicals. Dyes can be
incorporated into capsule walls to increase
UV protection, also stickers and wetters can
be adsorbed to their surfaces to improve
retention on the target. Microcapsules are
usually formed by three methods:
Phase separation. A suspension of the organ-
ism (core phase) is emulsified in a solution
of the wall material in an immiscible
continuous phase. A change, such as pH
or temperature, makes the wall material
separate and deposit around the organisms
in the emulsified core phase. Spray drying/
fluidized bed spray coating can also be
used where a film-forming polymer, dis-
solved in a liquid phase, is sprayed on or
with the organisms. The polymer coats the
organisms and dries to form a solid micro-
capsule.
Interfacial reactions. Reaction between chem-
icals in an organic phase and those in an
aqueous phase forms a wall at the interface
between the two phases. Wall formation
can be initiated by heating or by adding a
catalyst. Particle size, wall thickness and
permeability can be altered by changing
variables such as the amount of an organ-
ism or the amount of wall-forming agent.
Physical methods. Multi-orifice centrifugation
and electrostatic encapsulation. A solution
of the capsule wall is fed through special-
ized nozzle orifices with a suspension of the
organism. Fluid 'rods' are formed sheathed
in a fluid shell, which breaks to form micro-
capsules. Size of the microcapsules is
determined by flow rates of solution and
suspension. For electrostatic encapsulation,
two aerosols are produced separately with
opposite charges on the core and coating
particles. These coalesce and solidify on
cooling. A problem with these physical
methods is that some of the organisms
may be on the outside of the microcapsule.
'Encapsulation' of the Bacillus thuringiensis
toxin has also been achieved by genetically
engineering and fixing other bacteria (section
3.3.5)
Depending on the material used for encap-
sulation, the wall is broken to release the
organisms by crushing, pressure within the
capsule, dissolution or hydrolysis. Unlike
chemicals, diffusion cannot be used to release
the organisms.
Once formed, microcapsules need to be
formulated as liquid suspensions, e.g. flow-
able concentrates, or as powders, granules etc.

2.4 CONCLUSIONS
This chapter illustrates the wide range of
approaches to formulation and the many tech-
niques available. There are many additives,
some with dual or triple roles. It is often diffi-
cult to recognize or measure effects of indi-
vidual additives, so the performance of
complexes should be tested in terms of the
end product under conditions of intended
use and compared with minimal mixtures.
Where possible, tests should also be carried
out over a range of ambient environmental
conditions. The overriding principle for any
successful formulation, however, is that it
must be effective, economical and practical.
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PART TWO

ORGANISMS WITH A PERORAL MODE OF

ACTION

Microorganisms that attack insects through the gut must be eaten to take effect.
The application target is the insect's food, not its outer body surface. Formulated
survival stages of the organism must be resilient and palatable, and must be
spread evenly over the food to await chance consumption. These organisms
cannot attack sucking insects that feed from within the plant unless formulated
systemically, as with transgenic plants.
FORMULATION OF BACTERIA, VIRUSES 3
AND PROTOZOA TO CONTROL INSECTS
H. Denis Burges and Keith A. Jones

CONTENTS
3.1 Introduction 34
3.2 Optimization of production and stabilization for
formulation 36
3.2.1 Fermentation of Bacillus thuringiensis 36
3.2.2 Production of baculoviruses 38
3.3 Formulation types 40
3.3.1 Formulations applied dry: dusts, granules and
capsules 42
3.3.2 Spray technology 44
3.3.3 Liquid formulations for sprays 48
3.3.3a Efficacy 48
3.3.3b Composition 50
3.3.4 Wettable powders and water-dispersible granules 51
3.3.5 Improvement of sprays by encapsulation 53
3.3.6 Causes and timing of deterioration 54
3.4 Additives 55
3.4.1 Wetters 56
3.4.2 Stickers 59
3.4.3 Phagostimulants 62
3.4.4 Sunscreens 67
3.4.4a Damaging wavelengths in sunlight 67
3.4.4b Effect of sunlight on different pathogens
in different situations 67
3.4.4c Mode of action of sunlight 74
3.4.4d Effect of conditions of test 74
3.4.4e Effectiveness of different sunscreens 75
3.4.5 Additives to counter foliage factors 77
3.4.6 Synergists 78
3.5 Systemic action 86
3.5.1 Toxin-producing plants 86
3.5.2 Toxin-producing endophytes 87

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
34 Formulation of bacteria, viruses and protozoa to control insects
3.6 Application to water 87
3.6.1 Problems of the aquatic environment and its
target insects 87
3.6.2 Suspension concentrates and wettable powders 89
3.6.3 Penetration of foliage for mosquito control 90
3.6.4 Floating and slow-release products 91
3.6.5 Encapsulation 95
3.6.6 Monomolecular surface films 96
3.7 Future trends and research in formulation technology 97
3.7.1 Pathogen production 97
3.7.2 Trends in formulation for use on land 99
3.7.2a Products applied dry 99
3.7.2b Products applied as sprays 99
3.7.2c Additives 102
3.7.3 Products for use in water 104
3.7.3a Suspension concentrates and wettable
powders 104
3.7.3b Granules, pellets and briqu.ettes 104
3.7.3c Monomolecular layers 106
3.7.3d Bacillus sphaericus 106
3.7.3e Products without live spores 106
3.7.4 Systemic expression of microbial factors 106
3.7.5 Novel strategies for control 108
3.7.6 Research priorities 108
References 109

3.1 INTRODUCTION trast, survival stages of pathogens produced

Three major groups of insect pathogens - bac-
teria, viruses and Protozoa - have one feature
in common: they normally infect or poison the
insects perorally when they contaminate
insect food. Thus to control an insect infesta-
tion they must usually be eaten, and need to be
spread evenly over the food environment and
be hardy enough to stay alive whilst waiting
for the insect to eat them. Formulation is vital
to ensure the efficiency of these processes. The
first task is to make the pathogen-treated food
palatable and able to cover the food evenly. On
ingestion, the pathogens invade the haemo-
lymph and target tissues, grow within and
kill the insects; then in nature survival stages
of the pathogen are protected inside the cada-
vers, which eventually disintegrate. In con-
for insect control have to be applied as sprays
or solids spread thinly over the surface of the
insects' food, and so are more exposed to the
environment. Thus, formulation must replace
the natural protection afforded by insect cada-
vers. The chosen pathogens need to be highly
virulent to kill by the smallest possible
dosages, a requirement also facilitated by
formulation. The modes of action and the
main features of the three peroral groups of
pathogens are summarized in Table 3.1.
Among the spore-forming bacteria, the
crystal-formers also produce durable crystals
of toxic protein, which make them virulent
and rapidly lethal (Table 3.1). From the
formulation aspect, these crystals behave in
some respects similarly to stomach poison
insecticides. Spores and crystals are eco-
 3.1 Introduction 35
Table 3.1 Mode of action of bacteria, viruses and Protozoa that infect perorally

Main pathogens and hosts Mode of action

Bacteria
Lepidoptera, mosquitoes, Proteinous toxin crystals (0.5 x 1.0pm) of Bacillus thuringiensis and B.
blackfIies (Simuliidae), sphaericus dissolve in gut, destroy epithelium, and stop insect feeding,
some beetles while spores (0.8 x 1.7pm) germinate as pH falls, replicate, penetrate gut
wall, cause septicaemia, eventually sporulate, each spore with a crystal
Protozoa
Lepidoptera, beetles, Typical genus Nosema. Spores (3 x 5f.Lm) germinate in gut and live in cells of
locusts and other gut, fat body and other tissue, replicate; eventually sporulate as organs are
Orthoptera destroyed. Often chronic, debilitating. Transmitted by fouled food,
cannibalism and transovarially
Viruses
Lepidoptera, a few beetles Mainly baculoviruses with virions protected in protein to form occlusion
bodies. Comprise nuclear polyhedrosis viruses (with many virions/large
polyhedral body 0.5-1.5 f.Lm in diameter) and granulosis viruses (with one
virion/small body, 0.5 f.Lm). Infection and reproduction as for Protozoa but
often virulent with acute infections

nomically manufactured by fermentation. For
all these reasons, the spore- and crystal-
forming bacterium Bacillus thuringiensis is
used in the leading products now available,
accounting for more than 90% of the microbial
insecticides currently used. In 1990 its annual
sales worldwide were estimated to be up to
US$80 million (van Frankenhuyzen, 1993).
Sales are projected to more than double by
the year 2000 (Bernhard and Utz, 1993).
Rigby (1991) lists 12 manufacturers and 55
current commercial products and their uses.
Compared with most chemical pesticides, the
toxin is somewhat unstable, but it is much
more stable than the distributive stages of
most pathogens used for pest control.
The biology and ecology of B. thuringiensis
and its toxins, their mode of action and cur-
rent status of research and application, have
been reviewed in a book edited by Entwistle et
al. (1993) and the economics have been out-
lined by Baker and Henis (1990). Other ento-
mopathogenic bacteria have only minor uses,
including B. sphaericus for mosquito control in
developing countries (Jones et al., 1993b).
Some Protozoa have durable spores, produ-
cible only in living insects, but their virulence
is relatively low (Table 3.1). There has been
little practical application, using mainly
Nosema Iocustae to control grasshoppers (Capi-
nera and Hibbard, 1987).
Baculoviruses are highly virulent and pro-
duce a tough inclusion body, a survival stage
comparable to a spore (Table 3.1). In these
bodies, virions are protected in an inert pro-
tein matrix. Viruses are used much less than
B. thuringiensis, but are applied to over a mil-
lion hectares of land annually (Jones et a!.,
1993b). Winstanley and Rovesti (1993) list 31
commercial viral products produced by 1992
and able to give 85% control of 18 different
pest species; all are baculoviruses used largely
due to their safety for humans and the envir-
onment. The biology, production and applica-
tion of baculoviruses have been described in
two volumes edited by Granados and Federici
(1986a, b) and in a recent book by Hunter-
Fujita et al. (1997a). A few other viruses with
protective inclusion bodies have been
employed for insect control, but not commer-
cially (Tiong and Munroe, 1976; Katagiri,
1981; McGuire et a!., 1991).
The current dominance of B. thuringiensis in
the microbial insecticide market has resulted
36 Formulation of bacteria, viruses and protozoa to control insects
in an advanced and diverse liquid and solid
formulation technology, which will be used as
the leading example in the present chapter.
Production of the viruses in the living insect,
and the moderately robust nature of the virus
protected in the proteinous inclusion bodies,
have resulted in data that extend this techno-
logy. There is little information about the
Protozoa.
The survival stages and toxin crystals of
entomopathogens are particulate (Table 3.1),
so formulation must be designed to handle
particles, not solutions. Their targets are
mainly insects that feed on open leaf surfaces
on land or that filter out fine particles of food
from water. Thus, single pathogen bodies, or
dumps of many in an optimum size range of
5-200 {fm, need to be spread evenly over
leaves or suspended evenly in water. They
can be formulated dry, or as a suspension in
a liquid. More information about the target
insects and their feeding environments will
be given in section 3.3 (Table 3.4 in section
3.3.1 and Table 3.19 in section 3.6.1 below),
where the different types of formulations
made with pathogens are described - after a
consideration of production technology.
3.2 OPTIMIZATION OF PRODUCTION AND
STABILIZATION FOR FORMULATION
The active ingredient in a formulation - the
pathogen - needs to be produced either in
vitro by fermentation, as with B. thuringiensis,
or in vivo, as for Protozoa and viruses. An
exception is the case of transgenic plants,
which form the toxin of B. thuringiensis sys-
temically within their tissues (section 3.5).
Within the limits of the biological require-
ments of the microorganism, as well as the
economic reality of achieving the most effi-
cient production for the lowest cost, it is
necessary to tailor the production process to
suit the needs of subsequent formulations.
This tailoring aspect will be the main theme
of this section. B. thuringiensis is used as an
example of production by in vitro fermenta-
tion. The baculoviruses are used as an ex-
ample of in vivo production.
3.2.1 FERMENTATION OF BACILLUS
THURINGIENSIS
At the end of a B. thuringiensis fermentation,
the formulator is faced with a complex mix-
ture of toxin crystals, spores, medium solids
and solutes with a high moisture content.
There are two alternative technologies, semi-
solid and deep liquid.
In semi-solid fermentation, bacteria are
grown on a sterilized, wetted, aerated, friable,
solid substrate, illustrated by Dulmage and
Rhodes (1971). This may be inert, e.g. vermi-
culite, or itself a nutrient, e.g. wheat bran.
When most cells have completed sporulation,
although ~ few vegetative cells will remain
capable of further growth, fermentation is
stopped and the technical product stabilized
by drying with hot air to a moisture content of
5% or less (section 2.2.1); this is critical for
good storage. Caking of the substrate creates
problems during fermentation, hinders even
aeration of the fermenting mass and makes
drying difficult. After drying, the solid sub-
strate itself acts as a carrier-extender and the
technical product is ground - an energy inten-
sive process - to the required particle size.
Grinders that do not produce excessive heat
must be used. Substrates that swell when
wetted for spraying, e.g. bran, must be
ground fine enough to prevent swollen par-
ticles from blocking spray nozzles. Additives
are mixed with the technical product to make
dusts and granules (sections 2.3.1a, 2.3.1b,
3.3.1) and wettable powders (sections 2.3.1c,
3.3.4). The physical problems of aeration, dry-
ing and grinding make scale-up of semi-solid
fermentation difficult. Prevention of nozzle
blocking is an awkward problem and techn-
ical products made from semi-solid media are
more suitable for the manufacture of dusts
than wettable powders. Little modification to
semi-solid fermentation can be made to
improve formulation. The method is now
 3.2.1 Fermentation of Bacillus thuringiensis 37
Table 3.2 Production of a Bacillus thuringiensis technical powder by centrifugation and spray-drying
fermenter broth (adapted from Lisanky et aI., 1993)

Ingredients (Appendix l) Percentage (w/w) Function Cost ($/kg product)

Centrifuged broth 97 Agent 28.00
Aqueous 40% Bevaloid 211 2 Dispersant 0.03
Gum arabic 1 Drying protectant 0.03

Preparation
1 Stabilize broth by adjusting pH to 4.1 with 5 M H2S04
2 Spin sample of broth in bench centrifuge at 2: 8000g; supernatant should be clear*
3 Sample for later tests on pH, solids, microbiological purity and cell count
4 Centrifuge at 2: 8000 g and < 35C with continuous stirring/ agitation of the feed
5 Sample concentrate as for step 21
6 Add 2% dispersant and 1% gum arabic or 5% lactose slowly while stirring for 5 min, ensuring that no
lumps remain
7 Spray-dry+, using a rotary atomizer, to 6-7% water content; lower levels may cause loss of potency;
higher levels will allow caking and contaminants to grow
8 Sieve to < 50lIm and mill oversize material at < 35 DC
9 Bioassay potency of product. The relationship between potency of a 20 gil solids broth and that of the
technical powder is about x5 for ssp. kurstaki and x3 for ssp. israelensis

* If cloudy, cool sample to 5'C to clear possible interference by antifoam and check microscopically. If there are> 1-2
spores and crystals per field, add 4 mill Superfloc C 577 to bulk broth, but with ssp. israelensis dilute bulk broth to 1.5%
with cold water, then add 6 mill of Superfloc to flocculate.
t If the concentrate is not to be spray dried immediately, stabilize by adding 2 gIl potassium sorbate and adjust pH to
4.1 0.1 with 5 M H 2 S04 , or cool to 5C.
t Spray driers vary. Check residence time of product in drier as over-exposure to heat will lower potency. Typical
temperatures are inlet 180-210 C and outlet 85C. Experiment with temperatures and throughput rates.

used for B. thuringiensis only in developing
countries on a relatively small scale.
Aerated deep liquid, in contrast, is easy to
scale-up in large fermenters of 60 000 1 capa-
city or more, of which some 97% is water
(Dulmage and Rhodes, 1971). At the end of
fermentation, this is concentrated by centri-
fugation or filtration through bags to a slurry
containing about 80% water. Centrifugation
may result in some loss of the smallest cry-
stals. The technical fermenter slurry may be
stabilized as a suspension concentrate (section
3.3.3b).
The usual alternative method of stabilizing
the technical product is energy-intensive
spray drying (section 4.6.2). The whole pro-
cess of making a technical powder is illu-
strated in Table 3.2. A cook's recipe style has
been used to list the ingredients and bring the
process to life by describing how to use them,
including some detailed but critical points
gleaned from experience, so essential to prac-
tical success. Many other tables in the same
style have been used in this book to illustrate
formulation processes. Despite high air tem-
peratures in the drier, spores and crystals
survive due to short exposure and the cooling
effect of water evaporation, although typically
there is a small loss of activity. Lactose (5%) is
a common alternative to the gum arabic
(Table 3.2) as a protectant against the heat
and may have the advantage of partial screen-
ing against sunlight in the field (Lisansky et
al., 1993).
As another alternative after centrifugation, a
technical slurry may be stabilized and dried by
lactose-acetone co-precipitation (Dulmage
and Rhodes, 1971). Aqueous lactose solution
and acetone, which is miscible with water, are
added to the slurry and filtered. The resulting
powder is washed twice with acetone, then
air-dried. On a laboratory scale this is a
38 Formulation of bacteria, viruses and protozoa to control insects
valuable alternative harvest technique that
avoids exposure to heat and washes away
water-soluble metabolites. It provides a check
on possible heat damage during spray drying
or on the presence of soluble toxic factors,
whose action may be confused with that of
the 8-endotoxin crystal in spray-dried pre-
parations. In many fermentation media, Ca 2+
ions precipitate f3-exotoxin, so much of this
toxin is removed by centrifugation. In conjunc-
tion with the right fermentation medium, the
co-precipitation method of harvest usually
gives a friable powder easy to formulate, but
it has not been used on a commercial scale.
After optimization of media to obtain max-
imum yield, there are sometimes formulation
problems that may require modifications to be
made. For example, it is sometimes difficult to
grind spray-dried powders down to particles
less than 20 Jim diameter and friability may
need improvement. Also, some powders are
very hygroscopic, which may require changes
of ingredients. All dry products absorb water
from moist air and should be stored in water
vapour-proof packs. Aleshina et al. (1986)
claim that addition of KCl to the fermentation
medium not only improves insecticidal activ-
ity but also physico-chemical properties (wet-
ting and stability of the formulation).
3.2.2 PRODUCTION OF BACULOVIRUSES
The most cost-effective method of producing
baculoviruses is in living insects. They can be
produced in cell culture on a small scale, but
there are problems in scaling-up production.
Currently, cell culture is at least x 10 more
expensive than in vivo production (Monnet et
aI., 1994; Weiss et aI., 1994), but it would have
the great advantage of virtually eliminating
contamination.
An example of producing virus in insect
larvae is given in Table 3.3, an in vivo process
also described by Shapiro (1986) and others. A
major problem that affects formulation is lim-
itation of the bacterial content arising from the
gut of dead larvae (Podgwaite et aI., 1983;
Grzywacz et al., 1997). This must be below
the upper limits set by registration require-
ments, but with no primary human patho-
genic species present and a limit of 107
colony-forming units (d. u.) / g in some coun-
tries [and in the USA before 1983; the Envir-
onmental Protection Agency now accepts
more, recently as many as 2 x 108 (N. R.
Dubois, US Forest Service, Hamden, Connec-
ticut, personal communication)]. Whilst insect
debris and contaminant microorganisms can
be separated from the virus occlusion bodies
by techniques such as differential gradient
centrifugation (Harrap et al., 1977), this increa-
ses costs by x4 (McKinley et aI., 1989). Modifi-
cations at the virus replication stage aimed at
improving formulation are mainly curbs on
spore-forming bacteria, such as use of antibio-
tics in larval food, good hygiene and stabiliza-
tion of larval cadavers as soon as possible after
maximum virus production or death of larvae,
e.g. by deep-freezing. Bacterial invasion can be
minimized by harvesting virus-infected larvae
just before death (McKinley et al., 1989).
Ignoffo and Shapiro (1978) found that virus
harvested from live insects was less virulent
than that harvested from dead insects; how-
ever, D. Grzywacz (Natural Resources Insti-
tute, Chatham, UK, personal communication)
found no evidence of this. Bacteria might be
curbed by low-temperature incubation of
large larvae, so that virus replication and cell
lysis continue with much less bacterial growth
(McKinley et aI., 1989). Vegetative bacteria can
be killed by manipulation of subsequent
spray-drying (Huber, 1986; A. J. Cherry, Nat-
ural Resources Institute, Chatham, UK, perso-
nal communication).
Freeze-drying, although expensive, has
been the commonest method of stabilizing
virus (Table 3.3; Martignoni, 1978; Shapiro,
1982; Young and Yearian, 1986). It gave the
most active and most stable technical concen-
trate out of a number of methods compared
by Ignoffo et al. (1976b) for Heliothis virescens
nuclear polyhedrosis virus (NPV). Clumping
during freeze-drying may create storage and
 3.2.2 Production of baculoviruses 39
Table 3.3 Production of a technical powder (Gypchek) of gypsy moth nuclear polyhedrosis virus
(LdMNPV) from in vivo culture in larvae (J.D. Podgwaite, USDA, Hamden, Connecticut, personal
communication)

Ingredients (Appendix l) Percentage (w/w) Flinction

LdMNPV' 14-18 Pathogen
Insect body parts 82-86 Inert

Preparation
1 Egg masses of a laboratory strain (New Jersey) of the gypsy moth are held for 150 days at 6 'C to
complete diapause
2 Eggs are dehaired (Cosenza et al., 1963; Tardif and Secrest, 1970, modified by J. D. Podgwaite), surface-
treated for 1 h (10% formalin, v Iv), exhaustively rinsed and mechanically placed onto diet t (10-15 per
6 oz cup)
3 Larvae hatching from eggs are reared for 14 days at 26 c C in HEPA-filtered air chambers
4 When larvae reach early instar IV, cups are inoculated with 1 ml of a suspension containing 5x106 viral
occlusion bodies (OB) per ml and reared at 29C
5 Larvae (2: 70% mortality) are harvested 14 days after inoculation and held at -20 'C until processed
6 Frozen larvae are thawed for 24 h at 4 'C and then blended (1.0 g larvae: 5 ml sterile water) at high
speed for 10 s to release OB
7 Blended cadavers are poured through a 100-mesh vibrating separator (Sweco Inc., Florence, Kentucky)
and two layers of cotton cheesecloth (Type II, American Fiber & Finishing Inc., Colrain, Massachusetts)
to remove large body parts and urticating hairs!
8 The concentrate is centrifuged (Sharples AS-16VB, AHa Laval Separation Inc., Warminster,
Pennsylvania); continuous flow, 60 l/h at 15500 rev/min
9 Solids are removed, layered onto trays and frozen at -35 'C
10 Frozen solids are freeze-dried (Tri-philizer, FTS Systems, Stone Ridge, New York) for 24-36 h and then
ground (Braun Model KSM2, Braun Inc., Lynnfield, Massachusetts) to a fine powder (3-4% moisture)
that contains ca 15% (w /w) OB
11 The powder is subjected to microbiological quality assurance testing (standard plate counts, mouse
injection) before packaging in Scotchpak heat-sealable water vapour-proof pouches (Kapak Corp.,
Minneapolis, Minnesota)
12 The technical powder is formulated (tank-mixed, Table 3.15) at the application site in either a
lignosulphonate-molasses carrier or a commercial spray adjuvant (Carrier 038)"
Connecticut USA isolate; mixture of 12-15 closely related genotypes.
t Ingredients per litre final mixture: raw wheat germ (Mennel Milling, Fostoria, Ohio), 120 g; casein, industrial grade
(New Zealand Milk Products, Petaluma, California), 25 g; Vitamin Mixture 26862 (Hoffman-LaRoche, Fresno,
California), 10 g; Wesson Salt mixture (United States Biochemical, Cleveland, Ohio), 8 g; methyl parahydroxybenzoate
(Kalama Chemical, Seattle, Washington), 1 g; sorbic acid (Dirigo, Boston, Massachusetts), 2 g; agar (Moorehead and
Company Agar Products, Van Nuys, California), 15 g; water, 800 ml.
+ Hairs passing the screen and cheesecloth 1%) float to the surface of the concentrate and are removed by aspiration
prior to centrifugation.
Lignosite AN (Table 1.12) sunscreen 6% w Iv; Mo-Mix (Table 1.7) phagostimulantlantievaporant, 12.5% v Iv; Bond
(Table 1.6) sticker 2% w Iv; water, 85% v Iv .
.. Carrier 038 (Table 1.1).

tank-mixing difficulties, but these can usually
be prevented by suitable additives (McKinley
et al., 1989).
The NPV cream can be mixed with attapul-
gite clay or other diluent and spray-dried to
yield a microencapsulated formulation (Bull,
1978) as in the product Elcar (Shieh, 1989).
Clay increased activity of Spodoptera littoralis
NPV. Drying conditions are critical. Spray-
drying destroyed most of the activity of
Choristoneura fumiferana NPV (Young, 1989),
while with S. littoralis NPV under optimum
40 Formulation of bacteria, viruses and protozoa to control insects
conditions, no adverse effect has been ob-
served (A. J. Cherry, personal communica-
tion). It reduced the shelf-life of Cydia
pomonella granulosis virus (GV) (Huber,
1986) and a commercial product (Sandoz
406) was less stable than NPVs, even when
refrigerated,(Young and Yearian, 1986).
Air-drying has been used in some develop-
ing countries. For example, powders of Antic-
arsia gemmatalis NPV are made by pouring a
thin layer of virus suspension onto large
tables and leaving it to dry at room tempera-
ture for several hours, then scraping it off.
Bacteria obviously multiply during drying,
but speed of drying can be increased by fans
and warm air (Moscardi, 1989; F. Moscardi,
EMBRAPA-CNPSO, Londrina Parana, Brazil,
personal communication).
Lactose-acetone co-precipitation (section
3.2.1) has been used experimentally with
viruses (Ignoffo and Shapiro, 1978). NPV and
GV formulations mixed relatively easily into
water, but some activity was lost during the
process and shelf-life was poor (McGaughey,
1975; Hunter et al., 1977; Ignoffo and Couch,
1981; M. A. Parnell, Natural Resources Insti-
tute, Chatham, UK and K. A. J., unpublished
results).
Filtration can produce semi-purified virus
and microfiltration a more purified product
(M. A. Parnell and K. A. Jones, unpublished
results) as can Sodium Omadine (Dubois,
1976). A recent, inexpensive, novel and
experimental method of decontamination
uses high pressure and temperature. A pres-
sure of 550-580 MPa for 30-120 min at 50C
reduced microbial contamination of an aqu-
eous suspension of codling moth GV from
8 x 109 to 4 X 103 c.f.u. without lowering
potency of the virus, with similar results
with B. thuringiensis (Butz et al., 1995); later
work on GV was less promising (G. Zimmer-
mann, Institute for Biological Control, Darm-
stadt, personal communication). This process
may be feasible industrially, but would still
need inclusion of microbial suppressants in
liquid aqueous formulations.
Thus the virus technical concentrate most
often needing formulation is a mixture of
virus active ingredient, contaminant micro-
organisms which need to be prevented from
multiplying, and insect debris consisting of
fats, proteins, carbohydrates, etc.
3.3 FORMULAnON TYPES
Bacteria, viruses and Protozoa attack pero-
rally and are applied to control pests feeding
in four habitats: foliage, soil, food stores and
water. These environments present different
major formulation problems. On foliage, the
most intractable problem is young foliage
growing rapidly - Jones and McKinley (1987)
report a doubling of leaf area of cotton within
a week, and even greater rates of growth were
noted for cotton in Thailand (K. A. Jones,
unpublished results) - so effectively diluting
a surface spray coverage and necessitating
frequent costly resprays. In soil, many pests
feed below the surface, a difficult position to
reach with particulate microorganisms be-
cause the soil is an efficient filter of suspen-
sions applied to its surface. In stored bulks of
food, it is similarly hard to place pathogens
much below the surface of the bulk. In water,
it is hard to maintain the microorganisms sus-
pended for long in the feeding zone of pests
such as mosquito and blackfly larvae. By far
the most effective ploy that solves these major
problems in all environments is to apply the
active ingredient systemically to the pests'
food plants, especially by means of transgenic
plants (section 3.5.1). Here, we will discuss the
various alternative approaches taken to pro-
vide optimum formulation of products for use
in land environments. Treatment of water
bodies will be described in section 3.6.
The pest insects on land and key behaviour
factors that influence control are illustrated in
Table 3.4.
Larval behaviour, summarized in Table 3.4,
determines the amount of feeding on patho-
gen deposits, which are greatest on upper
surfaces of the leaves highest on the plants.

Table 3.4 Target insects on land, grouped primarily by habitat as it affects control by Bacillus thuringiensis,
baculoviruses and Protozoa, and secondarily by host plant
Pest insect
Lymantria dispar, gypsy moth, forest
defoliator; Plutella xylostella,
diamondback moth; Pieris rapae, P.
brassicae, white butterflies, cabbage
worms; Bombyx mori, moth, domestic
silkworm
Archips pomonella, small moth, leaf
roller
Choristoneura fumiferana, spruce
budworm
Trichoplusia ni, Sabulodes aegrotata,
moths, looper larvae
Spodoptera spp., Anticarsia gemmatalis,
Agrotis spp., Mamestra brassicae,
Lacanobia oleracea, Pseudaletia (=
Mythimna) unipuncta noctuid moths,
cutworms, armyworms
Heliothis (=Helicoverpa) spp., noctuid
moths, budworms, bollworms,
fruitworms
Cydia (Laspeyresia) pomonella, codling
moth
Ostrinia nubilalis, moth, European com
borer
Leptinotarsa decemlineata, Colorado
potato beetle
Galleria mellonella, wax moth
Newly hatched larvae (neonates), the most
susceptible larval stage, are too delicate to
chew completely though leaves. They browse
mainly on the softer lower surfaces, behaviour
which shields them from the heaviest patho-
gen deposits on the upper surfaces. These
they do not reach until later in life, when
they chew through the whole thickness of
3.3 Formulation types 41
Key ecological features
Young larvae feed singly or clustered mostly on lower
surfaces of open leaves (e.g. oak, brassica), leaving upper
epidermis intact. Older larvae eat through leaves, but do
not burrow
Weaves tree leaves together with silk, also feeds between
touching fruit
Larva I spins silken tunnel and feeds on the surface of
conifer needles until instar III, when it bores into buds
Young larvae feed on under-surfaces of open cotton and
vegetable leaves. Older larvae eat through the leaves.
Only the largest larvae bore into tomato fruits and
cabbage heads
Pests of cotton, vegetables, field crops and cereals. Young
larvae as Pieris spp. (above). Older larvae eat through
leaves, bore into growing points, hearts, fruits and stems,
often at soil surface. Hide in daylight
Young larvae browse on under-surface of leaves, as they
move up to young cotton bolls, tomato fruits and
growing tips of tobacco and other crops, into which they
bore while still in the early instars
Larva I feeds briefly on open foliage and surface of young
fruit on apple, pear and nut trees before boring into the
fruit, eventually emerging to find a pupation site
Young larvae feed on under-leaf surfaces and soon
migrate to spaces within leaf whorls, silks and young
com cobs. Older larvae bore into stems and growing com
seed
Adults and larvae feed on open potato foliage and on
tubers below ground
Larva I very mobile, settles after ca 6-12 h on bee-comb
and forms silken tunnel. Larger larvae span many comb
cells, harming brood and leaking honey
the leaves. Some species spin silken tunnels
or weave leaves together with silk. This prev-
ents the pathogen reaching part of the larval
feeding area. Boring and tunnelling abruptly
takes many species away from pathogen
deposits. Some species browse extensively
before tunnelling, others escape deposits by
tunnelling very soon after hatching, leaving
42 Formulation oj bacteria, viruses and protozoa to control insects
only a short window of opportunity for these
pathogens that act perorally.
Application techniques aim to maximize
deposits in the areas where the most suscept-
ible stages are found (Appendix II). All
formulation types likely to be used with
microorganisms are defined in Appendix III.
3.3.1 FORMULAnONS APPLIED DRY: DUSTS,
GRANULES AND CAPSULES
Products for application undiluted, in a dry
state, are used much less than sprays because
of the difficulty in handling and generally less
effective methods of application (Young and
Yearian, 1986; section 2.3.1).
Details of the preparation of a dust from a
technical powder are illustrated in Table 3.5.
Dust can be mixed with local carriers on site
to avoid transport and storage of bulky car-
riers. Formulation involving a sunscreen is
most effective if the screen is mixed in at the
liquid concentrate stage before drying to
ensure that it is as close as possible to the
organisms; this makes the required concentra-
tion of screen independent of the eventual
degree of dilution with the carrier (section
3.7.2b). Dust is the method of choice in
applying certain pathogens in particular
environments. Thus, in nature, cadavers of
soil-dwelling beetle larvae killed by Bacillus
popilline become virtual bags of spores which
disintegrate and wait for fresh larvae to eat
the spores. The first commercial microbial
product on sale - and still used - for insect
control contains this bacterium and mimics
nature by formulating spores in clay to form
a dust for spreading in small heaps spaced
over the soil surface (Dutky, 1963). Protozoa
(Nosema) and B. thuringiensis have been mixed
into attractive carriers as dry baits broadcast
over the soil surface against other pests, but
were only briefly available commercially (part
5 of Table 3.14 in section 3.4.3,). In the
storage environment, commercial dusts of
B. thuringiensis in a wheat flour carrier are
applied to the top IDcm of stored bulk grain
to control surface-dwelling lepidopteran pests
(McGaughey, 1985). Newly hatched larvae
feed selectively on such fine soft food particles
and the germs of wheat grains in preference to
the harder epidermis (H. D. B., personal com-
munication). Dusts of granulosis virus were
also applied experimentally to potato tuber
stores to control Phthorimaea opercullela (A.
Farghaly, Plant Protection Research Institute,
Cairo, personal communication). Nuclear
polyhedrosis virus (NPV) technical powder
can be mixed with a carrier for hand applica-
tion to maize leaf whorls, to control pests such
as Spodoptera jrugiperda. Early work reported
superior control of loopers, semi-loopers and
cabbage pests with B. thuringiensis dusts when
compared with conventional sprays (Hall,

Table 3.5 Production of a Bacillus tlwringiensis dust (500 IU/mg)*

Ingredients (Appendix l) Percentage (wlw) Function Cost ($/kg product)

Technical powder 0.6 Pathogen 0.18

Talcum powder 98.9 Carrier 0.18
Wessalon S 0.5 Free-flow agent 0.03

Preparation
1 In the ratio 5:4 mix technical powder (80000 IU Img) and silica powder, e.g. Wessalon S
2 Add mixture to carrier and mix in a powder mixer, e.g. Morton, Z-Blade or Paddle, or in a cement
mixer, until all aggregates have dispersed. Preferably mixture is delivered to the carrier in an airstream
while mixing. To avoid bulk transport, the above technical powder mixture (1) can be marketed for use
with a cheaper local carrier, e.g. another clay or gypsum
* From Lisarshy et al. (1993).
 3.3.1 Formulations applied dry: dusts, granules and capsules
1964; Sundara-Babu and Krishnan, 1970). This
was believed by Yearian and Young (1978) to
be due to more uniform coverage in optimum
conditions, especially on the underleaf surface
where young larvae generally feed and where
the virus is protected from sunlight. In con-
trast, under windy conditions sprays perform
better.
By far the greatest commercial develop-
ment of dry products has been for application
by specially equipped tractors (Table 2.2) to
the moist environment of leaf whorls of corn
plants, in order to kill corn borer larvae before
they burrow into stems. B. thuringiensis was
more effective and persisted longer when
applied dry than in sprays (Lynch et al.,
1980). Granules are larger than dust particles
and are preferred for corn borer control
because they are easier to handle and apply.
Clay granules can be made by spraying or
mixing with technical concentrates, preferably
with the aid of a sticker (Table 3.13 in section
3.4.2). This leaves the pathogens largely
exposed on the granule surface. For greater
protection they can be incorporated within
granules by agglomeration (section 7.7.3a)
and compaction (section 7.7.3c). Granules are
also made by mixing into a liquid carrier,
which gels and can be dried. Since this
embeds the pathogens into a matrix, these
products are usually termed capsules. The
distinction between granules and capsules is
a confused area. Logically a capsule has a
protective layer around the pathogens but
often matrices, which leave some pathogen
exposed on the surface, are called capsules.
The various terms are defined formally in
the glossary (Appendix III); the critical dis-
tinctions can be regarded as:
Capsule. An inner core containing active
ingredient, surrounded by a coat (e.g. algin-
ate capsule).
Matrix. Formed by a gelling process entrap-
ping active ingredient, but leaving some
exposed on the surface, e.g. pregelatinized
starch.
43
Granule. Carrier treated on the outside with
an active ingredient, e.g. clay, sand, corn
grit.
Water-dispersible granule. Made to disinte-
grate rapidly in water to form a spray (sec-
tion 3.3.4).
In this book, we usually use the term
adopted by the authors of the work cited,
with the result that capsules are sometimes
included in paragraphs discussing primarily
granules, and vice versa. The embedding mate-
rials must be soluble or freely permeable in
the insect gut to release the pathogens. Early
work by Raun and Jackson (1966) with cap-
sules of undisclosed composition gave good
control of corn borers but not substantially
better than other formulations. This work
was not followed up industrially, probably
due to the high cost of manufacture.
Recent work with corn starch preparations
has been much more promising. Starch con-
sists of amylose, which has a linear structure
with film-forming properties of great strength
and flexibility, and amylopectin, a highly
branched polymer. Cooking in water causes
starch to gelatinize, then to become insoluble
on cooling. Food-grade starch is freely avail-
able and inexpensive, ca US$1.32/kg for the
pregelatinized (soluble) cornflour Miragel in
the USA, and US$0.12/kg for nixtamalized
cornflour in Mexico. Early processing me-
thods involved harsh chemical or temperature
conditions, lethal to microorganisms. These
conditions were avoided by simply mixing
one part of pregelatinized starch and technical
B. thuringiensis with two parts of cold water,
drying the resultant gel, grinding and sieving,
or passing through an extruder then drying
(Dunkle and Shasha, 1988; Bartelt et al., 1990;
McGuire et al., 1990; McGuire and Shasha,
1995). On the shelf, no deterioration was
detected in 4 months. Benomyl was included
to prevent fungal growth in the moist condi-
tions within the corn leaf whorl (Dunkle and
Shasha, 1988). On a large scale, so much water
would make processing demanding. Water
44 Formulation of bacteria, viruses and protozoa to control insects
can be reduced to an equal part by mixing it
with substances such as isopropanol (30%)
which prevent or delay gelling, producing
friable granules that eliminate the need for
grinding and sieving. By altering the percent-
age of alcohol, granule size can be controlled
(McGuire and Shasha, 1992). Adding high
concentrations of a salt, e.g. CaCl 2, or a sugar
solution, e.g. molasses, allows further water
reduction and so less drying (Shasha and
McGuire, 1992). The sugar improves palat-
ability and addition of Coax improves it
even more (section 3.4.3), so that the bacterial
content can be reduced by 75% without loss of
insecticidal activity (Bartelt et al., 1990). These
developments tentatively push starch cap-
sules toward commercial feasibility.
In field tests, corn starch capsules preserved
B. thuringiensis activity in wet years better
than corn grit granules and gave 2: corn
borer control; both gave 2: control than the
normal chemical insecticide (McGuire et al.,
1994b). While Coax greatly improved palat-
ability and control (section 3.4.3; part 4 of
Table 3.14 in that section), control was not
improved by a sunscreen (sections 3.4.4b and
3.4.4e; Table 3.16 in section 3.4.4). Both types
of additive were used successfully in an
experimental formulation of starch-encapsul-
ated entomopox virus against grasshoppers
(part 5 of Table 3.14 in section 3.4.3).
Capsules can be made adherent to wet foli-
age. On drying they serve as effective bait for
corn borer larvae with considerable rainfast-
ness (McGuire and Shasha, 1992). In a com-
parative study, capsules were made by
mixing B. thuringiensis powder with the sur-
plus or waste agricultural materials gelatin,
pectin, colloidal chitin, alginate or corn starch,
involving a variety of reagents and additives
in an aqueous phase, then drying, grinding
and sieving. Chitin capsules were not accept-
able, while gelatin, pectin and corn starch
capsules stored well at room temperature (ca
26C) for 12 months, although no account was
taken of storage moisture content. A phagos-
timulant (section 3.4.3) is desirable to improve
palatability. Gelatin and pectin capsules
resisted wash-off by rain better than the
others (Morales-Ramos et a/., 1998). Biotrol 2,
an early granular B. thuringiensis bait, gave
control of budworms and hornworms as
good as or better than conventional pesticide
sprays (Creighton et a/., 1961).
3.3.2 SPRAY TECHNOLOGY
Spraying is the commonest method of ap-
plication of bacteria and viruses. Since these
pathogens act perorally, the spray target is not
the insects themselves, the surfaces over
which they move or the air they breathe, but
rather the insects' food, for which the opt-
imum droplet diameter is 40-100/lm (Table
2.4 in section 2.2.2; Appendix Table II.3). The
different types of formulation and application
equipment are described in Chapter 2; here
only their efficiency and their effects on the
pathogens are considered.
Most early attempts to obtain good spray
cover used high-volume (HV) sprays, formu-
lated with water as carrier. Spores, toxin crys-
tals, insect debris, fats etc. in unpurified virus
preparations are all hydrophobic, while virus
occlusion bodies have pH-dependent hydro-
phobicity, so wetters are needed to obtain
efficient mixing (sections 3.4.1, 4.3.6). The
pathogens are not damaged by the high pres-
sures in sprayers. Chlorinated water should
be avoided or aired for at least 8 h before use
because of its disinfectant properties. After
mixing, the spray must not be left to stand
for long periods before use because then the
pathogens deteriorate (section 3.3.6). Since
they are particulate they soon settle in spray
tanks, a problem solvable only by frequent
shaking or continuous agitation because HV
sprays are dilute, although additives slow
down settling (sections 2.3.2, 3.3.6).
A wide variety of HV sprayers have been
used, the choice being dictated by the acreage,
terrain and crop involved, as well as by what
machines are already available locally, par-
ticularly in developing countries (section

2.2.2 and Table 2.2 in that section; Topper et aI.,
1984; Jones, 1993). Target stages of foliar pests
mostly feed on the undersurfaces of leaves,
necessitating upward-and side-directed noz-
zles and presenting severe problems in dense
canopies, particularly where sprays must be
applied from above (Appendix Table 11.7).
This stimulated the use of low-volume (LV)
and ultra low-volume (ULV) with controlled
droplet application (CDA) technology, par-
ticularly for large inaccessible areas, or
where water was in short supply (section
2.2.2; Cunningham et al., 1975; Lewis and
Yendol, 1981; Topper et al., 1984; Renou,
1987; Reardon, 1991). Novel machines have
been used experimentally, e.g. Spodoptera
littoralis NPV has been sprayed through elec-
trostatic spray machinery without harm to the
virus (Jones, 1994).
Fogs have been used with viruses and B.
thuringiensis. There are two types of fogging
machines, cold and thermal. With a cold aero-
sol generator, cottonseed oil was a superior
carrier to water containing skimmed milk for
treating cotton with Heliothis NPV (Falcon,
1974). On single ash trees, an oil-water (1:2)
carrier, applied through air-shear nozzles
(11.7 J.Lm mass mean diameter) with fan-assist-
ance, gave better deposits of B. thuringiensis
and control of tent caterpillars (Malacosoma
disstria) than water alone (Johnson and Mor-
ris, 1981). With thermal fogging machines, B.
thuringiensis has been used in pulse-jet type
foggers without significant heat damage
(Burges et aI., 1979), but early exhaust-type
foggers were harmful. Water can be success-
fully used as carrier in pulse-jet foggers, at
least in greenhouses. With water, addition of
the VK2 carrier, used with chemical pesti-
cides, should be avoided because of phyto-
toxicity above certain spray volumes and
because it contains methanol and ethoxyetha-
nol (P. Jarrett, Horticulture Research interna-
tional, Wellesbourne and H. D. B., personal
communication).
Reduction of spray volume with conven-
tional sprayers also reduces droplet size
3.3.2 Spray technology 45
(Appendix II, Fig. II.7), which in turn
increases the importance of evaporation after
the droplet leaves the sprayer (section 2.2.2).
Evaporation can reduce droplet size too
much, making the spray prone to drift
(Appendix Table II.3), with poor impaction
and settling on target foliage. Formulation
with humectants and anti-evaporants may be
used to slow evaporation of droplets of LV or
even ULV water sprays, e.g. 0.5% of a hydro-
lysed polyvinyl alcohol, a refined algin mate-
rial (Keltose) (section 2.2.2; Appendix Table
1.4). More frequently, the rate of evaporation
is reduced by oil or oil-in-water emulsions as
carriers (Table 3.10 in section 3.3.3 below).
Entrapment of pathogens in emulsion dro-
plets in sprays of water has the additional
advantage of reducing settling in spray tanks
because the buoyant oil partly counteracts the
high density of the pathogen particles. Oils
used for chemical pesticides tend to be suit-
able because they are selected as having only
low phytotoxicity, a quality which reduces the
likelihood of harm to the pathogens. No harm
has been experienced during the short expo-
sure period involved in the spraying opera-
tion, but some oils tended to inactivate NPV if
used for long storage of products (Table 3.6;
sections 3.3.6, 3.3.3b). Some workers have
observed that viruses and B. thuringiensis
tend to move to the outside of oil drops,
which could lead to undesirable clumping of
the pathogens and more exposure to sunlight.
Some solvents, used to lower the viscosity of
spray in oil, damaged NPV but this did not
show in field trials (Table 3.7).
The effects on efficacy of drop size reaching
the forest canopy and of particle size within
drops, which disintegrate by splashing on
impact with foliage, are described by Entwis-
tle and Evans (1985), Fast et al. (1985) and Fast
and Reginiere (1984). With virus, less active
ingredient is required in a drop than with
B. thuringiensis, because the lethal dosage is
lower. In an extensive USDA effort to develop
spray machines, nozzles and specifications
for agriculture, NPV caused the highest
46 Formulation of bacteria, viruses and protozoa to control insects
Table 3.6 Oils (Table 1.1) used with Bacillus thuringiensis (Bt) and nuclear polyhedrosis virus (NPV)
Oil
Actipron
Bivert (emulsifiable crop oil)
Span 80 and No.2 fuel oil compared with water +
latex + dried blood
Mineral oils: Isopar M, Isopar V, Norpar 12, Norpar
13, Norpar 15, BP mineral seal oil, BP light
paraffinnic mineral oil, BP 50 spindle oil, BP 150
solvent neutral, Actipron
Vegetable oils: Seaton rapeseed oil refined and
deodorized, Seaton rapeseed oil refined, Seaton
soya oil refined and deodorized, Seaton
cottonseed oil refined and deodorized, Shell
Risella oil (L), Shell Risella oil (EL), BP Arachis
oil (Seaton)
Top oil (contains spreader penetrant)
Volek Spray Oil (Chevron): water (1:2) in fan-
assisted cold fogger
References
1 Entwistle, 1986; Barnett, 1992; Topper et al., 1984
2 Jones, 1994
3 Couch and Ignoffo, 1981
4 Smirnoff et aI., 1962
5 Cherry et aI., 1994, 1996
6 McKinley, 1985
7 Weinberger and Greehalgh, 1984
8 Smith et aI., 1977a
9 Johnson and Morris, 1981
Note: Vegetable oils are better than mineral oils, but need to mix mineral oil with vegetable oil in order to reduce
viscosity: quality of vegetable oils can be variable.
mortalities of Heliothis spp. in the combination
of small drop size, high drop density and
high NPV concentration, particularly with
increased viscosity caused by thickeners,
such as 0.5% polyvinyl alcohol (Table 3.8;
Appendix Table 1.4; Smith et al., 1977a,
1978a, b). Similar conclusions were drawn
from a laboratory study of Heliothis NPV
(Barnett, 1992).
However, lowering the spray volume has
often reduced efficacy. This is attributed to
Effects
Oil used at 20% in water for spraying NPVs of
Heliothis spp. and Srodoptera littoralis and for
aerial NPV spraysl,
Has been mixed with baculovirus spray3
Oils better for low-volume aerial sprays of NPV,
atomized more readily and effectively controlled
sawflies 4
Storage over 18 months at 4C resulted in ca x5
reduction in potency of S. lit/oralis NPV
in all except Arachis oil, in which all
activity was lost in 32 months but some
retained in Risella.
Vegetable oil mostly palatable to insects. Mineral
oils, except Norpar 12, generally reduced
feedings. Arachis oil/Shellsol T mix used
successfully in field trial (Shellsol required to
reduce viscosity)6
Used at 45% in sprays7. Can be phytotoxic8
Better deposits of Bt and caterpillar control on ash
trees than water alone9
poor coverage, particularly of underleaf
surfaces where the most susceptible larval
stages feed (Appendix Table 11.7). For
example, control of S. littoralis was poor on
cotton treated with NPV by a hand-held
spinning disc sprayer, compared with a
knap sack sprayer with a tail boom, which
directs the spray underneath the leaves
(Topper et al., 1984). Yearian and Young
(1978) report that with NPV against Heliothis
spp., optimum spray volumes were in the
 3.3.2 Spray technology 47
Table 3.7 Solvents used with nuclear polyhedrosis virus (NPV)

Solvent
Ethyl acetate
Petroleum spirit, Shellsol T,
isophorone, ethyl acetate
Toluene, petroleum distillate, methyl-
ethyl ketone combinations
Effect
Solvent for polymer did not harm NPV]
Petroleum spirit, Shellsol T inactivated Spodoptera littomlis
NPV, variable results with ethyl acetate. Isophorone
inactivated NPV at low (LDlQ), not at high doses (LD9d,
successful in field trials 3
Reduced activity of NPV by x 1.4 to x8, results erratic 4

References
1 Bull et aI., 1976
2 McKinley, 1985
3 Jones, 1994
4 Ignoffo and Batzer, 1971
Note: ethyl acetate and isopJiorone are best, but all can inactivate NPY.

range 90-400 l/ha, and yields were better
(though not significantly) with increasing
droplet size from 50 to 200 jim, partially con-
trollable with thickeners (Table 3.8). Yet inac-
cessibility of targets and the need to reduce
operator costs make volume reduction essen-
tial, thus much research has been directed to
improving LV and ULV formulation (section
3.3.3).
Tracer dyes, selected for their lack of harm
to microorganisms and insects (Appendix
Table 1.9), are added to microbial formula-
tions in trials to assess spray coverage (Morris
and Moore, 1975).

Table 3.8 Thickeners (Table 1.4) used with aqueous Bacillus thuringiensis (Bt) and nuclear polyhedrosis
virus (NPV) sprays

Thickener
Cargill Insecticide Base Concentrate
(stabilized molasses), Dowanol TPM
Carboxymethylcellulose, Kelzan (xanthan
gum)
Hydroxypropylmethylcellulose, sorbitol
Molasses and sugar solutions (thickener, anti-
evaporant)
Nalcontrol, Bivert
Effects
Increased drop deposition]
Deposits of Bt and control of spruce budworm
increased 2
Compatible with Spodoptera littoralis NPV, no anti-
feedant effece
In NPV tank mixes. Increase deposits on row crops and
forests 4
Deposit and drop size increased with Bt and NPV.
Form polymers or invert emulsions which reduce
evaporation. Effect of Nalcontrol minimal 5

References
1 Maksymiuk and Neisess, 1975
2 Morris, 1977a,b
3 Jones, 1988a
4 Young and Yearian, 1986
5 Couch and Ignoffo, 1981
Note: Best, molasses.
48 Formulation of bacteria, viruses and protozoa to control insects
3.3.3 LIQUID FORMULAnONS FOR SPRAYS
3.3.3a Efficacy About half of the present pro-
prietary B. thuringiensis products on the
market are oil-in-water emulsions of ever-
increasing efficacy. Larvae of some insect
species vomit after rapidly eating a high
dose, resulting in an interruption of feeding
and possible escape from death after the first
poisoned meal. Spruce budworm are less
likely than these species to escape from large
doses in this way. van Frankenhuyzen (1990)
and Payne and van Frankenhuyzen (1995)
concluded that the key to the success of B.
thuringiensis treatment of the spruce bud-
worm in the forest environment is to use
VLV with highly concentrated formulations
to deliver a lethal dose in the first spray dro-
plet encountered by a feeding larva. A lethal
droplet for larva VI on coniferous (Pinopsida)
leaves has a diameter of 80 pm at 95 billion
international units (IV) per litre, stronger
than any concentrated flowable yet on sale
(van Frankenhuyzen and Payne, 1993). The
droplet should be lethal because the crystal
toxin rapidly inhibits feeding and makes lar-
vae irritable so that they wander. It is thus
some time before a sub-lethally dosed larva
can feed again and eat more toxin. The resi-
dual toxicity half-life of high potency pro-
ducts was 1-2 days under rainy conditions
(van Frankenhuyzen and Nystrom, 1987).
Thus additives that increase persistence (sec-
tions 2.2.3, 3.4) not only increase the chance of
further dosage and death when larvae start to
feed again, but also render timing of B. thur-
ingiensis sprays less critical in terms of bud
and insect phenology. Additives also reduce
adverse effects of post-spray precipitation,
two constraints believed to contribute to
inconsistent efficacy.
The lethal dose for a larva has been
measured by placing precision droplets
individually with single larvae on leaves in

Table 3.9 Production of Bacillus thuringiensis water-based flowable concentrate (adapted from Lisansky et
al., 1993)

Ingredients (Appendix I) Percentage (wlw) Function Cost ($11 product)

Fermentation solids 10 Pathogen 2.80
Bevaloid 211 3 Dispersant 0.03
Veegum 0.45 Suspender 0.05
Xanthan gum 0.06 Suspender 0.03
Surfynol TG-E 0.5 Dispersant 0.06
K sorbate 0.2 Fungistat 0.03
5 M H 2 S04 to pH 4 Bacteriostat 0.03
Water to 100% Carrier 0

Preparation
1 Centrifuge broth as in steps 1-4 of Table 3.2, concentrating enough to ensure meeting required potency
of marketed product
2 Stabilize concentrate by adding 2 gil potassium sorbate, adjusting the pH to 4 with 5 M H 2 S04 and
adding 3% aqueous Bevaloid 211, mixing for at least 10 min in a paddle stirrer until sorbate dissolves
3 Bioassay stabilized concentrate and adjust with water to desired concentration for market
4 Dry mix enough Veegum and Xanthan gum powders in the ratio 7.5:1 to make a 10-fold strength pre-
gel, then slowly add to water while homogenizing with a Silverson high shear mixer at high shear and
< 60 DC for 30 min or until fully hydrated
5 Add pre-gel to concentrate (3). Mix at low shear
6 Check pH is 4. Adjust with 5 M H 2S04 or NaOH
7 Add 0.5% Surfynol TG-E last. Stir well, avoiding incorporation of air
8 Dispense into containers with continuous gentle stirring
 3.3.3 Liquid formulations for sprays 49
Table 3.10 Production of oil-based flowable concentrate with Bacillus thuringiensis ssp. kurstaki for ULV
application undiluted (Lisansky et al., 1993)

Ingredients (Appendix 1) Percentage (W/W) Function Cost ($/1 product)

Technical powder 24.0 Pathogen 6.72
Edelex 13 oil 74.4 Carrier 1.14
Bentone 38 1.2 Suspender 0.09
Propylene carbonate 0.4 Activator 0.05
Preparation
1 Mix suspender into a x 10 strength pre-gel for 45 min in 10% of the oil at maximum shear in a high-shear
mixer. Add activator and suspender (ratio 1:3, neat) rapidly while mixing at high shear. A very thick gel
forms in 5 min. Move head of mixer around in mixing tank to ensure uniform mixing
2 Transfer gel to main mixing tank
3 Use remaining oil to wash residual pre-gel into main mixing tank. Mix oil and pre-gel for 10 min to
ensure even mixture
4 Re-grind technical powder if necessary to achieve a particle size of < 50j.lm. Mix in powder of minimum
potency of 80000 IV/mg in batches until all lumps disperse
5 Filter through 125 j.lm sieve
6 Dispense into containers

the laboratory and retaining only the larvae
that eat all their droplet. Calculations from
these data to give droplet sizes for the field
are only a guide, which must be tested in field
trials. There are a number reasons for this
uncertainty. Oil droplets impinge better
onto leaves than water droplets (Appendix
II, Fig. 11.6), possibly because evaporation
from water creates a hard wall on the droplet
making it more prone to bounce-off (Sun-
daram et aI., 1993). Aston (1989) found a
disproportionate partitioning of toxin crystals
into droplets formed by spinning disc rotary
atomizers in proportion to the square of
droplet diameter, rather than the cube as
might be expected, an effect also reported
with NPV inclusion bodies by Killick (1990).
In addition, 40-70% of the crystals appeared
to be lost from a droplet during its formation
(Aston, 1989), while increasing pressure
through hydraulic nozzles in the field
decreased biological effectiveness - even
though plant coverage increased - possibly
as a result of a greater loss of crystals as
shear forces increased (Smith et al., 1977b).
Aston (1989) reported yet another factor, feed-
ing avoidance, which increased as the close-
ness of droplets decreased. A consensus of
literature on the biological effectiveness of B.
thuringiensis spray deposits suggests an opt-
imal guideline of 90 mm volume median dia-
meter droplets of high potency product at 20
droplets/cm 2 on flat, as opposed to needle-
shaped, leaves.
Suspension concentrates are ideal for pre-
paring concentrated products and they can be
formulated ready to use (illustrated in Tables
3.9 and 3.10). This started a remarkable chain
of cost reductions and increase in use of B.
thuringiensis for forest pest control. Viscosity,
kept high by the particulate pathogen and
accompanying solids, is a limiting factor.
This must be low enough for easy pumping
and application, ca 1500 centipoises for all
storage and use temperatures. Any increase
in volume should be made by adding pro-
prietary formulant carrier to avoid altering
the product viscosity. In the 1980s, potency
of products was at least doubled by minimiz-
ing the solid content of fermentation media to
reduce viscosity. In 1985-87, trials with ULV
for aerial application against the spruce bud-
worm achieved good control of moderate
infestations using only 21 (final volume) of
50 Formulation of bacteria, viruses and protozoa to control insects
50 - r - - - - - - - - - - - - - - - - - , bulk and small particle size, because particles
13.5
are not aggregated by drying.
45
12 The formation of emulsions from the aque-
40 ous concentrate improves stabilization of the
10.5 physical state of a product. It reduces sedi-
35
mentation of particles during storage and in
$ 9 S'"
~ 30 Application
,,
...................... \T()tal.c()st.
Q) the spray tank, because the buoyancy of the
, "
, oil counteracts the high relative densities of
'Q;"
~ 25
cost ... .....
,,
7.5 c
0
.~ the particles. In general, stabilization of activ-
c. +. 6 .~
gj 20 0.
c.
ity in aqueous emulsions (Table 3.9 in section
II
.... .+. ,, 3.3.3a) is more difficult and shelf-life is shorter
4.5
151~~~~ti~id~~-~~ .......<~
than with dry products. It mainly involves the
cost '
10 ''"-'- 3 prevention of microbial growth and the action
+.
5
Mixing/handling
~ . 1.5
of enzymes, most of which have alkaline or
near neutral optima. It is achieved by lower-
o 0 ing the pH of raw ex-fermenter B. thuringiensis
1980 1981 1982 1983 1984 1985
material to ca 4, and by adding preservatives
Figure 3.1 Cost components for Bacillus thllrillgien- such as .xylol (now discontinued, section
sis control of spruce budworm in forests in Maine, 3.7.2c, Appendix Table 1.8), sugar concen-
USA (from Cibulski et aI., 1993). trates or antibiotics, and preservatives com-
mon in the food and cosmetics industries
spray /ha (at least 22 x 109 IV /ha). Some (sections 4.6.6b, 7.6.3; Appendix Table 1.8).
products were supplied ready formulated to Suitable preservatives include sodium benzo-
spray, requiring only the addition of the ate, benzalkonium chloride, sorbic acid and
Chevron sticker (0.06%) at the airfield. This proprionate. On a small scale, Ejiofor and
minimized spraying costs by eliminating mix- Okafor (1991) added only molasses and palm
ing time and maximizing payload of the spray olein. They claimed no loss of activity after 2
aircraft (Valero, 1989). Costs have been further years in storage at 34 ec. Xylol was success-
reduced by transport ready-to-use in bulk fully used in early B. thuringiensis products,
tankers. The net result of these improvements and no deterioration was detected by bioassay
arising from advances in formulation has with Galleria mellonella in an exceptionally
been much lower costs, the component for stable ssp. galleriae oil-in-water emulsion dur-
application falling from highest to second ing storage at 2-5 ec for 18 years (H. D. B. and
highest (Fig. 3.1). In 1990, more than 70% of P. Jarrett, personal communication). Lacey
all North American forest Lepidoptera control (1985a) exposed B. sphaericus in buffer solu-
sprays were made with B. thuringiensis com- tions to pH levels from 3 to 10.8. The best
pared with less than 5% in 1981 (Cibulsky et preservation of insect activity and spore
aI., 1993). viability, 308 days, was at 4 ec and neutral
pH. At 4 ec, both values remained relatively
3.3.3b COMPOSITION high for 3 months at pH 3-10. At pH 10.8,
activity was lost in 1 week but spore count
B. thuringiensis liquid suspension concentrate remained high. Decline was faster at 21 than
is formulated from ex-fermenter slurry (sec- at 4 ec, the difference being significant for
tion 3.2.1). It avoids the cost of spray-drying activity in 2 months at neutral pH (see also
and the measurable loss of activity that occurs section 3.3.6).
at that time. Minimal solids need to be added In general, with both species of bacteria,
to the slurry, so it has the advantage of low manufacturers have difficulty in maintaining
 3.3.4
good storage of formulated products contain-
ing water for longer than 18 months without
refrigeration. Generally, oil-based concen-
trates are more stable than those containing
water, e.g. 2 as against 1.5 years at 25 DC (Devi-
setty, 1988).
Occluded viruses can be very stable in
water when purified. For example, S. littoralis
NPV was stored at room temperature in dark-
ness for 8 years without loss of activity
(McKinley, 1985). However, water-based for-
mulations have rarely been marketed, except
in developing countries, because of the diffi-
culty of curbing growth of contaminants in
less pure preparations. Growth is easily
arrested by drying in wettable powders (sec-
tion 3.3.4). However, currently there is more
interest in developing flowable concentrates
and two are at present on the market (R.
Georgis, ThermoTrilogy, Columbia, Mary-
land, personal communication).
In contrast to emulsions, formulation of a
concentrated bacterial or virus product by
suspension of finely ground dry technical
powder in oil alone (Table 3.10 in section
3.3.3a) increases sedimentation because of the
relatively larger size of particles and the low
density of the oil carrier. However, this pro-
blem can be reduced by grinding the particles
very finely, so that they form a colloid when
suspended. Oil has the advantages of low visc-
osity and minimal evaporation during spray-
ing, as well as easier stabilization of activity
due to low moisture content, the moisture con-
tent of the oil itself being removable by heating
before use. Table 3.6 and Couch and Ignoffo
(1981) describe some oils that have been used
with both B. thuringiensis and viruses. Veget-
able oils are preferable to mineral oils for
spraying because they are less phytotoxic and
possibly more palatable. However, freeze-
dried Spodoptera littoralis NPV stored equally
well at 26 DC in a mineral oil alone and a plant
(Arachis) oil for 15 months, then rapidly dete-
riorated in the Arachis oil to only <1-16%
activity after 32 months, compared to >90%
in the mineral oil (Cherry et al., 1998).
Wettable powders and water-dispersible granules 51
3.3.4 WETTABLE POWDERS AND WATER-
DISPERSIBLE GRANULES
Prototype and early B. thuringiensis and NPV
commercial products were wettable powders,
because they are relatively easy to produce. It
was soon realised, however, that these formu-
lations have two disadvantages: difficulty
with mixing into water and comparatively
large particle size.
Wettable powders consist of technical pow-
ders (Table 3.2 in section 3.2.1 and Table 3.3 in
section 3.2.2) plus additives to make them
readily miscible with water and stable during
r:;torage on the shelf. Details of formulation
with B. thuringiensis are illustrated in Table
3.11. Up to 80% of the product can be techn-
ical powder (section 3.2), the remainder being
fillers (Appendix Tables 1.1.2, 1.1.3), surfac-
tants (Appendix Table 1.5) and dispersants
(Appendix Table 1.4) included to improve
application and handling. The ratio of patho-
gen to filler is critical. The filler maintains
flowability and prevents agglomeration of
hydrophobic pathogen particles during sto-
rage, which would reduce wettability and
clog spray nozzles (Couch and Ignoffo,
1981). The filler (Table 3.11) can be the same
as that in powders applied dry (section 2.3.1)
but it must be hydrophilic and disperse
quickly in water as a uniform suspension; ca
33% lactose, e.g. in skimmed milk (which con-
tains 50% lactose), has been used very suc-
cessfully as filler. The insecticidal activity of
any toxin that becomes bound or adsorbed
to filler clays is retained and sometimes
enhanced (Tapp and Stotzky, 1995). With
virus, a wettable powder of S. littoralis NPV
contained 50% freeze-dried virus-infected
insects, 30% Speswhite china clay (English
China Clays Ltd, Stoke-on-Trent, Derbyshire)
plus 20% of a 1:1 mix of a synthetic silica
(Neosyl) as dispersant/ filler and a surfactant
(Etocas 30). The silica was included to prevent
clumping as well as to aid flowability (McKin-
ley et a/., 1989). Formulation as a wettable
powder improved short-term (15 month)
52 Formulation of bacteria, viruses and protozoa to control insects
Table 3.11 Production of wettable powder with Bacillus thuringiensis (adapted from Lisansky et al., 1993)

Ingredients (Appendix I) Percentage (W/W) Function Cost ($!kg product)

Technical powder 20 Pathogen 5.60

Kaolin China Clay 75.25 Carrier 0.44
Wessalon 5 0.75 Free-flow agent 0.03
Bevaloid 116 2 Dispersant 0.09
5urfynol 1045 2 Wetter 0.26

Preparation
1 From the bioassayed potency of the technical powder batch, calculate percentage, P, of powder needed
for required potency of marketed powder
2 To P% technical powder* add 0.75% (by weight of final product) silica powder, e.g. Wessalon 5, 2%
dispersant and 2% wetter iin a powder mixer (e.g. Morton, Z-Blade or Paddle). Mix at < 35C until all
silica aggregates are dispersed
3 Add (l00-P-4.75)% carrier. Mix thoroughly at < 35C
* If the fermentation ingredients have not conferred enough stickiness to the product, a sticker (e.g. maize dextrin) is
desirable.
, Alternative wetters are Air Products Acetylenic Surfactant 5485, Tween 20 and Montanox 80.

survival of the virus in store but had no long-
term advantage over unformulated technical
powder (Cherry et aI., 1998). When mixing a
spray, it is desirable to wet the powder with a
little water, then pre-mix to make a smooth
paste or cream before adding to the spray
tank. Surfactants should normally be either a
solid incorporated in the on-shelf product, or
a solid or liquid added to the tank-mix. Excep-
tionally they can be adsorbed to the surface of
one of the other ingredients, as was done with
Etocas 30 by McKinley et al. (1989). However,
adsorption to a solid will increase the amount
of time and agitation required to wet and
suspend the powder in water. Dispersants
ensure that particles of the technical product
do not attract each other and tend to distrib-
ute uniformly in a water column without set-
tling.
Water-dispersible granules have been
recently introduced to improve handling and
mixing, and to reduce the amount of fine dust
that puffs up into the air. Particles are held
together by binders (Appendix Table 1.4),
which may also serve as stickers during appli-
cation. In contrast to powders" the granules
mix instantaneously into water where dis-
persants (Appendix Table 1.4) in the granule
rapidly release particles on stirring. Dispers-
ants include low-foam surfactants (which
may later function as wetters) and water-solu-
ble additives such as sugars (which later act as
phagostimulants). At the end of processing a
free-flow material such as Supernat 22 is
added to ensure that the granules pour easily
and do not compact. Finally, light sieving may
be needed to remove dust. The extra proces-
sing and additives may increase manufactur-
ing time and costs, but this may be offset by
the useful dual functions of some of the addi-
tives and time savings when mixing spray.
During storage, B. thuringiensis and baculo-
virus have the great advantage of being the
most stable of the biocontrol agents. Shelf-life
of dusts, wettable powders and water-disper-
sible granules with satisfactory moisture
content exceeds 18 months, the absolute mini-
mum time required for trouble-free commer-
cialization. For example, most commercial
leaflets quote a shelf-life of 2 years for dry B.
thuringiensis products at a yearly average
temperature not exceeding 25C. At lower
temperatures, experience has shown that
powders lose little activity even after many
years in storage, provided that they have
been processed and stored correctly.
 3.3.5 Improvement of sprays by encapsulation
With viruses, most species of NPV remain
infective for several years at room temperat-
ures (Huger, 1963; Lewis and Rollinson, 1978),
and tussock moth NPV for at least 5 years in a
cool, dry place (Martignoni, 1978). In contrast
to NPV, the GV of Pieris brassicae survived
better as an aqueous suspension than a dry
powder (David, 1978). Loss of efficacy
occurred with Erinnyis ella GV after being
stored frozen for 3 years (CIAT, 1987).
At higher temperatures, activity is soon
reduced. The crystal of B. thuringiensis is
stable for 8 h at 80C but is inactivated by
15 min at 120C (Gaugler and Finney, 1982).
As long as containers were moisture-proof,
40C and 90% RH had no effect on the viabil-
ity of spores for 12 weeks (Pinnock et al.,
1977). With baculovirus, at 50C or above
activity is lost in hours or minutes (Jaques,
1977). At 38-42C, significant loss in activity
can occur in a few months or even weeks
(David and Gardiner, 1967; Hunter et al.,
1973; Jaques, 1977).
3.3.5 IMPROVEMENT OF SPRAYS BY
ENCAPSULATION
B. thuringiensis can be tank mixed with starch
powder and sugar to form a spray formula-
tion in water that autoencapsulates on drying.
The best of seven mixtures tested by McGuire
and Shasha (1990) was equal parts sucrose
and a commercial cold water-dispersible pre-
gelatinized corn starch, Mirasperse. In the
spray water, it did not clump to block nozzles
and viscosity was satisfactory. After drying
on leaves, the capsules held molecules in
juxtaposition to spores and crystals, complet-
ely surrounding most of them. The sugar
acted as a dispersant while in solution and,
together with the starch, as a sticker and
phagostimulant -after drying. On exposure to
simulated rain in a greenhouse, 70% remained
on cotton leaves after 2 weeks, compared
with 4 days or less for the six other mixtures.
In assays after 7-8 days, ca 80% of larvae
were killed compared with ca 20% by non-
53
encapsulated B. thuringiensis. Autoencapsulat-
ing starch-based sprays are not yet on the
market and have the disadvantage that they
need to be applied in a specified volume of
spray to achieve the correct concentrations.
This disadvantage can be overcome by dry-
ing into granules as a formulated product,
either to apply dry (section 3.3.1) or to use in
a spray. Fine products, encapsulated by
spray-drying, can be sprayed in any volume
because the protective matrix tightly holds the
pathogen in close juxtaposition to additives
such as sunscreens, i.e. in the optimal posi-
tion, to avoid waste. The functional and eco-
nomic significance of this is discussed in
section 3.7.2b. Tamez-Guerra et al. (1996)
spray-dried a mix of sugar (48%), pre-gelatin-
ized cornflour (24%), corn starch (24%), techn-
ical B. thuringiensis powder (3%) and citric or
lactic acid (0.3%). Insecticidal activity was
increased ca x2 compared with technical
powder, probably as a result of improved
palatability. Shasha et al. (1995) spray-dried
B. thuringiensis in kraft lignin solution, cross-
linked with CaCh to form insoluble capsules,
without reducing insecticidal activity. The
process of Tamez-Guerra et al. (1996) has
been adopted for use with baculoviruses.
Multiple-embedded nuclear polyhedrosis
viruses (MNPVs) including Autograplw califor-
nica MNPV (AcMNPV) have been spray-dried
without loss of activity, and preliminary
laboratory and field experiments demon-
strated improved solar stability (M. R.
McGuire, National Center for Agriculture
and Utilities Research, USDA-ARS, Peoria,
Illinois, personal communication). Nuclear
polyhedrosis virus, encapsulated in the
water-insoluble polymer styrene maleic
anhydride half ester with sunscreens, was
4-10-fold less active in bioassays than the
non-encapsulated virus. This is believed to
be due to inactivation by ethyl acetate (Table
3.7 in section 3.3.2) during encapsulation and
not to failure of gut juices to release the virus
in the insect gut. During encapsulation, the
NPV was dispersed in an ethyl acetate
54 Formulation of bacteria, viruses and protozoa to control insects
solution of the polymer in a high-shear stirrer
and spun into predominantly 10-30 pm dia-
meter capsules with a high velocity rotating
disk (Bull et al., 1976; section 2.3.2c). Addi-
tional materials for trial for encapsulation
included polycationic biopolymers, chitosan,
gelatin types A and H, and polyglycosamine.
Epiphytic microorganisms, transformed
with B. thuringiensis toxin genes, offer another
approach to protect the toxin from factors that
cause it to deteriorate. A number of organisms
have been investigated as carrier capsules, e.g.
Bacillus subtilis, B. sphaericus, Pseudomonas
cepacia, Agrobacterium, Erwinia, Klebsiella,
Flavobacterium and Alcaligenes (Rigby, 1991).
Some are aggressive colonizers of foliage.
For example, toxin-carrying Pseudomonas can
protect lettuce (Rigby, 1991) and toxin-carry-
ing Bacillus megaterium can protect cotton for
several weeks, far longer than conventional B.
thuringiensis sprays (Bora et al., 1994). Jacobs
(1989) reported on a programme to insert B.
thuringiensis toxin genes into a fluorescent
pseudomonad to control the sugarcane stem
borer, Eldana saccharina. The aim was to
increase persistence and coverage through
the ability of the carrier organism to colonize
plants; unfortunately the organism was
rapidly lost to the soil. Even if these organ-
isms are technically successful, they have yet
to pass the hurdle of registration of transgenic
microorganisms that can multiply in the
environment.
With CellCap products the registration
problem has been solved by killing the encap-
sulating carrier bacterium (Pseudomonas spp.)
and fixing the cell wall and cytoplasm by a
physical and chemical process (Barnes and
Cummings, 1986; Gelernter and Schwab,
1993). These capsules are formulated in the
same ways as conventional mixtures of spores
and crystals. In field tests the activities of the
resulting products were x 2-3 that of conven-
tional products and the persistence of activity
on the crop was extended, e.g. to give 76%
mortality after 7 days compared with 44%
for Dipel and 32% for Javelin, neither of
which contained sunscreens (Soares et al.,
1993). Activity in CellCap is due to the protein
toxin alone and, presumably, any loss of activ-
ity due to the absence of live B. thuringiensis
spores was compensated by synergism with
phylloplane bacteria (section 3.5.1) and out-
weighed by protection afforded by the cap-
sule. The CellCap products are a commercial
success, marketed mainly as liquid concen-
trates containing many of the major toxins of
B. thuringiensis.
3.3.6 CAUSES AND TIMING OF
DETERlORATION
There are many causes of deterioration of
bacteria, baculovirus and Protozoa because
they have live components and complex pro-
teins, such as crystal toxin and polyhedrin
(Table 3.1 in section 3.1.1). This section com-
pares deterioration at the different stages of
production, storage and use of the pathogens.
It highlights places where formulation can
help. The principal causes of deterioration
are high temperature, length of time of expo-
sure to causative factors, presence of free
water (as opposed to molecularly bound
water), adverse pH, enzymes (particularly
proteases), surfactants and combinations of
these factors. Some substances, e.g. certain
sugars and amino acids, stimulate bacterial
spores to germinate and lose their innate
resistance to adverse factors.
During its passage from production
through formulation to ingestion by target
pests, a pathogen passes through a number
of critical points. At different times, particular
deteriorating factors assume significant
importance. Some time elapses between stop-
ping the production process and preservation
by drying. Deterioration in fermenter liquors
can be minimized by cooling and lowering
pH to a minimum of 4 to curb not only dis-
solution of the B. thuringiensis by high pH and
the action of proteases and autolysis, but also
the growth of both contaminants and the
remaining vegetative B. thuringiensis cells. To

avoid brief extremely acid conditions during
the concentration involved in drying, the pH
of the liquors must not be lowered too much,
or must be partially raised again. For viruses,
near neutral pH is best (Ignoffo and Garcia,
1966). It protects the polyhedrin and those
virions that lie at or near the surfaces of occlu-
sion bodies, and also inhibits alkaline protease
(of insect origin) associated with the viruses at
harvest and capable of attacking occlusion
bodies. However, Guillon (1995) recommends
buffering at pH 4-4.5 by addition of food
grade stabilizers, e.g. ascorbic acid, to
simultaneously prevent growth of contami-
nant microorganisms.
During spray-drying, substantial time at
critically high temperature must be avoided,
e.g. the time particles adhere to drum sur-
faces, and high temperature in the mass of
particles accumulating in the collecting vessel.
For grinding the product after stabilization
by drying below a moisture content of 7%, a
machine must be selected that does not gen-
erate excessive heat, e.g. one with a chopping
action. Alternatively a 'wet' mill can be used
before drying.
The above conditions established during
stabilization and harvest allow stable shelf
storage of B. thuringiensis for 18 months to
many years, depending on formulation type
(sections 3.3.3b and 3.3.1). With viruses im-
purities are important. Purified spray-dried
or freeze-dried Spodoptera littoralis NPV lost
no activity during 32 months at 26 cC, but
unpurified virus lost 87-97% activity. This
deterioration was reduced by storage under
vacuum or mineral oil but not under a plant
(Arachis) oil. It was probably due to autoxida-
tion of fats as a result of exposure to oxygen,
not to the presence of bacteria (Cherry et al.,
1998). Moisture content was not measured, a
factor critical to the survival in store of many
organisms. Dempah and Coz (1980) observed
sharp decreases in activity of dry preparations
of B. thuringiensis var. israelensis stored under
humid tropical conditions in open containers.
Jones et al. (1991) reported that a wettable
3.4 Additives 55
powder of S. littoralis NPV stored unsealed
in the laboratory in Egypt lost 22% activity
in 1 month, 52% in 3 months and 90% in 1
year. High humidity caused premature germi-
nation of B. thuringiensis spores and autolytic
spoilage of products (Couch and Ignoffo,
1981), the effects being more rapid the higher
the temperature. Thus, water vapour-proof
containers are essential for storage of dry pro-
ducts which absorb moisture rapidly when
the containers are opened. Hygroscopic addi-
tives are best avoided if possible.
Before application, when a wettable pow-
der is mixed into water to form a spray fluid,
soluble substances are dissolved. These
include enzymes, bacterial nutrients, addit-
ives such as sugar (which may stimulate
spore germination), and surfactants (section
3.4.1). If the spray is allowed to stand, dete-
rioration is significant in 1-2 days and may be
accelerated by some surfactants that may
slowly solubilize the toxin crystal and poly-
hedrin at ambient temperatures. Standing
should therefore be minimized.
On application to foliage, deterioration is
accelerated by a number of factors. The
water evaporates, concentrating all the sub-
stances present and accentuating pH. Further
substances are dissolved from the leaf surface
(section 3.4.5) and the pathogens are exposed
to sunlight, rainfall, etc. (sections 3.4.4 and
3.4.2).
Formulation counteracts most causes of
deterioration by using a series of additives,
which are the subject of the next section.
3.4 ADDITIVES
Additives help a spray to reach its target and
improve performance once it is there. Wetters
facilitate leaf coverage, phagostimulants
improve palatability, and synergists increase
the effect on larvae. Of the post-spray haz-
ards, rain is countered by stickers, sun by
sunscreens and allelochemicals in leaves by
phagostimulants and neutralizing additives.
The following sections assess the importance
56 Formulation of bacteria, viruses and protozoa to control insects
of each factor and then the efficiency of each
type of additive in functionally increasing
the potency of a spray or decreasing the
adverse effects of the hazards. Available data
have been assembled in tables to indicate the
order of magnitude to be expected of each
type of improvement. For example, a synerg-
ist may lower the LC so up to 1000-fold in the
laboratory, but less in the field, where the
required pathogen dosage to achieve pest
control may still be reduced. This advantage
has to be assessed against availability, cost
and any disadvantages of adding the syner-
gist. Finally, indications are given of where
and when each additive is best used - is it of
general applicability and value as an insur-
ance to cover the worst-case usage, or should
it be applied just to tank mixes in varying
amounts for different pests, and sprayed
only on some crops and at some times only?
The units of quantification need some expla-
nation. Ratios are used. The effect of a factor
such as rain is expressed as the persistence
ratio of a pathogen with and without rain.
Similarly, the effect of an additive, such as a
sticker, is given by the ratio of persistence with
and without the sticker. The most valuable
ratios are obtained from LC so values. Fre-
quently, only percentages of mortality at single
dosage points are available. These are much
less useful, whether expressed as ratios or
just as differences between two percentages.
3.4.1 WETTERS
Wetters are used for three reasons.
They improve spray coverage on hydrophobic
leaf surfaces.
They facilitate mixing into water of hydro-
phobic spores and toxin crystals, as well
as NPV occlusion bodies which have pH-
dependent hydrophobicity (section 3.4.2).
They are also used to form emulsions between
oil and water by reducing interfacial ten-
sion and surface tension (see also sections
2.3.1c, 2.3.2b, 4.3.6 and 4.6.6b).
Table 3.12 summarizes uses, qualities and
performances of many wetters, as well as the
concentrations at which they are applied.
They are used at 0.01-0.5% in tank mixes
(Table 3.12; Table 3.15 in section 3.4.3 below)
and at 2-18% as dispersants in commercial
products subject to storage, including use at
4-5% as emulsifiers (Table 3.12, also Table 3.2
in section 3.2.1, Table 3.9 in section 3.3.3, Table
3.11 in section 3.3.4, Table 4.7 in section 4.6.3).
Dispersants in the products double up as wet-
ters on reaching the spray tank.
The best wetters can be judged by a general
assessment of the varied data in Table 3.12
(see Note at foot of Table) and the frequency
with which they have been used in practice.
Briefly, the best include the Tritons, Tweens
and the new organosilicone superspreaders:
non-ionic wetters are preferable. Liquid Tri-
tons and Tweens are unpopular with spray
operators because they are difficult to mix
into water.
Generally, B. thuringiensis and baculo-
viruses are compatible with many commercial
surfactants (Table 3.12). Little information is
available on the effect of surfactants on Proto-
zoa. Some wetters harm or inhibit pathogens
(Appendix Table 1.5). In the only truly crit-
ical study to mimic the effect of wetters
during spraying, B. thuringiensis was exposed
to wetter solutions in buffer for 24 h, then
accurately bioassayed on leaf or sheet bees-
wax (second group in Table 3.12). Only
Teepol L reduced potency during this severe
exposure. The non-ionic Triton X-100 and
Tween 80 have been widely and successfully
used in sprays, insect bioassays and viable
spore counts (Table 3.12; Burges and Hussey,
1971). Morris (1975) reported that Triton X-
100 impaired germination and! or bacterial
growth when added to broth in shake flasks
inoculated with B. thuringiensis spores. How-
ever, it is not phytotoxic on plants at opera-
tional spray concentrations (H. D. B., personal
communication) and it has been included in
experimental application of Helicoverpa
(Heliothis) armigera NPV in Thailand (K. A. J.,

Table 3.12 Wetters tested in aqueous preparations of Bacillus thuringiensis (Bt), B. sphaericus and nuclear
polyhedrosis virus (NPV) in alphabetical order of the first entry in each group
Surfactant (composition, Appendix Table 1.5)
Acetylenic Surfactant 5485, Tween 20,
Montanox 80
Agral; casein; Manoxol OT; Plyac; Sovix;
Spreader; Spreadite; Teepol L; Tenac; Triton
GR5, B-1956, X-100; 1% exposed to Bt
(Thuricide, serotype V) 24 h, 30C, pH 7.2 in
buffer
AL-1246, 1280, 1364, 1403; Atlox 848, 849,
3404/849; Atplus 300, 448; Plurafac A-24;
Triton GR7M; Triton X-35, X-45, X-363M, X-
N60; Witconol H-31A
Alkylphenols; Arlacel 'C'; Colloidal X-77;
Igepal CO-630; Later's surfactant; Novemol;
Petrol AG; Sandovit; Triton X-45, X-100, X-
114, X-ISS; Tween 20; Vatsol OT
Atlox 848, 849 and 3404/849, anionic and
non-ionic; Triton X-lOO, non-anionic; 0.1 %
Cargill Insecticide Base Concentrate
Crodofos N3N (4%)
Cyclamines, hexylamine, butyl-, octyo-, de-,
dodecylamine hydrochloride and cetyltri-
methylammonium bromide; cationic
Lovo 192, 0.4%; Later's surfactant, 0.1%;
Igepal CO-630, 0.1%; Triton B195b, 0.1%;
Plyac, 0.4%; Pinolene 1882,4%; Folicote 351;
Hi-Spread-Casein 10% + lime 90%, 4%
Maywood surfactant
Maywood surfactant; whey; corn oil
surfactant (corn oil emulsified with
Maywood surfactant)
Multifilm Buffer-X, 0.04%; Triton X-lOO,
0.04%, X-152, 0.02%, X-l72, 0.02%, B-1956,
0.03%
Organosilicones in tank-mixes
Petro Morwet EFW, 1%
Span-85, Biofilm, Triton B-1956, 0.1%; non-
ionic
3.4.1 Wetters 57
Effects
Used in commercial dry Bt products l
Accurate assays on cabbage against Lacanobia oleracea,
Pieris brassicae and on beeswax against Galleria mellonella.
Only Teepol2 increased LC so (x 6.5)
Satisfactory with Bt3
Good results with Bt, virus and fungus. No indication
that additives reduced effectiveness of the pathogens 4
Inhibited Bt fermentation and growthS
More acidic than polyethylene glycol; did not decrease
evaporation or improve sticking; consistent drop
spectra 3
Compatible with Heliothis armigera and Spodoptera exigua
NPVs for formulation in Arachis oil 6
0.03% enhanced and 0.1% depressed NPV infection of
Pseudaletia unipuncta larvae, possibly by attachment to
NPV surfaces, so neutralising their negative charges at
pH 8 and overcoming repulsion by negative charges on
membranes of midgut cells?
Did not affect germination of Bt spores, nor prevent
growth in media 8
Increased spread of Bt on foliage 3
Thickened and physically stabilised Bt tank mix; did
not decrease evaporation or improve sticking3 ;
consistent drop spectra 3
Heliothis NPV unharmed by 24 h storage at 30C 9
Excellent spread of Bt to cryptic parts of leaves. No harm
to BtlO
With 10% lactose in B. sphaericus slurry during spray
drying, an effective product concentrate l l
No adverse effects on Bt fermentation and growthS
58 Formulation of bacteria, viruses and protozoa to control insects
Table 3.12 (Contd.)

Surfactant (composition, Appendix Table 1.5) Effects

Teepol, Triton X-IOO, Pitsulin, Etocas 30
Surfynol TG-E, 0.5%; Bevaloid 211, 3%
Triton AG-98
Triton CS-7 spreader-binder
Triton CS-7; polyvinyl alcohol
Tween 80
Tween 80 (5%), emulsion in oil
4% Tween 80 + Span 80 emulsions of 6%
water in no. 2 furnace oil, 7 days storage
with Bt
Compatible with S. littoralis or H. armigera NPYs, no
reduced effectiveness of NPy 12
Used as dispersants in Bt flowable formulations 1
Recommended in tank-mix with Collego 13
Decreased activity of aqueous NPY tank-mix in
laboratory14
Compatible with Heliothis NPy 15
3% for water-based and 18% for oil-based Bt flowable
formulations 1
Successful with H. armigera and S. exigua NPYs in
Arachis oil 16
No loss of toxicity, germination and growth on agar not
inhibited, sprayed foliage readily eaten by budworm
larvae)?

References
1 Lisansky et aI., 1993
2 P. Jarrett, personal communication
3 19noffo and Couch, 1981
4 Angus and Luthy, 1971
5 Morris, 1975
6 K. A. J., personal communication
7 Yamamoto and Tanada, 1978a, 1980
8 Morris, 1969
9 Ignoffo and Montoya, 1966
10 Green et aI., 1996; C. F. Green, personal communication
11 Lacey et al., 1988
12 Topper et aI., 1984; Jones, 1988a; K. A. J., unpublished data
13 Industrial literature Ecogen, Langhorne, Pennsylvania 1990
14 Smith et al., 1978a
15 Smith et al., 1978b
16 K. A. J., unpublished data
17 Angus et aI., 1961
Note: Best, organosilicone. Non-ionic wetters preferable. Triton X-lOO and Tween 80 tested more than any other wetter
(section 3.4.1), but difficult to mix into sprays in the field. Side effects listed in Appendix Table 1.5.

personal communication). It is not regarded have appeared. Experience of use suggests

as harmful to fungal spores, which are less
well protected than those of bacteria (sections
4.3.6, 4.6.6b). There is some evidence of the
possibility of harm by Triton CS-7, as well as
the Altox and cyclamine groups of wetters
(Table 3.12). Organosilicone superspreaders
have been used with microbiaIs only recently,
but no relevant studies on bacteria and
viruses comparing them with other wetters
that they do not harm microbials when
added at the spray tank stage but they may
cause on-shelf deterioration if included in
proprietary microbial formulations. There is
some evidence that Silwet L-77 damages
fungal herbicides (section 6.5). Organosili-
cones are believed to carry water-soluble pes-
ticides into open stomata (Green et aI., 1996),
so they may do the same for pathogens. They

should carry particles very effectively even to
the feeding areas of cryptic larvae, e.g. those
that feed under silken tubes (Table 3.4).
Manufacturers of microbials will advise on
suitable wetters for their products.
In practice, wetters are best incorporated
into commercial products in quantities that
give adequate dispersion into the spray car-
rier and good cover of tractable foliage. More
should be added to tank mixes for very
hydrophobic, waxy or hairy foliage.
3.4.2 STICKERS
Measurement of physical persistence of
unprotected pathogens has produced conflict-
ing results. Burgerjon (1989) reports that the
activity of preparations of B. thuringiensis
spores and toxin crystals is not rapidly lost
when subjected to artificial rain; however,
Krieg (in Huber, 1989) concluded that rain
rapidly removed crystals from foliage. This
is much the commoner effect, e.g. 3 mm of
rain removed 46-72% of the original deposits
on leaves from 12 Dipel products (Sundaram
et al., 1993).
Of the baculoviruses, GVs and to a lesser
extent NPVs adhere strongly to leaf surfaces,
and are not easily washed off by rain (David
and Gardiner, 1966; Richards and Payne,
1982; Richards, 1984; Entwistle and Evans,
1985; Young and Yearian, 1986; Burgerjon,
1989). Acquisition of polyhedral inclusion
bodies (PIBs) of NPV from a spray involves
long-range forces such as electrostatic forces,
van der Waals' forces and hydrophobic inter-
actions. Retention of PIBs involves shorter-
range forces, i.e. various types of chemical
bonds (Small, 1985). At neutral pH in most
conditions, PIBs are negatively charged and
the hydrophobicity is inversely related to pH,
thought to be due to a charge-masking effect
(Small et al., 1986; Small and Moore, 1987).
These relationships are likely to be similar
for GVs. Attachment will be weaker if prod-
ucts contain particles large enough to impair
contact between inclusion bodies and the leaf
3.4.2 Stickers 59
surface (Appendix II, Fig. II.6). Entwistle and
Curruthers (1989) report that cabbage leaves
did not readily retain PIBs because of epicuti-
cular wax and that Neodiprion sertifer NPV was
acquired more readily by pine and cotton
leaves than was S. littoralis NPV. Also, S. lit-
toralis NPV was acquired more readily by cot-
ton than pine.
Jones and McKinley (1987) and Jones
(1988a) reported that S. littoralis NPV was
physically lost from cotton leaves in Egypt.
This happened also with H. armigera NPV in
Thailand (K. A. J., unpublished results). Over-
all, loss of NPV was greater from the upper
surface of leaves than from the lower surface.
In both countries, this loss occurred during
periods of no rain and was attributed to the
abrasive action of wind and dust or sand,
which can remove epicuticular waxes. It is
unlikely that any sticker could prevent this,
or could adhere so strongly to pathogens as to
prevent release in the insect gut. Purification
of virus suspensions reduces adhesion to the
leaf, because the insect protein and debris acts
as an effective sticker Oones, 1988a; Entwistle
and Curruthers, 1989).
Stickers (Appendix Table 1.6) improve
adherence of pathogens to foliage. They
reduce wash-off by x2 to x 10 after up to
13 cm of rain (Table 3.13). Specialist stickers
are usually used at rates of 0.1-2% and the
multifunctional molasses up to 25% (Table
3.13; Table 3.15 in section 3.4.3 below). Effec-
tiveness varies from the delaying action of
water-soluble materials, such as molasses, to
the fastness of materials such as resins, which
dry to become insoluble. Jones (1988a) tested
13 stickers on viruses, including celluloses,
vegetable gums and molasses. Gum Guar
was the most effective. None of the gums
inactivated the virus or inhibited feeding.
Stickers may double up as thickeners, i.e.
additives to increase spray viscosity and
increase the size of spray drops, e.g. gums
and molasses, or as phagostimulants, e.g.
molasses. Details of the characteristics and
performance of individual stickers are given
60 Formulation of bacteria, viruses and protozoa to control insects
Table 3.13 Stickers tested with Bacillus thuringiensis (Bt) and nuclear polyhedrosis virus (NPV) sprays in
alphabetical order of first entry in each group
Sticker (composition, Appendix Table 1.6)
Acrylic polymer, Rhoplex AC33NP, all 2% +
tracer dye Erio Acid Red XB 400, 0.2%.
Efficient ULV cover of leaves
Biofilm; High Tack Fish Glue; Nacrylic
X4260, X4445; NuFilm 17; Plyac; X-Link 2873
Bond, 2% on lettuce with no pathogen
Bond. Recommended for Bt Dipel 8L and
Thuricide 48LV by air, not for Foray
Bond, 5%; Acrylocoat, 3%
Carboset
Casein 0.05% cross-linked enhanced
mortality by x3.8, 0.05% casein x2.8 and 1%
gluten x2.2 with Bt
Chevron spray sticker in shaken flasks of
broth
Chevron sticker, 0.6% in ULV aerial spray, 2
l/ha on moderate spruce budworm
populations
Chevron, 0.6-0.1%; Rhoplex B60A, 2%;
Dupont spreader-sticker, 0.06%; Biofilm,
0.1% with Bt; Biofilm, 0.1% with NPV
Chevron spray sticker with Cargill
Insecticide Base concentrate (stabilized
molasses) 25%
Chevron spray sticker with sorbitol and
chitinase
Chevron sticker (with Shade), 6%; molasses,
25%
Corn starch (pregelatinized) + sucrose 1:1 in
spray
Dextrin (maize)
Geon Latex (colloidal vinyl chloride
polymer), 10% on mulberry foliage
Gluten (1 %) needs pH > 10 or < 5 for
dispersion in water
Gluten (1.3% at pH 10) in spray on young,
washed cotton plants
1% gluten x 1.53 = 0.5% casein x 1.49 > 2%
flour / sucrose x 1.23
Effects
Superior to Chevron Sticker, no effect on toxicity of Bt,
stable on pine needles to manual handling and heavy
rain (dye leached out of drops)1
Best protection of Bt among 18 stickers in 2.5 cm rain
while tank-mixes without stickers lost ca 30% activity2
No effect on feeding of gypsy moth larvae 3
At 2%, retained 88-100% original activity after 6 mm
rain, c.f. 29% without Bond on oak4.5
Highly effective with Bt and NPV against gypsy moth
on oak seedlings6
Depressed Bt activity2
With Ostrinia nubilalis after 13 cm of simulated rain on
pre-washed cotton plants in spray chamber?
Did not impair germination and growth of Bt8
Effective control with ready-to-use Bt suspension
concentrates requiring only mixing in of sticker9
Good results in forest against spruce budworm and
gypsy moth; Rhoplex initially blocked flow meter and
spinning disc cage with Bt6
Results of LV sprays of Bt generally favourable lO
Acceptable spruce budworm control with Bt; sorbitol
improved spray deposit 11
NPV effectively controlled gypsy moth larvae 12
Some evidence 4% solids improved Bt field rainfastness,
1-2% did not, nor 4% under 6 cm artificial rain 13
Used as sticker in some commercial wettable powders 14
Insoluble in water. Palatable to silkworm larvae,
protected Sudan IV for 6 weeks outdoors, Bt from 3 min
daily hosing for 2 weeks, or 12 h in moving water, or 12
h washing under a shower (toxicity retained)15
Lower bulk replacemant for sugar and starch (2-4%).
Enhanced mortality c.f. commercial fomulation (20%
against 90%)16
Enhanced mortality of O. nubilalis due to Bt by x 10.1
after 5 cm simulated rain in greenhouse 1?
Increased mortality of O. nubilalis due to Bt after 3.2 cm
simulated rain on cabbage in field 18
 3.4.2 Stickers 61

Lignin, kraft (0.5%) crosslinked with CaCh,
exposed to 5 cm simulated rain on cotton.
Encapsulated, or not cross-linked
Lignosulphonate, 0.5%
Lysine KKL increased persistence x7;
polyglucine x6: citric acid by-product x4;
molasses of peat x3
Methyl cellulose. Water soluble
Molasses, egg albumen, whole milk, larval
extract
Plyac
Polyvinyl sticker
Rhoplex B60A, x2.1; Acrylocoat, x1.9;
NuFilm 17, x1.3
Skimmed milk, OSlo
Skimmed milk, 2%; methyl cellulose, 2%
Sutro (25%) with molasses (25%)
Topwet spreader sticker, 0.1%
Sorbitol, 1%; gum guar, 1%; gum xanthan,
1%; gum tragacanth, 1%; gum karaya, 1%;
acacia gum, 1%; locust bean gum, 1%;
gelatin, 1%; molasses, 0.1-10%; casein, 1%
Did not harm Bt and NPV and protected 52-98%
original activity from wash-off19
No reduction of Bt wash-off19
No reduction of Bt wash-off19
3 days after sEraying apple trees with Malacosoma
nellstria NPV
In field 4 cm rain reduced spore count of Bt by 80%15
Compatible with NPVs in field trials 21
Recommended for Bt in Foral and Dipel5 by air.
Compatible with Heliothis NPV 22
Use abandoned as it adhered semi-permanently to car
finishes 23
Improved rainfastness of Bt on oak seedlings after 2.5
cm rain 24
Aqueous NPV (2.4 l/ha), excellent control of pine
sawfl y 25
NPV controlled Lymantria dispar on hardwood trees 26
Rain washed off more Bt than with water plus Sutro
alone 2
No effect on germination of Bt spores, good control of
He/iothis punctiger in field test27
Compatible with S. Jittoralis NPV. Gum guar best in field
tests, but still some physical loss of virus. All palatable
to larvae. None more effective than unpurified virus 28

References
1 Fast et al., 1985 16 McGuire et al., 1994a
2 Neisess, 1979 17 Behle et al., 1997a
3 Farrar et al., 1995 18 Behle et al., 1997b
4 McLane, 1991 19 Shasha et al., 1995
5 Devisetty, 1988 20 Jankevica and Zarins, 1997
6 Morris, 1985 21 Jones, 1994
7 Behle et aI., 1996 22 K. A. J., unpublished data
8 Morris, 1975 23 Nichols, 1985
9 Valero, 1989 24 Cibulsky et aI., 1993
10 Lewis et al., 1974 25 Bird,1953
11 Smirnoff, 1977 26 Magnoler, 1974
12 Yendol et al., 1977 27 D. J. Cooper, Waite Agricultural Research Institute,
13 McGuire et al., 1996 Australia, personal communication, 1984
14 Lisansky et al., 1993 28 Jones, 1988a
15 Angus, 1959
Note: best, acrylic polymers, Biofilm, Chevron sticker, skim milk, gum guar, cross-linked lignin.
62 Formulation oj bacteria, viruses and protozoa to control insects
in Table 3.13; the best are listed in a Note at
the foot of the Table. Lignin (very good),
casein, flour, albumen, gluten, milk and
molasses are also sunscreens (Table 3.16 in
section 3.4.4 below).
Thus, degrees of acquisition and retention
of pathogens are likely to be related to differ-
ences in pathogen species, formulations and
environmental conditions (plant species, type
of leaf surface, position on leaf, precipitation,
wind, etc.). A formulated product should
contain enough sticker for most treatment
situations, unless specialist products are for-
mulated to retain strong natural adhesion on
tractable foliage, e.g. codling moth GV on
apple. Extra sticker should be added to the
tank mix for water-repellent foliage. Also, a
sticker may benefit a pathogen in other ways,
e.g. 4 days after application, B. thuringiensis
alone on red oak seedlings without rain killed
89% of test gypsy moth larvae, whereas
applied with 1 or 3% Bond it killed 97-100%
larvae, whether or not exposed to 0.6 or 1.2 cm
of rain (McLane, 1991).
One sticker, Carboset, harmed a pathogen.
Other reasons for unsuitability have also been
reported (Appendix Table 1.6). A polyvinyl
sticker was withdrawn because it fouled car
paintwork, and some stickers are incompat-
ible with individual formulations because
precipitation from suspension blocks on-line
filters and nozzles (Table 3.13).
3.4.3 PHAGOSTIMULANTS
In the field, phagostimulation is a combina-
tion of attracting an insect to an area bearing
the stimulant and encouraging it to eat more
once it is there. The latter is the effect usually
measured in the laboratory by placing insects
on food either with or without the stimulant.
A few experiments allow the insects to make a
choice (Table 3.14).
Phagostimulants (Appendix Table 1.7)
encourage pests to eat a maximum amount
of pathogen before it deteriorates on the
foliage or, with B. thuringiensis, before the
crystal toxin arrests feeding (section 3.3.3a).
Researchers sought gustatory recipes for cot-
ton pests (Table 3.14, part 1), later incorpo-
rated into commercial adjuvants (Appendix
Table 1.7), and then tried each recipe on
other pests and crops. Some principles are
evident from the data assembled in Tables
3.14 and 3.15. The most stimulatory materials
were plant extracts and materials made from
seeds, particularly cottonseed flour and oil,
cornflour, corn oil and soybean flour. Com-
binations of these materials were more sti-
mulative than single materials. Although
stimulating many pest insects, no single mate-
rial (Table 3.14, part 1) or combination (Table
3.14, part 2) does so universally. These prin-
ciples are not surprising: plant pests have
evolved to fill niches created by the defences
of individual plant species, mainly in the form
of allelochemicals (section 3.4.5) and mechan-
ical difficulties presented to feeding insects.
The adult insects choose the niche plants
for oviposition and the resulting larvae
choose where and on what to feed, often in a
crop monoculture. The attractive materials
described above are almost free from allel-
ochemicals and are easy to eat.
Some pitfalls are apparent in experiments
with phagostimulants, partly explaining the
variability of the data in Table 3.14. In the
laboratory, it is not easy to mimic the insect
in natural free-ranging competition between
micro-areas hit or missed by a spray or bait
on fresh plant material of its chosen food
plant. The dose of pathogen eaten on evenly
treated leaf is proportional to the amount of
leaf eaten, but the resultant mortality is not
always so. With B. thuringiensis, food intake
was generally lower in four insect species
given treatments causing the highest mortal-
ity, possibly a result of stimulants speeding
up ingestion of a lethal dose before the
antifeeding action of the crystal toxin stopped
further feeding (Yendol et al., 1975; Farrar
and Ridgway, 1995; section 3.3.3a). Effective
concentrations of stimulants tend to be high,
altering the physical performance - and hence

Table 3.14 Phagostimulants used with microorganisms Bacillus thuringiensis (Bt), nuclear polyhedrosis
virus (NPV) and Protozoa
Phagostimulants and ranking
(composition, Appendix Table 1.7)
1. Cotton insects on cotton
Corn, water extract with NPV
Corn and other plant extracts impregnated
in paper
Corn extract with NPV
Cottonseed oil, sugars, extracts of parts of
cotton plant mixed in agar
Cottonseed flour (Pharmamedia and
Proflo), soya flour, dried-extract of corn seed
and extracts of various vegetables all at 5%,
or cottonseed oil 10% in agar
Cottonseed oil sprayed bait with NPV in the
field
Soybean oil and soybean flour> corn oil and
corn flour
Grassmeal > wheatgerm > 'groundbait'
(grassmeal, Celacol M2500, bran,
wheatgerm, molasses) > molasses
Citrus pulp bait (Griffin Corp.); Coax;
Gustol; soybean adjuvant (8% soybean
flour, 5% soybean oil, 1% sucrose)
Lactose, maltose, glucose, sucrose in
laboratory soybean baits
Molasses (10%) added to cottonseed meal-
Shade bait sprayed on leaf
Coax 1.2-12%; cottonseed oil 1%; molasses
2% in spray with Bt
Coax, 3.4 kg/ha in trials with NPV and Bt
Molasses (e.g. 10%) 0.6% + NPV
Sugar + Heliothis NPV
2. Commercial adjuvants compared
Pheast, Coax, Gusto and Entice with
Lymantria dispar, H. zea, Ostrinia nubilalis
and Plutella xylostella
3. Gypsy moth and other species feeding on open leaf
Molasses (12.5%) on lettuce leaf discs
3.4.3 Phagostimulants 63
Effects and activity
Increased Heliothis feeding on cotton leaf, also mortality
by x2.4 1
Increased feeding of Heliothis spp. on the paper up to
x24 to x30 2
Increased mortality in leaf assay x 1.7. In field increased
cotton yield x 1.2, reduced damage x 1.5-3.62
Pink bollworm neonates fed on cottonseed oil, sucrose,
extracts except leaf extract, galactose, raffinose agars 3
Heliothis virescens neonates favoured Pharmamedia
agars most, and mixtures more than single materials 4
Heliothis spp. control better than NPV alone and
equivalent to cotton standard chemical controls
Mortality with NPV in laboratory6
Compatible with Spodoptera littoralis NPV. Field tests: all
except molasses increased feeding of s. littoralis larvae,
grassmeal by 32%7
Soybean adjuvant and Coax gave higher mortality with
Heliothis armigera NPV than citrus pulp and Gustol, all
gave better control c.f. no adjuvant6
No significant difference in mortality between sugars
with NPV 6
xLI potency with NPV in Heliothis zea under simulated
suns
Only Coax significantly increased mortality (by 13-25%)
of Heliothis spp. on cotton leaf 9
Decreased Heliothis damage (x2.1) on cotton and
increased yield (x 1.3) 10
x2 potency with NPV against H. armigera on cotton 11
Increased yields of cotton in some tests 12 , but not
others 1314
Stimulants variably (0-8%) increased mortality with Bt
on leaf in Petri dish and greenhouse with all species;
stimulants were not consistently different in all insects
and host piants 1S
Increased gypsy moth larval feeding x1.7 and x2.3 16
64 Formulation of bacteria, viruses and protozoa to control insects
Table 3.14 (Contd.)

Phagostimulants and ranking

(composition, Appendix Table 1.7) Effects and activity

Coax and 16 spreader stickers,
phagostimulants, oils, etc.
Sugar (10%) added to Keltose-Shade or
PVA-Shade sprays of Bt
Molasses
Heliothis NPV on potted gram (legume);
stimulant unspecified
4. Com borer
Molasses > fresh corn leaf
= CaCh + Coax> CaCl z, all at 17-18%,
except Coax, 8%. In granules in corn whorls
Coax (4.7%) in granules> plain granules by
x 320 and leaf by x 12; corn oil 1% < leaf by
xO.22, sugar = leaf
Coax (25%) > homogenized fresh leaf (16%)
> CaCh 35% + Coax (15%) > CaCh (18%) in
flour granules
Coax (1-10%) in corn starch granules
Coax> corn oil by x5, corn oil = plain
capsules
5. Dry baits
Wheat bran, Nolo Bait with Nosema Iocustae
Wheat bran bait with Bt
Molasses (16%) in corn starch granules with
entomopox virus
Only Coax increased control of the Colorado potato
beetle by Bt ssp. tenebrionis 17
x 1.6 and x2.6 potency in Trichoplusia ni on leaf under
simulated sun, c.f. no sugar8
xlO more deaths of P. xylostella GV in greenhouse tests 18
Instar III larvae ate x2.1 LC 90 dosage with stimulant, c.f.
x1.5 without19
Significant differences with corn borer in both
greenhouse and field. Molasses improved potency of Bt
by x1.3 20
Feeding; stimulants in starch granules with O. nubiialis
and fresh untreated corn leaf in Petri dishes without
pathogens Z1
Mortality of corn borer on cotton leaf and preference in
feeding choice tests. Coax increased Eotency of Bt x4.
Larvae rejected granules with CaCh
Reduced corn borer tunnel length in field by x2.2 to
x3.0 with 400 ru Bt and by 0 to x2.4 with 1600 ru Z3
In capsules on corn in greenhouse with Bt at the LCso for
O. nubiialis 21
Improved grasshopper suppression z4 ,zs
Successful control of Agrotis ipsiIon 26
Increased grasshogfer mortality x 1.1 to x 1.2 in assays
with rye seedlings

References
1 Allen and Pate, 1966 15 Farrar and Ridgway, 1995
2 Montoya et al., 1966 16 Farrar et al., 1995
3 Bell and Kanavel, 1975 17 Riethmiiller, 1990
4 Bell and Kanavel, 1978 18 K. A. J., personal communication
5 Andrews et al., 1975 19 Ignoffo and Couch, 1981
6 Smith et al., 1982 20 McGuire et al., 1994
7 Jones, 1990 21 Bartelt et al., 1990
8 Smith et al., 1980 22 Gillespie et al., 1994
9 Luttrell et al., 1982 23 McGuire et al., 1990
10 Bell and Romine, 1980 24 Caudwell, 1993
11 Roome, 1975 25 Johnson and Henry, 1987; Meneley and Sluss, 1988;
12 Stacey et al., 1977, 1980 Lockwood and Debrey, 1990
13 Bull et al., 1976 26 Salama et al., 1990c
14 Pfrimmer, 1979 27 McGuire et al., 1991
Nole: best, Coax and some plant extracts (with or without sugars).

Table 3.15 Tank-mixes used in forest and field with B. thuringiensis (Bt) and nuclear polyhedrosis virus
(NPV)
Tank-mix (ingredient details, Appendix 1)
Forest
Molasses 25% + Shade 3-6% + spreader-
sticker 0.1 % with Bt
Molasses 25% + Shade 3% against N. lecontei
(1) 19.3% Molasses + 2.3% or 4.6% Shade +
1.7% Chevron oil + 76.7% water; (2) 12.3%
Protec and 87.7% water. Both 20 l/ha
Molasses (ProMo, feed grade) 12.5% +
Orzan LS 6 or 10% + Rhoplex B60A 2% +
stream water (pH 5-8) for aerial sprays
Molasses (MO-MIX) 12.5% + Lignosite 6% +
Bond 2% + stream water (pH 5-8) for
ground spray
Carrier 038 95%, water 5% + Gypchek
(NPV) against gypsy moth
Carboxymethylcellulose 0.2% +
polyvinylpyrrolidone 1% + Erio Acid Red
XB 0.1% + Chevron Spray sticker 0.1%
acephate 0.5% (organophosphate
insecticide, 6% of normal rate)
(1) Uvinul DS49 1% + Uvitex ERN-P 1% +
Chevron Spray Sticker 0.1 % + acephate 0.6%
(10% of normal rate)
(2) Xanthan gum 0.02% + (1) above
Latex + dried blood in water, or fuel oil +
magnabentonite + Span emulsion
Field crops
Molasses 10%, Teepol 0.5%, Tinopal RBS 200
0.001%
Nutrisoy 7B soybean flour 8% + crude
soybean oil 0.5% + sucrose 1% + Triton CS-7
0.01%
Cottonseed oil + sucrose + Dacagin
hydroxycellulose
Cottonseed oil bait modified with sucrose +
Dacagin hydroxycellulose + glycerin
3.4.3 Phagostimulants 65
Effects and insect control
Effective against sawfly (Neodiprion lecontei)I,2,3. Tank-
mix superior to commercial formulation (Sandoz
285WP) against spruce budworm 4
Effect of NPV in the mixture equalled effect in water
alone 5
NPV (Gypchek wettable powder) gave x2 better gypsy
moth larva reduction with Beeconmist than with flat fan
nozzles with (1); vice versa with (2)6
Standard in North American tests with Gypsy moth
NPV (Gypchek) in 1987-92. Handling on site slow.
Frequent superior results d, Gypchek alone or in
simpler mixes7,8
Gypsy moth larvae fed x 1.56 d. NPV alone on lettuce
leaf discs 9 . Standard in field tests of Gypchek from 1993.
Frequent superior results of Gypchek alone or in simpler
mixes 8
Recommended 1996,25-60 xl09 OB/ha is x2 or more
the dosage in the molasses mixes to minimize the
volume per ha, Has better sun screening and less
droplet evaporation lO
With Dipel 36B liquid (Bt). Some clogging of Micronair
emission system11. Strong effect of evaporation. High
population reduction and very good foliage protection
due to superior deposit rates and resistance to
weathering l l
With Dipel16B liquid (Bt). Clog-free spraying l l ;
superior retention of activityl\ high population
reduction and very good foliage protection due to
superior deposit rates and resistance to weathering l l
With Dipel 36B liquid. Clog-free etc as (1) above l l
NPV effective against small sawflliarvae (Neodiprion
swanei), not against older larvae 1
Best formulation of purified S. littoralis NPV of a
number tested in the field 13
Suggested after studies on individual additives l4 ,15
Has several desirable properties but spray qualities poor
against Heliothis on cotton l6 ,17
With NPV attracted bollworms and reduced
population 16
66 Formulation of bacteria, viruses and protozoa to control insects
Table 3.15 (Contd.)
Tank-mix (ingredient details, Appendix 1)
Polyvinyl alcohol 99% hydrolysed + Shade
Speswhite china clay 30%, Neosyl 10%,
Etcos 3010%
Egg albumen 0.3%, Teepol 0.01%, soybean
flour 5%
References
1 Cunningham et al., 1975
2 Knapp and Cunningham, 1977
3 de Groot et aI., 1979
4 Cunningham et al., 1978
5 de Groot and Cunningham, 1983
6 Lewis and Yendol, 1981
7 Reardon et aI., 1992
8 Reardon and Podgwaite, 1994
9 Farrar et al., 1995
10 J.D. Podgwaite, USDA, personal
communication
effectiveness - of sprays in the field. Stacey et
al. (1977) found the most effective sprays had
the largest drops (235 pm diameter) and the
most deposit on the upper zone of cotton
plants. These results were not repeatable
later, even with larger drops of (600 J.lm
(Stacey et aI., 1980). It is debatable whether to
regard bulky, stimulant-laced sprays as
sprays or as baits. Stacey et al. (1977) sug-
gested that increases in cotton yield recorded
in the presence of sugars may have been due
to physiological effects of the sugar on the
plant. Sucrose is routinely used with B. thur-
ingiensis on grape against grape berry moth.
The value of a phagostimulant with NPV
was clearly shown with gypsy moth on lettuce
leaf discs by Farrar et al. (1995). Technical
virus had no effect on feeding, but a com-
mercial wettable powder was strongly deter-
rent (x 0.10), as was the synergist Tinopal
LPW (= Blankophor BBH) (x 0.43). Molasses
strongly stimulated feeding (Table 3.14, part
3) and its use with the wettable powder in a
tank mix (NPV+12.5% molasses+2% Bond
+6% Lignosite AN), with or without 1%
Effects and insect control
Best of several tank-mixes with NPV for Heliothis
control 18
Wettable powder with freeze-dried S. littoralis NPV.
Effective field control of larvae 19
Increased mortality by NPV in lab, but not in field d.
NPV + Teepol2o
11 a.N. Morris, Agriculture Canada, personal
communication
12 Ignoffo et al., 1976b
13 Topper et al., 1984
14 Smith et al., 1978a
15 Smith et al., 1982
16 McLaughlin et al., 1971
17 Andrews et aI., 1975
18 Smith et al., J978b
19 Jones et al., 1994
20 Pawar et al., 1992
Tinopal LPW, restored feeding to the level
with water alone. Concurrently with the feed-
ing, potency (LDso) of virus alone was less
than that of the tank-mix (by x 20), but the
synergism of the Tinopal was so great that the
LC so ratio, (with/without it; x 42 in the tank
mix and x 214 in the wettable powder) was
remarkable, as was the LCso ratio (x 1671) of
Shapiro and Robertson (1992); the synergism
swamped the feeding inhibition. Stimulants
improved potency of both virus and B. thur-
ingiensis in other insects feeding on expanded
leaves (Table 3.14, part 3).
Cotton pests have responded to the best
phagostimulants, which partially masked
the cotton leaf surface alkalinity and allelo-
chemicals (Table 3.14, part 1). Coax, devel-
oped specially for cotton, is the most widely
used and successful stimulant adjuvant.
Results with viruses and single stimulants
(including sugars) with Heliathis spp. have
varied. Molasses did not stimulate S. littaralis
(Table 3.14).
Starch is being developed as a carrier for
corn borer control. Alone, it is less attractive

than fresh corn leaf. The difference disappears
on addition of sucrose, and starch+Coax is
more attractive than fresh leaf (Table 3.14,
part 4). Starch can be formulated as a dry
granular corn borer bait (section 3.3.1) or as a
self-encapsulating spray (section 3.3.5).
Dry baits for other pests commonly consist
of pathogen-treated cereals or cereal products.
These are shown to be attractive and stimulat-
ory to pests by virtue of successful pest
control. Bran, corn meal, cottonseed meal
and wheat stimulated various insects, and
molasses made corn starch palatable to grass-
hoppers (Table 3.14, part 5).
There has been no evidence that any of the
phagostimulants (Table 3.14) have been harm-
ful to, or incompatible with, pathogens. Com-
paring results in different publications is
difficult, but a general view of the literature
in these tables gives the impression that the
most effective phagostimulants have been
Coax, molasses and plant extracts.
3.4.4 SUNSCREENS
Data on field persistence of microbial in-
secticides reveal that sunlight is the most
destructive of the environmental factors
(Ignoffo and Hostetter, 1977; Ignoffo et al.,
1977; Jones and McKinley, 1987; Jones,
1988a). A subjective assessment of the value
of different sunscreen additives is presented
in Table 3.16. Factors involved in this assess-
ment are elaborated below.
3.4.4a Damaging wavelengths in sunlight Of
light reaching the earth's surface, that with
wavelengths mainly up to 380 nm, with a
peak at ca 300 nm, kills B. thuringiensis spores,
while light of mainly 300-380 nm damages
crystals O. Mitchell, Cranfield Biotechnology
Centre, Cranfield University, UK, personal
communication; Pusztai et al., 1991). How-
ever, Griego and Spence (1978) found that
the greatest kill of spores occurred at 400 nm
in the visible range because of its much
greater amount of energy compared with
3.4.4 Sunscreens 67
light of shorter wavelengths. The medium-
wave or erythermal UV band (UVB, 280-
320nm) is the most important photoinactiva-
tor of baculovirus, with considerable but
slower effect in the near-UV region (UVA
320-360 nm), and in some cases some effect
above this (David, 1969; Timans, 1982; Griego
et al., 1985; Martignoni and Iwai, 1985; Killick,
1986; Jones et al., 1993b). Light of some longer
wavelengths, however, may be beneficial to
microorganisms (Jones et al., 1993b). The
wavelengths involved in experiments have
been taken into account for assessments in
Table 3.16.
3.4.4b Effect of sunlight on different pathogens
in different situations Insect bioassay showed
that a laboratory simulation of the UV radia-
tion in sun affected the potency of B. thurin-
giensis less than six other types of pathogen.
Exposure to UV for 4 h, that reduced the ori-
ginal activity of B. thuringiensis to 46%,
reduced the activity of entomopox virus to
18%, of Nomuraea rileyi conidia to 13%, of
NPV and cytoplasmic polyhedrosis virus
(CPV) to 8%, and of GV and spores of the
protozoan Vairimorpha necatrix to 4% (Ignoffo
et al., 1977). Work on protection of two types
of pathogen, B. thuringiensis and baculovirus,
is distinguished in Table 3.16.
In the field, the half-life of B. thuringiensis
and baculovirus varies greatly, from as little
as 10 h to 10 days (Entwistle and Evans, 1985).
In general, the half-life in full sunlight without
protective screens centres at ca 24 h. Thus
there appears to be much scope for improve-
ment. This scope varies in different situations,
being greatest where the pest feeds on the
upper surface of foliage and in the tropics,
and minimal on lower leaf surfaces deep in a
foliage canopy under cloud, where UV radia-
tion penetrates least. For example, the level of
UV radiation reaching undersurfaces of
leaves in the lower canopy of the cotton crop
in Egypt was only 1% of that at the top of the
plants (Jones, 1988a). In contrast, UV is not an
important degrading factor deep in cryptic
68 Formulation of bacteria, viruses and protozoa to control insects
Table 3.16 Effect* of sunscreens on Bacillus thuringiensis (B) and nuclear polyhedrosis or granulosis virus
(V) exposed to UV light

Sunscreens (composition, etc. Appendix References (orders

Tables 1.9 - 1.12) Percentage Effect* as under effect)
I ABSORBENTS
Amino acids
Adenine 1.0 ++ IV
Glutamic acid 1.0 + 2V
Histidine 1.0 ++ 3V
Lysine KK1 0.5 17+++ 4V
Phenylalanine 1.0-10.0 ++ 3V
0.1 + 3V
Proline 1.0 ++ 3V
Soya hydrolysate 5.0 a++++, 5V
a+++ 5V
Tryptophan 10.0 ++++ 3V
0.01-1.0 +++ 3V
0.001 ++ 3V
Tyrosine 10.0 ++++ 3V
1.0 +++ 3V
0.1 ++ 3V
0.01 + 3V
B Vitamins
p-aminobenzoic acid (PABA) 5.0 +++ 6V
1.0 +, s++ 2V,7B
0.1 s++ 7B
Amyl-dimethyl-p-aminobenzoic acid 5.0 +++ 6V
Ethoxylated p-aminobenzoic acid 5.0 +++ 6V
iso-Octyl p-aminobenzoic acid 5.0 ++ 6V
Choline chloride 1.0 ++ 2V
Folic acid 1.0 ++++, 2V,
s+++,+++ 7B,7B
0.1 ++, s++, 2V,7B,
++ 7B
Xanthopterin 1.0 +++ 2V
Inositol 1.0 + 2V
Nicotinic acid 1.0 ++ 2V
Pantothenic acid 1.0 ++ 2V
Pyridoxine 1.0 ++ 2V
Riboflavin 2.0 0 8V
1.0 +++ 2V
0.25 ++ 2V
Thiamine 1.0 + 2V
Cosmetic sunscreens
p-Aminobenzoic acid and derivatives;
see under B vitamins
Benzilidine sulphonic acid 5.0 ++++ 6V
Benzyl cinnamate 3.0 0 9B
2-Ethoxyethyl-p-methoxycinnamate 5.0 +++ 6V
2-Ethylhexyl salicylate 5.0 ++ 6V
Homomenthyl salicylate 5.0 6V
 3.4.4 Sunscreens 69
Eusolex 4360 5.0 + 6V
0.1 s++++, lOB,
+++, ++, 10V, lOB,
+++ 10V
Eusolex 6300 5.0 ++ 6V
0.1 s+++, ++ lOB,10V
Eusolex 8021 0.1 ++++ 10V
Uvinul D549 1.0 s+, +++, 9B,9B,
++ llV
Uvitex ERN-P 0.1 s+++ 9B

Dyes
Acid Black 48 1.0 ++ 12V
Acid Orange 8 1.0 ++ 12V
Acridine Yellow 1.0 ++++ 12V
Acriflavin 0.42 mmol/g ++++ 13B
0.06 mmol/g +++ 13B
Alcian Blue 8gx 1.0 ++ 12V
Alizarin Yellow R 1.0 ++ 12V
Alkali Blue 1.0 ++++ 12V
Astrazone Orange 1.0 ++ 12V
Astrazone Yellow 1.0 ++ 12V
Azocarmine 1.0 ++ 12V
Benzopurpurin 4B 1.0 +++ 12V
Bismark Brown 1.0 ++ 12V
Brilliant Blue g 1.0 ++ 12V
Brilliant Blue R 1.0 +++ 12V
Brilliant Yellow 1.0 ++++ 12V
Buffalo Black 2.0 c+++ 14V
Chrome Axurol 1.0 ++ 12V
Chrysophenine 1.0 +++ 12V
Cibachron Blue 1.0 ++ 12V
Cibachron Yellow 1.0 +++ 12V
Congo Red c.1. 22120 1.6 c++++ 15V
1.0 ++++, 16V,
++++, 12V,
s++++, 7B,
++++ 7B
0.5 ++++ 16V
0.25 +++ 16V
0.1 ++, s+++, 16V,7B,
++ 7B
0.04 0 l7B
Curcumin 1.0 ++ 12V
Direct Red 81 1.0 ++ 12V
Direct Yellow 8, 17 1.0 ++ 12V
Disperse Blue 14 1.0 +++ 12V
Disperse Orange II 1.0 +++ 12V
Erio Acid Red XB100 0.1 s++++ 9B
Fast Blue 1.0 +++ 12V
Haematoxylin 1.0 ++ 12V
Indigo Carmine 1.0 +++ 12V
Lissamine Green 1.0 ++++ 12V
Mercurochrome 1.0 ++++ 12V
Methyl Green 0.53 mmol/g ++ 13B
70 Formulation of bacteria, viruses and protozoa to control insects
Table 3.16 (Contd.)

Sunscreens (composition, etc. Appendix

Tables 1.9 - 1.12) Percentage Effect* References
Methyl Orange 1.0 +++ 12V
Methyl Red 1.0 +++ 12V
Mordant Brown 1,33 1.0 +++ 12V
Neutral Red' 1.0 ++ 12V
Nigrosin 1.0 +++ 12V
Orange IV 1.0 ++ 12V
Orcin 1.0 ++ 12V
Reactive Blue 4 1.0 ++ 12V
Rhodamine B 0.1 mmol/g + 13B
20 stains 1.0 + 12V
20 stains 1.0 0 12V
Miscellaneous
Aesculin 0.1 s+ lOB
Bentonite ? p+++ 4V
Carbon, carbon Rb, 5.0 ++, +++, 6V,5V
carbon black, +++ 18V
charcoal, India Ink 2.0 c++++, 14V,
++++,Y+ 14V,19V
1.6 c++++ 15V
1.0 0, ++, ++, 208, 21V, 21V
0, ++,0, 21V, 21V, 21V,
0, c++++, 0, 21V, 19V, 21V,
0.42 C++++, 19V,
yco, +++, 19V,19V
0.1 s++++ lOB
? yO,+++ 22V,22V
Citric acid by-product 0.5 p+++ 4V
Coax 6.0 ++++ 6V
5.0 ++ llV
Corn starch carrier sO, 0 7B,7B
Corn starch/ flour / sucrose carrier c++++,+++ 23B,23B
Flour 961/sucrose 2.0 +, ++++, 248,25B,
+++ 26V
Flour, corn pre-gelatinized 0.5-4.0 +++ 17B
Lignin, Kraft, + CaCh 0.5 ++++, 26V,
s++++, 26B,
+++ 26V
Lignin, Indulin, + CaCi z 0.5 s++++ 26B
Lignin, REAX, + CaCi z 0.5 s+++ 26B
Lignin, Kraft, + CaCh 43.5 (carrier) c++++, c++++ 26B,26V
Lignin, Kraft, + no CaCh 0.5 s++++ 26B
Lignosulphonate, Na 2.0 0 8V
0.5 s++ 26B
0.1 s++ lOB
Orzan LS 6.0 pO, ++++, 27V,llV,
++++ nv
Raymix L3 12.15 +++ llV
 3.4.4 Sunscreens 71
Raymix powder 6.28 ++++ llV
1.05 ++ llV
Melanin 0.0003 s++++, 28B,
++++ 28B
Molasses 25.0 ++++ llV
5.0 ++, +++, 6V,6V,
+++ 6V
Dri-mol 5.0 + 6V
Sucrose 5.0 ++ 6V
Molasses of peat 0.5 p+++ 4V
Polyglucine 0.4 p++++ 4V
Protec-100 5.0 ++ 6V
Shade 6.0 +++, PY++, llV,29V,
PyO 29V
5.0 +++, ++, 6V,15V,
0,+++ 19V,18V
2 ++, ++++, 30B,30B,
0,++ 31V,31V
1.6 c++++ 15V
1 +++ 32V
0.5 +,+Y 32V,26V
0.25 +++ 32V
0.1 sO, 0 10B,lOV
? + 33V
? +,Y+ 21V,22V
Sulisobenzone 0.25 32V,
+,+Y 32V,32V
Talc carrier + 18V
Yeast, brewer's 5.0 +++ 5V
Nitrogenous metabolic products
Allantoin 1.0 ++ IV
Guanine 1.0 ++ IV
H ypoxanthine 1.0 0 IV
Urea 1.0 0 IV
Uric acid 10.0 ++++ IV
5.0 +++ IV
1.0 ++ IV
Xanthine 1.0 + IV
Optical brighteners
Aclarat 8678 1.0 +++ 34V
Calcofluor LD, RWP 1.0 + to ++ 34V
Columbia Blue 1.0 0 34V
Intrawite ABL 1.0 0 34V
Intrawite CF 1.0 ++++ 34V
0.1 +++ 34V
0.01 + 34V
Intrawite EBF, ERN 1.0 +++ 34V
Leucophor BS 1.0 +++ to 34V
++++
0.1 +++ 34V
0.01 + 34V
Leucophor BSB 1.0 ++++ 34V
72 Formulation of bacteria, viruses and protozoa to control insects
Table 3.16 (Contd.)

Sunscreens (composition, etc. Appendix

Tables 1.9 - 1.12) Percentage Effect* References
0.1 +++ 34V
0.01 + 34V
Leucophor EfIB 1.0 + 34V
Leucophor EFR, KNR, PAB, PAL, PAT, 1.0 +++ 34V
WGS
Phorwite AR 1.0 +++ to ++++, 34V,
p+++ 35V
0.1 ++ to +++ 34V
0.01 +++ 34V
0.001 ++ 34V
Phorwite BLK, BRU 1.0 +++ to 34V
++++
0.1 ++ to +++ 34V
0.01 ++ 34V
0.001 0 34V
Phorwite Cl 1.0 +++ 34V
0.1 ++ 34V
0.01 + 34V
Synacril White 1.0 0 34V
Tinopal CBS-X 2.0 8V
1.6 c+++ 15V
1.0 ++ llV
Tinopal DCS 5.0 ++++ llV
Tinopal LPW 1.0 ++++ 34V
0.5 p++++, 36V,
py+++, 37V,
p++++, 35V,
pyo, pyo, 38V,38V,
pyo 38V
0.1 +++, s+++, 34V,9B,
py+++ 37V
0.05 P++++ 35V
0.02 py+ 37V
0.01 +++ 34V
0.001 ++ 34V
Proteins
Casein 0.5 ++,++ 248,25B
Egg albumen 5.0 a+++,+++ 5V,39V
Gluten, wheat 1.0 +++,+++ 248,25B
Milk, peptonised 5.0 ++++,a+++ 5V,5V
Milk, skimmed 5.0 ++ 5V
1 ++ 32V
0.25 ++ 32V
II REFLECTORS
Aluminium powder 2.0 c+ 14V
Titanium dioxide 0.84 c++++, 19V,
e++++, yeO 19V,19V
0.084 +++ 19V
 3.4.4 Sunscreens 73

III ANTIOXIDANTS AND OXIDATIVE ENZYMES

Ascorbic acid 10.0 ++++ 40V
1.0 +++ 40V
0.1 ++ 40V
Na ascorbate 3.0 0 9B
Catalase 10.0 ++++ 40V
1.0 +++ 40V
0.1 ++ 40V
Peroxidase 10.1 ++++ 40V
1.0 0 40V
Phenylthiocarbamide 0.1 ++++ 40V
0.01 ++ 40V
Propyl gallate 0.1 ++++ 40V
0.01 +++ 40V
0.001 ++ 40V
Superoxide dismutase 10.0 +++ 40V
1.0 ++ 40V

References
1 Shapiro, 1984 21 Jaques, 1972
2 Shapiro, 1985 22 Ignoffo et aI., 1972
3 Ignoffo and Garcia, 1995 23 Tamez-Guerra et al., 1996
4 Jankevica and Zarins, 1997 24 Behle et al., 1997b
5 Jaques, 1971 25 Behle et aI., 1996
6 Shapiro et aI., 1983 26 Shasha et aI., 1995
7 Dunkle and Shasha, 1989 27 Webb et al., 1993a
8 Killick, 1990 28 Uu et aI., 1993
9 Morris, 1983 29 Stelzer et al., 1977
10 Krieg, 1975 30 Smith et al., 1980
11 Martignoni and Iwai, 1985 31 Smith et ai., 1978a
12 Shapiro and Robertson, 1992 32 FUchards, 1984
13 Cohen et aI., 1991 33 Ignoffo et aI., 1976a
14 Ignoffo and Batzer, 1971 34 Shapiro, 1992
15 Ignoffo et al., 1991 35 Webb et al., 1994b
16 Shapiro, 1989 36 Webb et al., 1994a
17 McGuire et aI., 1996 37 Webb et al., 1996
18 Ignoffo and Garcia, 1996 38 Reardon and Podgwaite, 1994
19 Bull et al., 1976 39 J. W. Klijnstra, Brueren and T. A. de Vlieger,
20 M. R. McGuire and B. S. Shasha, Agricultural University, Wageningen, personal
USDA, personal communication, communication
1994 40 Ignoffo and Garcia, 1994
Symbols: standard type, laboratory studies; bold type, field studies; --, statistically significant adverse effect; -, non-
significant adverse effect; 0, no effect; +, non-significant protection; ++, small but significant protection; +++, good
significant protection in studies with a series of screens; ++++ best protection; s, only spore viability studied; c,
encapsulated; p, field population study; y, field yield or defoliation; bold type without appended letter, laboratory
assay of field-exposed pathogen.
Note: best in rough order of merit, melanin, insect remains, Tinopal LPW and other optical brighteners, Orzan LS, Coax,
molasses, carriers (e.g. clay, flour), lignin, carbon, titanium dioxide, milk, albumin, Eusolex 4360, Uvinal DS 49, Congo
Red CI, Shade (in general, tests disappointing).
74 Formulation of bacteria, viruses and protozoa to control insects
positions for corn borer control, exposure
being reduced by 93% in corn whorls and
97% in leafaxils, where the insects normally
feed (McGuire et a/., 1994b).
3.4.4c Mode of action of sunlight Pusztai et al.
(1991) believe that toxin crystals are photo-
sensitized by exogenous (and possibly
endogenous) chromophores picked up from
fermentation broth after the lysis of bacterial
cells. The chromophores create singlet oxygen
species on irradiation by light. Decreasing the
exposure of crystals to oxygen, e.g. by use of
glycerol as a humectant, reduces photodam-
age. Pozgay et a/. (1987) found that tryptophan
in the crystal was destroyed. Cohen et al.
(1991) suggest that photoprotection is attained
with cationic chromophores such as acrifla-
vin, by transfer of energy from excited trypto-
phan moieties to the chromophore molecules.
The mechanisms of baculovirus inactivation
are not fully known, but are most likely to be
similar to those reported for mammalian
viruses. With these, inactivation is primarily
due to the formation of DNA cross-links
through pyrimidine dimers. This may be
related to the formation of peroxide radicals
(Ignoffo and Garcia, 1994). In Table 3.16, part
I, screens that function by preventing dam-
aging wavelengths from reaching the patho-
gens are distinguished from the antioxidants
and enzymes that act by scavenging hyperact-
ive oxygen.
3.4.4d Effect of conditions of test Test condi-
tions have great effects on assessments with
sunscreens. These effects are minimized in
Table 3.16 by making direct comparisons
between screens only within studies, i.e.
experiments within which conditions are
identical. However, differences between stud-
ies involving the same screen are often great.
These differences are partially due to the
parameters measured: studies with spores
alone (assigned as's' in the table) and the
whole pathogen complex of B. thuringiensis
(no letter assigned) are identified in the
table. Field studies (bold type) are more strin-
gent than laboratory work, because the effects
of screens are least likely to show up when
insect populations (p) or yield and defoliation
(y) are measured. Consequently, when using
the table different weights must be given to
the different types of results. In particular,
effects on B. thuringiensis spores are less
important than effects on the whole spore-
crystal complex and - even more pertinent -
effects demonstrated in the field, especially on
yield or foliage protection, are more meaning-
ful than laboratory results.
A number of studies have been undertaken
in the field where microorganisms have been
exposed to direct sunlight on glass slides or
Petri dishes. Under tropical conditions, Jones
et al. (1993a) found that glass surfaces on
which NPV samples were placed reached
60C within a few minutes; the temperature
alone would be sufficient to inactivate the
virus (section 3.3.4). This problem was
avoided by placement of samples on the sur-
face of a refrigerated tray. The same problem
occurs with some laboratory artificial sunlight
equipment and UV lamps. Again, heat should
be dissipated, particularly since light inactiva-
tion is influenced by temperature.
Different results may be attributed to less
obvious technical factors. For example, dusts
exposed on the surface of agar plates are
wetted, but those exposed on glass are not;
wetting and redrying increased the suscept-
ibility of Heliothis NPV in a talc-based dust by
x28 (IgnoffoandGarcia, 1996). Martignoniand
Iwai (1985) reported that the sunscreen Coax on
dry, non-wettable Teflon pads gave 61% of the
protection obtainable by the same concentra-
tion of Shade, in contrast to 113% reported by
Shapiro et al. (1983) on moist, porous agar-
based insect diet. Shade is soluble in water but
Coax is not, which may result in considerably
different distributions on the two surfaces.
Also, moisture on the agar surface would
allow diffusion of the singlet oxygen species
created by the irradiation, facilitating their
absorption by Shade, but the water-soluble

screen would dissolve and diffuse away into
the agar (Table 3.16). Silicobenzone inactiv-
ated 70% of codling moth GV as it dried on a
slide, probably because a 1% solution has an
acid pH of 2.5, but it did not increase the
speed of inactivation on apple trees, possibly
because of the buffering action of chemicals
(Richards, 1984). It is essential that final con-
clusions on the efficiency of UV protectants be
based on field tests.
A sunscreen may have other benefits in
addition to photoprotection. Thus it may be
difficult to apportion the cause of an observed
improvement. For example, molasses also
alters viscosity of a spray and hence spray
drop size and distribution, which in turn
influence the amount of the pathogen that
target larvae eat. Also, spray composition
determines the spread of a drop after impact,
which influences the thickness of the dried
deposit and so the amount of protective screen
above the particles (Appendix II, Fig. 11.6). As
another example of multiple action, milk has
moderate sun-screening capacity (Table 3.16),
is used as a wetter-sticker (Table 3.13 in sec-
tion 3.4.2; Richards, 1984) and humectant (sec-
tion 4.3.4), and also acts as a feeding and
growth stimulant to larvae of the torticid
moth, Archips pomonella, causing increased
damage to apple foliage treated with codling
moth GV since Archips is not susceptible to
this virus (Glen et al., 1984; Richards, 1984).
The position of a pathogen in a drop is
important (Appendix II, Fig. 11.6). Pathogens
have been observed to float on the surface of
an oil drop, where they would gain little
protection (Killick, 1986). This must be reme-
died by use of surfactants. In photographs of
deposits, some pathogens were virtually
unshielded by a screen, while others could be
totally hidden under bigger particles. On dry-
ing, some deposits of lignosulphonate devel-
oped cracks (Killick, 1986). Molasses, on the
other hand, appeared to cover all particles in a
deposit (K. A. J., unpublished results).
The efficiency of any sunscreen depends on
the depth of the layer of screen covering the
3.4.4 Sunscreens 75
pathogen (Appendix II, Fig. 11.6), which, in
turn, depends on drop size. Functional opti-
mization of spray design requires a balance
between larger drops to achieve the best
photoprotection and smaller droplets to
achieve the best spray cover.
The effectiveness of a screen covering the
organisms depends on the concentration of
both the screen and the organisms in a prod-
uct. For example, 5% (of final weight) Shade
added to a technical concentrate of Heliothis
NPV, before freeze drying and grinding,
improved survival after exposure to UV radia-
tion (peak at 365 nm) by x 1.9 (ratio of OARs
with and without Shade: an OAR is an original
activity ratio), but by x 5.7 in a talc-based dust
(Ignoffo and Garcia, 1996). This difference is
not surprising because the amount of Shade
per polyhedral inclusion body (PIB) in the
dust was x 25 that in the concentrate due to
the different PIB concentration.
3.4.4e Effectiveness of different sunscreens
Sunscreens act by selectively absorbing,
blocking or reflecting UV radiation, or by
negating active oxygen radicals. These mate-
rials are classified by mode of action in Table
3.16 and Appendix Table 1.9-12, and further
described in section 4.3.3c. Absorbents con-
vert damaging UV light to harmless visible
wavelengths.
Natural absorbents that accompany the
pathogens in microbial products confer vari-
able UV protection. This is explained by the
demonstrated protective action of amino
acids, B vitamins, nitrogenous metabolic pro-
ducts, oxidative enzymes and proteins (Table
3.16). These materials occur in residues of fer-
mentation solids in B. thuringiensis products
and in insect remains in unpurified or par-
tially purified virus. Many materials are
protective at 1% or more, and tyrosine, tryp-
tophan, folic acid, ascorbic acid and catalase
give some protection at low concentrations of
0.1% and less. Remarkably, the insect pigment
melanin is highly effective at only 0.0003%
(Table 3.16; discussed in section 10.9) and it
76 Formulation of bacteria, viruses and protozoa to control insects
is the active pigment in a UV-resistant B. thur-
ingiensis mutant (Patel et al., 1996). Unpurified
NPV has frequently proved to be more photo-
stable than purified NPV (e.g. Manjunath and
Mathad, 1982; Shapiro, 1984). Surprisingly,
Jones (1988a) found that none of 12 recog-
nized screens tested gave better protection
than the debris in unpurified NPV. Some of
the naturally occurring absorbents in Table
3.16 can be considered as candidate screens
in practice. None of the nitrogenous metabolic
products is suitable. Although tryptophan
and tyrosine are good protectants, an amino
acid would not be economically feasible to use
(Ignoffo and Garcia, 1995). Some B vitamins
protect by absorbing UV (Table 3.16). The best
are folic acid and riboflavin, which also func-
tion as biochromes in insects and other ani-
mals. Some other fluorochromes are also
effective (Table 3.16). The commercially avail-
able xanthopterin, which is chemically close
to pteridine, a constituent of folic acid, was
almost as effective as the vitamin. Among the
proteins (Table 3.16), albumin and milk prod-
ucts are already used as spray stickers (section
3.4.2) and sometimes as additives during
harvest of pathogens (section 3.2.1). Gluten
makes sprays autoencapsulate (section 3.3.1),
as well as being a feeding stimulant (section
3.4.3) and sticker (section 3.4.2). Their protec-
tion from sun is a valuable additional asset.
Three good natural products, used as
screens in practice, are listed under 'miscel-
laneous' in Table 3.16. Molasses is a multi-pur-
pose additive which was once popular and is
still used at high concentrations, at which it is a
good sunscreen. However, it is cumbersome
and cannot be easily incorporated for product
storage; while freeze-drying or spray-drying
could make this possible, these processes are
difficult with molasses. Carbon products have
been tested extensively as blocking screens
with good results. This is thought to be due,
at least partly, to the ability of carbon to act as
an oxygen sink, preventing the formation of
free active radicals (Ignoffo and Garcia, 1978).
The phagostimulant Coax is also an excellent
sunscreen and - being a dry powder - can be
mixed into dry stored products; however, it
can be difficult to suspend in water (Jones,
1994). All three materials are bulky, a big dis-
advantage for aerial application.
The need for an easy-to-handle water-solu-
ble protectant, effective at low concentration
and miscible with most products during stor-
age, led to the special marketing of Shade, a
polyflavanoid (Table 3.16, miscellaneous;
Appendix Table 1.12). This absorbs radiation
maximally at 285-290nm (Krieg et al., 1980b),
as well as peroxide radicals (Ignoffo and
Garcia, 1978). Also, it was an effective buffer
on cotton, and reduced foliar pH below 8.6
which may have reduced inactivation of virus
by high pH (Young and Yearian, 1976). As
Shade was taken off the market, many other
materials were evaluated from the late 1980s.
The new materials include cosmetic sun-
screens, dyes, lignin, lignosulphonate (by
products of wood pulping) and optical bright-
eners (Table 3.16). Of the cosmetics, Uvitex
ERN-P and the Eusolex series at 0.1%, also
benzilidene sulphonic acid at 5%, were rated
best. Both water- and oil-soluble dyes include
good protectants; Congo Red is the best.
Some, e.g. the alkaloid berberine, are best
avoided due to mammalian toxicity.
A number of studies directly compared the
new with the older materials. Shapiro et al.
(1983) and Jones (1988a) found the best from
both to be Coax, benzilidene sulphonic acid,
Eusolex, Indigo Carmine, molasses and clays
(Table 3.16). One of the best comparative
laboratory studies is that of Martignoni and
Iwai (1985), who took cost into account. They
rated two lignosulphonates, Orzan LS and
Raymix Powder, as best, and Tinopal DCS
considerably behind as second best on
grounds of effectiveness and cost. Orzan and
Raymix Powder have excellent properties as
spray additives: they easily dissolve in cold
water and the solution is free from insolubles,
with low surface tension and pH near neutral.
Both substances were less expensive than
many cosmetic sunscreens.

Over the last 10 years, most progress in field
testing sunscreens has been made with NPV,
the type of pathogen more in need of protec-
tion. In 1986, Orzan LS was demonstrated to
be effective in North American gypsy moth
NPV field tests (Podgwaite and Shapiro,
1986; Table 3.16). It was used in the standard
Gypchek formulation in subsequent trials.
Recently, a number of stilbene optical bright-
eners have proved effective at low concentra-
tions (Table 3.16). They have been most
effective with some moderately active viruses
due primarily to their spectacular synergistic
effects (section 3.4.6). Tinopal LPW (= Blanko-
phor BBH) at only 0.05% was outstanding in
the laboratory and promising in the field
(Table 3.16). It has been possible to lower the
current recommended dose rate for ground
application of Gypchek using brightener by
x10 (Reardon et al., 1996). Other superior
brighteners include Intrawite CF, Leucophor
BS and BSB, and Phorwite AR (Table 3.16).
User-friendly commercial gypsy moth NPV
products now being developed in trials from
1992 are expected to contain a sunscreen.
Although Orzan LS is no longer commercially
available, the main choices are likely to be
from the lignosulphonates and optical bright-
eners.
Use of UV protectants at 1-10% in low-or
high-volume tank-mixes is wasteful, and
they can be more efficiently used in microcap-
sules (section 3.3.5). Carbon black and tita-
nium dioxide gave good protection in the
laboratory and on field cotton when mixed
with NPV, and excellent protection when
encapsulated in a polymer (Table 3.16).
McGuire et al. (1991) incorporated Congo
Red into matrices of starch (the matrix itself
is a blocker); incorporation may partly solve
the dye's unfortunate property of staining
one's skin, as experienced by one operator
who finished brilliant red all over after apply-
ing an experimental field spray containing the
non-encapsulated dye. However, after storage
without exposure to UV it decreased grass-
hopper mortality, possibly by toxicity to the
3.4.5 Additives to counter foliage factors 77
virus, altering the infection cycle or deterring
feeding.
Both Shade and carbon are believed to
reduce free oxygen radicals, an alternative
mode of action to absorption and reflection.
Among possible antioxidants and oxidative
enzymes that scavenge or catalyse the de-
gradation of reactive radicals (Table 3.16,
part III; Appendix Table I.12), low concentra-
tions of propyl gallate might be used in tank
mixes because of low cost and common use as
a food additive (Ignoffo and Garcia, 1994).
Taking an overall view, the best sunscreens
are listed in approximate order of merit in the
Note at the foot of Table 3.16.
A few sunscreens have been critically
examined for possible harm to pathogens
(Appendix Tables 1.9-12). Sulisobenzone inac-
tivated GV on slides.
3.4.5 ADDITlVES TO COUNTER FOLIAGE
FACTORS
The chemical composition of plant leaves may
influence insect pathogens in various ways.
The cotton phylloplane is highly alkaline,
with pH values as high as 11. High pH dis-
solves crystals of B. thuringiensis and harms
baculoviruses, making them less stable on the
leaf surface. Standing NPV in phosphate buf-
fer at pH 4-9 for 24 h at 30 cC had no effect
but, at pH 10, virus was inactivated even
when encapsulated in the water-insoluble
polymer SMA-2625A (Appendix Table 1.3;
Bull et aI., 1976). Occlusion bodies of several
NPVs are solubilized - and hence inactivated
- at pH values close to 10 (Griffith, 1985).
Addition of Shade and molasses reduces the
pH of sprays (Young and Yearian, 1976) and
the pH can be made slightly acid with >0.06%
Sorba Spray Zip (Vail et aI., 1977, 1980). Buf-
fered sprays have had negative or indifferent
effects (Falcon, 1971; Young and Yearian,
1976). Elleman and Entwistle (1985a, b) sug-
gested that, rather than the effect of alkalinity,
free Mg 2 + ions in cotton dew prevented the
dissolution of polyhedra within the insect gut,
78 Formulation of bacteria, viruses and protozoa to control insects
an effect reversible by the addition of a chelat-
ing agent, e.g. ethylenediaminetetra-acetic
acid (EDTA) (Appendix Table 1.7).
The surface of some leaves is acid, e.g. pH 1
on chickpea due to maleic acid, which might
directly inactivate an organism, and also
lower the insect gut pH and impair the activ-
ity of both the crystal and spore of B. thurin-
giensis. Thus Gringorten et al. (1992) found
that reducing the alkaline pH of enzyme-
activated crystal toxin reversibly lowered
toxicity in assays on lawns of IPRI-CF-1 cells,
and in force-feeding assays with silkworm
larvae. Also, the principal activator of spores
in the gut of tobacco hornworm larvae is alka-
line pH (Wilson and Benoit, 1993). Dew of
<pH 4 on leaves could denature the crystal
or NPVs.
Allelochemicals, part of a plant's natural
defence against herbivores (an increasing
area of study) influence insect pathogens in
various ways. Some plant extracts inhibit bac-
terial growth (e.g. Morris, 1972; Morris and
Moore, 1975). Nicotine, an alkaloid, reduces
the activity of B. thuringiensis, probably that of
the crystal (Barbosa, 1988; Krischik et al.,
1988). Tannins (mixtures of polyphenols) are
general precipitators of proteins and inactiv-
ate the toxin, as shown by a reduction of
larval mortality after pre-reacting tannin
with crystals in various stages of activation
(Liithy et al., 1985). The presence of tannin
depends on the plant species: for example, it
depressed the activity of B. thuringiensis in
gypsy moth larvae on tannin-rich trees more
than on aspen, a tree low in tannin (Appel and
Schultz, 1994).
The amount of insect feeding on plants is
reduced by many allelochemicals, e.g. nico-
tine and tannins (Navon, 1992, 1993; Morris
et al., 1995). This decreases the amount of
bacterium or virus ingested, and may be
partly responsible for differences in LCso
values of the same pathogen on different
plants observed by Richter et al. (1987), San-
tiago-Alvarez and Ortiz-Garcia (1992). Allelo-
chemicals probably also have an effect in
other ways (Jones, 1988a; Navon, 1992, 1993).
Richter et al. (1987) suggested that differences
in the susceptibility to NPV of larvae reared
and dosed on different plant species was due
to stress. It is well established that a protein
present in mulberry leaves combines with a
protein in the gut of silkworms to form an
anti-viral protein (Uchida et al., 1984; Hou
and Chui, 1986). Although no similar mechan-
ism has been identified for other insect or
plant species, this example does illustrate
that such effects are possible. Felton et al.
(1987) found that two ortho-hydroxy phenolic
compounds, rutin and chlorogenic acid,
which are present in tomato plants and are
potential sources of insect host-plant resis-
tance, reduced the infectivity of Helicoverpa
zea NPV in tissue culture, but rutin had no
effect on Manduca sexta NPV on leaf (Krischik
et al., 1988).
Specific attempts to neutralize antibacterial,
antiviral and allelochemical substances have
been unsuccessful (the above example of
EDTA against free Mg 2+ ions is one exception,
although probably not a practical solution).
However, these substances may be partially
counteracted by the use of phagostimulants
(sections 2.2.4, 3.4.3). Also, additives regarded
primarily as stickers, sunscreens and syner-
gists (Table 3.13 in section 3.4.2, Table 3.16 in
section 3.4.4, Tables 3.17 and 3.18 in section
3.4.6 below) may owe part of their ability to
prolong the activity of organisms to anti-alle-
lochemical action. Problems associated with
extremes of leaf surface pH might be addres-
sed through encapsulation of the organism in
a polymer insoluble in either acid or alkali, or
with good buffering capacity, e.g. the sugar-
starch formulation (McGuire et al., 1996).
Alternatively, protection from both low and
high pH may be given by formulation in oil.
3.4.6 SYNERGISTS
Additives have a bewildering array of inter-
actions with pathogens, ranging from great
increases in activity to nearly total inhibitions

of host deaths. Benz (1971) describes eight
types of effect within this range. An additive
may be innocuous to the insect on its own, but
within the same host-pathogen system it may
have a spectacular effect with the pathogen as
a classical synergist, or its lethal effects may
do no more than add to the mortality caused
by the pathogen, a transition that can be
dosage dependent. For simplicity in this chap-
ter, all increases in mortality in combinations
will be called synergy.
All pathogens detailed in this chapter attack
perorally, so the dosage taken up by the insect
depends on the amount of food eaten. Thus
phagostimulants (section 3.4.3; Appendix
Table 1.7) synergize and feeding depressants
antagonize the pathogens, while malnutrition
due to feeding depression may itself kill, giv-
ing a synergistic increase of mortality. Table
3.17 relates the effects of synergists to their
suggested modes of action. Some synergists
may combine more than one mode of action,
e.g. acetamide, caffeine, tannic acid (a variable
mixture of complex phenolic acids) and sim-
pler phenolic compounds, e.g. chlorogenic
acid and polyphenol oxidase (Ludlam et al.,
1991), may synergize by reducing feeding,
while direct poisoning may also be a factor.
B. thuringiensis is synergized by protease
inhibitors, although MacIntosh et al. (1990)
have evidence that they do not prevent release
of toxins from the crystal in the gut. Activities
of all three main strain groups, active in Lepi-
doptera, Diptera and Coleoptera, were syner-
gized.
B. thuringiensis synergists may act at any
stage of the pathogenic process (Table 3.17).
At the very beginning, seed extracts stimulate
feeding (section 3.4.3) and increase the
amount of pathogen ingested, while some
also contain trypsin inhibitors (see above).
Alkalis increase gut pH, aiding protoxin solu-
bilization by breaking disulphide bonds.
Ca2+, K+, Na+ and Zn 2+ ions (Table 3.17,
part III) are cofactors of proteolysis, which
cleaves protoxins into active toxins. Additives
that damage the epithelium and impair secre-
3.4.6 Synergists 79
tion of alkaline gut juices lower gut pH,
enabling spore germination and bacterial
growth, although some synergistic additives
are known germination inhibitors - an activ-
ity that would have the opposite effect. Ascor-
bic acid is a common ingredient in insect diets
and influences insect health; larval mortality
of the codling moth was increased by sub-
and supra-optimal proportions (Pristavko
and Dovzhenok, 1974). Abrasion by boric
acid and erosion by chitinase may make the
peritrophic membrane more permeable to the
toxins. Detergents are lipid emulsifiers and
may increase the permeability of the epithelial
cells themselves to these toxins. Without
direct proof, ascribing causes to observed
effects, these suggestions are tentative. Some
of the above synergistic additives may, of
course, have other effects which may be con-
current, active in different circumstances, or
functional in different insects. Thus, Ca2+ is a
K-channel blocker that inhibits the action of
toxin on the apical membrane of epithelial
cells and causes inhibition. Tannic acid, a
strong synergist (Table 3.17, part VI), can
also inactivate the crystal toxin (section
3.4.5). After the toxin disrupts epithelial per-
meability, the blood is polluted and the pre-
sence of extra amino acids may alter the vital
amino acid balance in the blood, which could
explain both observed synergisms and inhibi-
tions (Table 3.17, part I).
Whatever their modes of action, some
synergists appear exciting in practice. With
B. thuringiensis in pests of only limited suscept-
ibility, i.e. species of Spodoptera and Agrotis,
Salama's group found that over 18 additives
in insect diets gave 2: la-fold synergism
(Table 3.17, part III, references 4, I, 5). In
these and other insects, the most promising
additives were CaC03 and CaO, CaS04,
CuC03 Cu(OHh, K2C03, Na2C03, ZnS03
and ZnS04, also the amino acids arginine,
asparagine, glutamine, ornithine, proline, ser-
ine, tryptophan and valine, as well as aceta-
mide, caffeine, tannic acid, Tween 60 and 80,
trypsin inhibitor, EDTA, sodium thioglycolate
80 Formulation of bacteria, viruses and protozoa to control insects
Table 3.17 Synergists of Bacillus thuringiensis (Bt) and of nuclear polyhedrosis virus (NPV) in assays on
artificial insect diet and, in bold print, in leaf assays and field tests

Synergist (Appendix Table 1.7) Concentration (%)* Effect (x fold)! Reference Suggested mode of action
I AMINO ACIDS AND AMIDES WITH Bt
DL-Alanine ? 4.6 1 Bt germination
D-Alanine + D-serine 0.12-0.48 R 2 inhibitors
L-Arginine 0.01 3.5 3 Many amino acids
0.05 3.5 3 affect Na+ and K+
0.1 1.8, 3.6, 6.5, 14 3,4,5,5 transport in gut. May
leaf alter toxin activity.
? 40 1 Balance in blood
important
L-Asparagine ? 26 1
DL-Aspartic acid ? 4.8 1
D-Cycloserine 0.02 1 2 Germination and
protease inhibitor,
bactericide
L-Glutamic acid ? 3.1 1
DL-Glutamine ? 29 1
Guanosine 0.05 1 2 Germination inhibitor
DL-Ornithine ? 22 1
L-Proline ? 31 1
L-Serine 0.05 1.7 3
? 7-40 1
DL-Serine ? 7.1 1
I.-Tryptophan 0.5 13 4
DL-Tryptophan ? 16 1
L-Valine 0.05 2.3 3
0.1 4.2,20 5,5
leaf
? 7-40 1
DL-Valine ? 7.3 1
D-Methionine, 0.1 0.52-1.19 5
L-phenylalanine,
DL-phenylalanine,
L-tryptophan
L-Cystine, L-histidine, ? 0.57-1.5 1
DL-isoleucine, L-Ieucine,
L-Iysine, DL-methionine,
DL-threonine, DL-tyrosine
II SURFACTANTS WITH Bt
Cetyltrimethylammonium 0.025 3.6 4 Damage lipid
bromide membranes of gut
Tween 40 0.5 3.1 4 epithelial cells

Tween 60 0.05 1.6,9.3 3,4

Tween 80 0.05 0.3,6 3,4
Sodium dodecyl sulphate 0.01 1.8 3
0.05 3.5 3
0.1 1 3
 3.4.6 Synergists 81
III INORGANIC SALTS AND ACIDS WITH Bt
(NH4 lzS 2O S 0.05 0 3
(NH4 lzHP04 0.05 1.4 3
0.5 7.3 5 leaf
Boric acid 0.01 1.1 3 Abrasive, damages
0.05-0.1 3.5,3.5 3,3 peritrophic membrane
1 1.3-1.8,4.0-7.0 6,6 and epithelial
Borax 0.1 6.6 5 leaf membrane
0.5 27 5
CaC03 0.05 1.6,10.1 3,3
0.1-0.25 4.6, 6.0, 4.3 1,4,5
0.5 1.2,1.2,1.4,5.7 7,7,7 all
yield, 15
leaf
CaO 0.05 1.7,7.6 3,4
0.1 15, 15 5,5leaf
0.5 0,1.2 7,7
Ca(OHlz 0.5 1,1.2 7,7
Ca(N03)2 0.01 1 4
CaS04 0.05 1.9,2.0, 3,3,
0.1 3.5,4.6 1, 1
0.5 1.1,1.3 7,7
both
yield
1 5.2 4
CuC03 . Cu(OHlz 0.05 1.5, 14.6, 20 3,1,4
0.5 1.1 7 yield
CuO 0.05 1, 13.5 1,4
Copper phosphate 0.05 6.7 13
CUS04 0.05 3.0 4
MgCl 2 0.01 3.5 3
MgS04 0.05 2.1 3
0.01 1.9 3
0.05 3.5,2.8 3,3
0.1 1.5 3
0.2 R 2
K2C03 0.05 2.2,4.1 3,3
0.075 1.3, 1.4, 1.1 8,8,7 all
yield
0.5 8.8; 4.8; 4.4; 1.1, 5,5,
1.2,1 5 leaf, 8,
8, 7 all
yield
KHC0 3 1 18 4
K2HP04 0.05 1.8 3
1 25,2.5 1,5
Na2C03 0.05 1.8,7.6 3,3
NaN03 1 3.9 4
NaN02 0.1 R 2
ZnS04 0.05 2.2,20 3,4
0.1 16 5
1 10 1
82 Formulation of bacteria, viruses and protozoa to control insects
Table 3.17 (Contd.)

Synergist (Appendix Table 1.7) Concentration (%)* Effect (x fold)t Reference Suggested mode of action

ZnS03 0.1 4.7,8.9 1,5 leaf

0.5 1,1.2,1.2 7,7,7 all
yield
IV MISCELLANEOUS WITH Bt
Ascorbic acid 0.05 2.3 3 Excess reduced
haemocytes and
phagocytes, increased
susceptibility
Caffeine 0.1 3.5,9.2 9,9 Feeding inhibitor
Dimethyl sulphoxide 0.05 1.4 3
Dodecyclamine 0.05 1 3 Some acylamines
increased NPV infection
Dipicolinic acid 0.02 R 2 Germination inhibitor
Enhancin protein from GV various 0.7-3.4 10
Neemazal-T 0.001 1.0-2.9 10
Salicylic acid 0.5 0.83 5
p-amino salicylic acid 0.1-0.2 2.9 2
0.5 1.6 3
Sodium salicylate 0.5 0.50 5
Sorbic acid 0.05 1.6 3
V OPTICAL BRIGHTENERS WITH NPV
Tinopal LPW 0.01 1,1-1.6 11,11 Causes virus
= Blankophor BBH 0.02 1-4.3 12 maturation in gut cells
= Brightener 28 0.05-0.1 15, 11,
= Calcofluor White M2R 1584, 11,
0.7--461 10
1-2.7 11,
2.1-5.7, 12
0.5 5.5-11.7 12
1.0 1.7-2.3 11,
42,214 13,13
0.25 4-16 14
1.0 41-214 15
VI ORGANIC ACIDS AND THEIR SALTS WITH Bt
Acetamide 0.01 1.5,5.0 3, 5 leaf Reduces feeding
0.05 3.5 3
0.10 2.3,21 3,5
1.0 24,25 1,4
Calcium acetate 0.05 1.7,3.7, 8.8 1,3,4
Fumaric acid 0.5 3.9 1
Lauric acid 0.05 1.5 3
Malic acid 0.05 1.8 3
0.5 3.8 1
K tartrate 0.1 3.8 1
Sodium acetate 1.0 3.4 1
Na formate 05 2.6 1
Uric acid 0.5 1.9 1
 3.4.6 Synergists 83

VII PHENOLIC COMPOUNDS WITH Bt

NH4 benzoate 0.1 1 2 Feeding inhibitors,
Methyl-p-hydroxybenzoate 0.88 1.83 16 direct poisons
1.76 0.82 16
Sodium benzoate 0.5 2.5,0.75 4,5
Gallic acid 0.001 0-1.4 16
0.01 0.66-0.75 16
0.72 3.4 17
Phenylacetic acid 0.1 1.1 5
Resorcinol 0.27 3.6 17
Tannic acid 0.0025 7.1,0 18,18
0.05 1.8 3
1.0 20 5
VIII PROTEASE INHIBITORS WITH Bt
20 seed extracts < 20 1.4-6.9 19 Phagostimulants
Metallo, sulphydryl, carboxyl 1.0 1 19
protease inhibitors, amylase
Trypsin inhibitors 2 x 10- 6 -0.6 2-40 19
0.05 1.8 3

IX PROTEIN SOLUBILIZING REAGENTS WITH Bt

EDTA 0.25 1 2 Germination inhibitor,
0.5 12 1 chelating agent,
protease inhibitor
Naz - ,a-glycerophosphate 0.05 6.0 4
0.1 2.0 5
1.0 12 5
Sodium thioglycolate 0.05 1.9, 16 3,4
1.0 3.4,3.0 1,5
Potassium phosphate, 1.0 5.6 4
K zHP03
Urea 0.5 2.7,2.3 1,4

References
1 Salama et aI., 1989 11 Zou and Young, 1996
2 Burges, 1977 12 Webb et aI., 1996
3 Morris et aI., 1995 13 Farrar et aI., 1995
4 Salama et aI., 1985 14 Vail et aI., 1996
5 Salama et aI., 1986 15 Dougherty,1996
6 Doane and Wallis, 1964 16 Dimetry and Matter, 1990
7 Salama et aI., 1990b 17 Sivamani et aI., 1992
8 Salama et aI., 1990a 18 Gibson et aI., 1995
9 Morris et aI., 1994 19 Macintosh et aI., 1990
10 El-Salamouny et aI., 1997
Percentage (wt/vol) in diet or spray.
t Statistically significant ratios of LC so or of percentage mortality for pathogen + synergist to LC so or percentage
mortality for pathogen alone. Non-significant ratios indicated by zero. Where actual values are not given in the orginal
publications, significant reduction in potency is shown by R, and significant enhancement by E.
84 Formulation of bacteria, viruses and protozoa to control insects
and sodium glycerophosphate. These results
were obtained with a range of B. thuringiensis
strains, including one of the newer strains, that
are more active than kurstaki against Spodoptem
spp.
Effects were even more impressive on leaf
(bold values in Table 3.17). Seven additives
gave greater synergism on cotton leaf than in
diet and one vice versa (Morris et al., 1995).
Most diets contain no inhibitory allelochemi-
cals, so these differences may reflect reactions
on leaf-to-plant allelochemicals (sections
3.4.5). With a virus, Jones (1988b) found the
LC so of NPV against S. littomlis to be lower on
diet than on cotton or lucerne, possibly due in
part to the presence of inhibiting allelochem-
icals in the leaves.
Synergists could improve some uses of B.
thuringimsis from marginal to highly eco-
nomic. With var. kurstaki against S. littornlis
on cotton leaf, for example, an LC so of
1222 {Lg/ml (0.1% by weight) could be
upgraded to 122 or even 12 {Lg/ml (Salama et
a!., 1986). In this species, rapid direct poison-
ing by the toxin may be synergized to replace
slow death by attrition, due to starvation and
prominent involvement of the spore. Many of
the synergists are inexpensive, e.g. individual
salts would increase the cost of sprays by only
2.6-7.0 cents/l (Morris et al., 1995). In field
tests, synergistic effects of some of the salts
have been demonstrated as x1.1 to x5.7
increases in crop yields (Table 3.17).
Combining synergists sometimes enhances
the synergism (Table 3.18). The two most
effective combinations both involved K2 C03 ,
one with borax and the other with ZnS04;
their collective synergisms were x3 the
expected sums of individual synergisms. Six
combinations increased synergisms by >20%,
five remained similar, but four combinations
resulted in less synergism by at least 20%
(Table 3.18).
Some of the synergisms are probably speci-
fic to certain plant and insect' species, since
results in Table 3.17 vary and refer to many
different insects, crops and trees. For example,
the addition of tannin might inhibit B. thurin-
giensis activity in some insect species not
adapted to feeding on tannin-rich plants (sec-
tion 3.4.5). Also, allelochemicals in some
leaves may prevent synergism of some sub-
stances active on diet. Thus a note of caution
is sounded, and these materials are likely to
be best used in specific tank mixes, rather
than added to the on-shelf products. Some
effects appeared to be B. thuringiensis-dosage
specific. For instance, sodium dodecyl sul-
phate and arginine showed significant syner-
gisms at the LD so , but not at the LD90 level
(Morris et al., 1995). This suggests further cau-
tion.
Viruses have even more exciting synergists.
Recent patented work by Shapiro and co-
workers vyith stilbene optical brighteners on
virus (Table 3.17, part V) has been spectacular
(Shapiro et a!., 1992). These are also sunsc-
reens (section 3.4.4e). Tinopal LPW (= Blan-
kophor BBH) enabled NPV to infect midgut
cells, a tissue where virus production of poly-
hedral bodies does not normally occur.
Within 24 h of application on insect diet, infec-
tion was irreversible and within another 24 h,
feeding stopped prematurely and the larvae
eventually died - exhibiting most of the
effects of starvation (Hamm and Shapiro,
1992; Shapiro, 1992; Shapiro and Robertson,
1992; Sheppard and Shapiro, 1994; Sheppard
et al., 1994).
The stilbene effect occurs on diet and leaf
(Table 3.17, part V), giving enhanced mortal-
ity and early oak foliage protection in 48 h
(Sheppard et al., 1994). The synergism is
most pronounced with the less active NPVs
(Dougherty et a!., 1996; Vail et a!., 1996) and
with low concentrations of virus (Vail et al.,
1996). There was no mortality in hosts not
susceptible to the virus alone (Adams et al.,
1994; Hunter-Fujita et a!., 1997b). Remarkable
enhancements were obtained, x118 at 0.1%
Tinopal LPW and x 1670 at 1% against
gypsy moth larvae (Shapiro and Robertson,
1992) and x164 to x303000 at 0.1% against
S. frugiperda (Hamm and Shapiro, 1992). Less
 3.4.6 Synergists 85
Table 3.18 Effect of combinations of synergists on the potency of Bacillus thuringiensis against Spodoptera
littoral is, compared (in parenthesis) with the sums of the effects of the synergists individually (Salama et
al., 1986)

Synergist combinations Concentrations (%) Effect (x fold)*

K2 C0 3 + borax 0.5 + 0.5 122(36)
Borax + tannic acid 0.5 + 1.0 23(47)
K2C03 + tannic acid 0.5 + 1.0 12(29)
Borax + tannic acid + K2 C03 0.5 + 1.0 + 0.5 60(56)
L-arginine + L-valine 0.1 + 0.1 17(34)
L-arginine + ,8-glycerophosphate 0.1 + 1.0 26(33)
L-arginine + L-valine + ,8 glycerophosphate + 0.1 + 0.1 + 1.0 + 0.1 24(51)
acetamide
ZnS04 + CaC03 0.1+0.5 16(15)
ZnS04 + (NH4)zHP04 0.1 + 0.5 20(21)
ZnS04 + K 2 C03 0.1 + 0.5 45(17)
ZnS04 + borax 0.1 + 0.1 39(15)
ZnS04 + K2C03 + CaO + (NH 4)zHP04 + borax 0.1 + 0.5 + 0.1 + 0.5 + 0.1 60(47)
CaC03 + (NH 4)zHP04 0.5 + 0.5 11(13)
CaC03 + borax 0.5 + 0.1 27(12)
CaC03 + ZnS04 + (NH 4)zHP04 +borax 0.5 + 0.1 + 0.5 + 0.1 42(28)

* LC so without synergist/LC so with synergist, on artificial diet and on (bold) cotton leaf.

spectacular results were obtained in forest
trials, from which 0.1% Tinopal LPW and
one tenth the normal rate of NPV has been
recommended for its ability to halve defolia-
tion with a visible but not unsightly Tinopal
deposit, speculatively estimated to reduce the
commercial materials cost of US$8 per large
shade oak tree for virus alone to US$3 (Webb
et al., 1996; Table 3.17, part V, reference 12).
Tinopal is stable at pH 3.0-1004, at 121 cC for
5 min and under UVA, UVE or UVC for 7
days (Shapiro and Argauer, 1995).
Among other synergists, early work
showed a 10- to 100-fold enhancement of
NPV by 0.03% hexylamine (C 6 ) and dodecy-
lamine (C l2 ), while higher concentrations
were inhibitory (Yamamoto and Tanada,
1980). These may alter the charge on virus
particles and hence their attraction to cell
membranes. Cationic detergents (cetyltri-
methylammonium bromide and dodecyla-
mine hydrochloride) enhanced infection;
anionic detergents did not (Yamamoto and
Tanada, 1978b). Boric acid and sodium tetra-
borate increased NPV potency (McKinley,
1985). In present industrial practice, the only
synergists (excepting chemical insecticides)
tentatively employed with viruses are stil-
benes, which permit a x 10 reduction in
amount of NPV applied against gypsy moth
(see above).
Numerous workers have tested combina-
tions of chemical insecticides with pathogens
in the field. The overall impression is that the
effect of these combinations is variable and
dependent on doses used, age of insect tested
and whether the chemical and microbial
insecticides were administered simultan-
eously or separately (e.g. reviews by Benz,
1971; Jones, 1994). This is particularly true
for chemicals with antifeedant activity either
before or after poisoning occurs. The most
useful results, therefore, are from field tests
against natural pest populations. These have
mostly been with tank mixes. However, some
products have been formulated to include
chemical pesticides, for example, Mamestrin+
includes deltamethrin at 10% of the normal
field rate and the normal concentration of
Mamestra brassicae NPV, synergism having
86 Formulation of bacteria, viruses and protozoa to control insects
been demonstrated in both laboratory and
field.
Both synergism and antagonism been noted
between different pathogens. Two NPVs were
synergistic in Trichoplusia ni (Lara-Reyna et al.,
1996). In four Lepidopteran species, GV cap-
sules contain synergistic factors that aid the
fusion of NPV with the cell membrane of the
midgut brush border (e.g. Tanada and Huku-
hara, 1971; Derksen and Granados, 1988).
However, caution is needed, because Hun-
ter-Fujita et al. (1992, 1997b) found that
another species, S. littoralis GV, antagonizes
the homologous NPV, although the effect
appears to be dose-related. This GV has been
found as a contaminant in batches of NPV
produced in Egypt and the UK. Variable
results have also been reported with mixtures
of NPV and B. thuringiensis (Chancey et al.,
1973; Lipa et al., 1975; McVay et al., 1977).
3.5 SYSTEMIC ACTION
To date, systemic action of insect pathogens
has been achieved only with the b-endotoxin
of B. thuringiensis. There are two methods of
making the endotoxin act systemically.
The toxin gene can be transformed into plants
to produce the toxin in plant tissue, which
has already had a huge impact on the use of
B. thuringiensis for pest control.
The plant may be inoculated with commensal
endophytic microorganisms, themselves
transformed with the toxin gene.
3.5.1 TOXIN-PRODUCING PLANTS
Transformation of B. thuringiensis toxin
genes into plants has been reviewed by Ely
(1993). Levels of toxin expression have been
progressively increased to 0.1% of the total
soluble plant protein (Hickle and Fitch,
1990), which is equivalent to about 0.003%
toxin in plant tissue, well above the LC 99 of
highly susceptible insects and sufficient to
kill at least some larvae of less susceptible
Lepidopteran pests normally targeted with
B. thuringiensis insecticides, such as Spodoptera
spp.
Once the transformed plant has been
perfected, no further action is necessary from
the user except possibly resistance manage-
ment (section 3.7.4). The systemic toxin pro-
tects the plant in the manner of a systemic
formulation, often for the whole life of the
crop, and afterwards biodegrades with the
plant remains. The gene is transmitted in
the seed. In 1996, transgenic varieties of corn,
cotton and potato were grown commercially
on over 2 million acres in the USA. Gelernter
(1997) states
'overall performance of the crops has been
superlative. By overcoming most of the pro-
blems that have plagued microbial insecti-
cides - most importantly, delivery to the
target insect - B. thuringiensis plants have
taken the quantum leap necessary for the
transformation of B. thuringiensis from a
novelty to a mainstream product.'
Other transformed strains and crops will be
produced. For cotton alone, it is projected that
the number of planted acres will increase to
2.4 million in 1997. In many Lepidoptera, pure
crystals entirely without spores applied to
insect artificial growth medium are less active
than spore-crystal mixtures (Li et al., 1987).
Luckily, this does not impair toxin activity in
transgenic plants, because phylloplane flora
assume the function of the spores: 14 out of
15 phylloplane bacterial species tested were
effective synergizers if formulated with pure
crystal toxin (Dubois and Dean, 1995).
Systemic action can be achieved in the
aquatic environment by transforming B. thur-
ingiensis toxin genes into algae in the food
chain of filter-feeding mosquito larvae
(Gelernter and Schwab, 1993; Yap et al. 1994;
Anon., 1995). Use of an ammonium-secreting
host organism adds the additional function of
a nitrogen fertilizer (Boussiba et al., 1992;
Ziniu et al., 1996). These have not been devel-
oped commercially.

3.5.2 TOXIN-PRODUCING ENDOPHYTES
Endophytic micro-organisms, which live
inside plants, can be transformed with toxin
genes. These were being developed as Incide
products by Crop Genetics International, but
were later dropped. A transformed maize
endophyte, Clavibacter xyli var. cynodontis,
gave some control of the European corn
borer (Rigby, 1991; Gelernter and Schwab,
1993). The endophyte did not survive outside
the host plant or in plant debris, was not seed-
transmitted and did not spread from inocu-
lated maize to adjacent non-inoculated plants.
There was a minimal 4% reduction in yield
(Rigby, 1991). The endophytes did not affect
substantially the extent of pest-crop residue
decomposition (Tester, 1992).
Endophytes can be applied as a spray to
plants or as a seed dressing. They require
formulation technology for delicate organ-
isms. Application to seed is the most conveni-
ent and efficient method. They are forced into
seed in a pressure chamber through micro-
scopic cracks that develop in the seed coat
during the seed-drying process. Very low
quantities are needed per acre compared
with a typical 1-5Ibs/acre/season for tradi-
tional B. thuringiensis products. Other advant-
ages are as for transformed plants, except for
the lack of seed transmission.
Strains of Rhizobium expressing the cry III
gene significantly reduced damage by Sitona
species feeding on root nodules of pea and
Table 3.19 Target aquatic insects and feeding behaviour that influences control by Bacillus thuringiensis
Insect
Blackfly larvae: inhabit fast-running water
Simuliidae, Simulium spp
Mosquito larvae: 'inhabit static and slow-moving water
Aedines, Aedes spp
Anophelines, Anopheles spp
Culicines, Culex spp
3.6.1 Problems of the aquatic environment 87
lucerne. These transformed strains competed
with wild strains but were less effective at
increasing plant biomass (Bezdicek et al.,
1994). They could presumably be formulated
for soil inoculation (section 7.3).
3.6 APPLICATION TO WATER
B. thuringiensis and B. sphaericus are used
extensively for mosquito and blackfly control
(Table 3.19) in a variety of different water
masses, ranging from fast-flowing rivers in
West Africa to public drinking containers in
the Far East (section 2.2.2c). Many different
formulations have been developed. The main
commercial products are suspension concen-
trates, followed by wettable powders and
much lesser quantities of large-grained for-
mulations. Both species of bacteria behave
similarly and can be regarded as interchange-
able from the formulation viewpoint. Neither
viruses nor Protozoa have been commercially
applied to water.
3.6.1 PROBLEMS OF THE AQUATIC
ENVIRONMENT AND ITS TARGET INSECTS
Unique problems are presented to the formu-
lator by water as a target surface (section
2.2.2c) and mosquitoes and blackflies as
target insects (Table 3.19). For control of
mosquito larvae, formulated bacteria are
sprayed or spread over the surface of static
Mode offeeding
Larvae adhere to static objects and filter passing
water in streams and rivers
Filter feed and scavenge along the bottom,
vertical surfaces and objects
Sweep under the upper water surface and filter
feed downwards relatively weakly
Filter feed strongly throughout a water mass
88 Formulation of bacteria, viruses and protozoa to control insects
or slow-moving water into which they sink at
a rate determined by the design of the formu-
lation. Blackfly larvae live in fast-moving
water courses and are controlled by pouring
bacterial suspensions into the water at conse-
cutive points, from which they are carried
downstreaflil
With mosquitoes, different feeding habits of
larvae of different species (Table 3.19) influ-
ence the effectiveness of the bacteria. Culex
larvae filter-feed up and down the depth of
the water; they are often termed column feed-
ers. Aedes larvae tend to scavenge along
substrate surfaces, particularly the bottom.
Anopheles larvae feed on buoyant material
trapped at or just below the water surface
and filter-feed downwards relatively poorly,
depending on availability of food at the sur-
face. In comparable conditions, two Anopheles
species filtered water at the rate of 33-34 and
49-55f.tljlarva/h, respectively, while Culex
quinquefasciatus filtered 490-590 and Aedes
aegypti 590-690 fll/larva/h (Aly, 1988).
Anopheles larvae were most susceptible to B.
thuringiensis held near the surface, e.g. formu-
lated in flour or lipid capsules. In wheat flour
+5% corn oil in 100-ml volumes of water in
plastic cups, B. thuringiensis was x2-4 more
effective against three species of Anopheles
than suspensions of very fine particles con-
taining the same amount of B. thuringiensis
applied to the surface and allowed to diffuse
downwards. The larvae ingested the buoyant
formulation in a short time (maximum
20 min). In larger volumes (1751), the differ-
ences were much greater, x39-68 (Aly et al.,
1987). When confined in buoyant lipid cap-
sules in test tubes 4.5 cm deep, purified crys-
tals were x20 more effective than in
suspensions against Anopheles larvae, but
only x2-3 more effective against Aedes and
Culex larvae. Rather than a position effect,
the latter result was probably due to the cap-
sules being of a more amenable size for feed-
ing than free crystals (Cheung and Hammock,
1985). When assayed with dissolved crystal,
toxicity was less than with intact crystals in all
three types of larvae because these larvae are
filter-feeders and do not ingest the protein
efficiently in soluble form (Schnell et aI.,
1984); the observed toxicity was similar in
larvae of all three types (Cheung and Ham-
mock, 1985). These observations accord in
showing that larval feeding habits partly
explain why species of Anopheles have consist-
ently appeared less susceptible to B. thurin-
giensis suspensions than the column- and
bottom-feeding Culex and Aedes larvae in
laboratory assays and field tests (for review
see Lacey, 1985b).
Thus differently formulated products are
required for mosquito larvae of different
feeding types. Buoyant products are required
for anophelines, but products should stay
in suspension below the surface for column-
and bottom-feeders. In natural waters, rapid
sinking should be avoided because steady
deposit of debris would soon cover the
particles.
The content of crystal toxin in products is
measured by bioassay with Aedes aegypti and
expressed in IV. The use of small volumes of
water, soon filtered clear of particles by the
assay larvae, largely negates the effect of par-
ticle size, texture and formulation to give a
true measure of toxin content. However, in
the field, even when comparing suspensions,
the effectiveness against target mosquito
and blackfly larvae depends more on the
rate of settling than on the IV (for review see
Guillet et al., 1982; Molloy et al., 1984; Lacey,
1985b; Lacey and Heitzman, 1985). Lack of
agreement between potency measurements
with Aedes aegypti larvae and field perform-
ance of products against blackflies in West
Africa has not caused serious difficulties.
There is some confusion about the quantita-
tive value of the IU according to different
definitions [1 international unit (lTV) = 2.5
Aedes aegypti units (AAV)], discussed by
Wassmer (1995) in relation to an excellent list
of current formulated proprietary products,
their costs per IV and application rates.
Universal acceptance of the World Health
 3.6.2
Organisation's recommended Aedes assay
technique and definition by industry - and
users also - would be a great advantage (Guil-
let et al., 1990).
The effectiveness of many bacterial formu-
lations against both mosquitoes and blackflies
is short-lived in the field, often only 1-2 days.
This is due to rapid settling, adsorption to
plants and other substrates (which also filter
particles out of the water), denaturing of the
crystal by sunlight and engulfment by filter
feeding fauna (for reviews see Lacey, 1985b;
Lacey and Undeen, 1986). A major objective of
formulation is to extend the effective period.
The UV component of sunlight is much less
important in water, where particle settling is
the key factor, than on land foliage; partly
because water filters out much of the UV
radiation. With B. thuringiensis only the effect
of sunlight on the crystal reduces larval mor-
tality, since the spore is unimportant in mos-
quito and blackfly larvae. In water 2.5 cm
deep, the equivalent of 6 days natural sunlight
inactivated B. thuringiensis ssp. israelensis
(Ignoffo et al., 1981), but shorter exposures
did not (Garcia and Des Rochers, 1979; Mulli-
gan et al., 1980). In much less than 6 days,
particles are mostly filtered out of fast-run-
ning water or largely sink out of reach of a
damaging dose of radiation in still water. B.
sphaericus is more susceptible to sunlight,
being inactivated in clear water a few centi-
metres deep in full sun (Mulligan et al., 1980),
while strong sunlight reduced its effective-
ness several-fold (Skovmand and Bauduin,
1998). A sunscreen might be beneficial with
B. sphaericus, particularly in formulations
designed to float. However, as with many
additives used in water, the screen must be
insoluble in water and must strongly adhere
to the particles. This would be facilitated by
encapsulation (section 3.6.5). Probably no pre-
sent proprietary product for water incurs the
expense of a sunscreen.
Sprays do not need to be fine for application
to water. Without this constraint the range of
product types has become wide.
Suspension concentrates and wettable powders 89
3.6.2 SUSPENSION CONCENTRATES AND
WETTABLE POWDERS
Suspension concentrates (= flowabies; section
3.3.3b) extend the effective period that the
product remains freely suspended in water
by attaining minimal particle size. This is
achieved best in aqueous flowabies by main-
taining harvested fermentation residues in a
wet state (Table 3.9 in section 3.3.3). In the
production of wettable powders, the drying
process aggregates the material and it is costly
to grind particles down to less than 10 p,m in
diameter.
The impact of particle size is well illustrated
by the work of Molloy et al. (1984). In the
laboratory, the ideal particle size for ingestion
by blackfly larvae was 35 p,m diameter (Mol-
loy et al., 1984). Larvae of mosquitoes ingest
particles ranging from <0.5 p,m (i.e. the size of
small toxin crystals) to a bit above 100 p,m (if
soft or flocculent) (Dahl, 1988). However, in
water courses the critical factor for blackfly
control (Table 3.19 in section 3.6.1) is the dis-
tance that particles are carried downstream
from each application point. A suspension
concentrate with finer particles than wettable
powders gave the greatest number of kills
because small particles held up best in the
water for the longest distance, although mort-
alities were similar for both types of formula-
tion near the application points. The mean
particle size in the suspension concentrate
was 2.1 p,m in diameter (range 0.5-28.0,
usually single spores and crystals), compared
with 5.2 (0.5-122.0) and 4.0 (0.5-98.8) for two
wettable powders. Several other workers have
shown that the rate of sinking of particles in
different products is positively correlated
with particle size (Guillet and Escaffre, 1979;
Guillet et al., 1980; Hinkle, 1983; Lacey and
Undeen, 1984; Guillet et al., 1990). Efficacy
against early instar larvae decreased sharply
as particles increased in size above their feed-
ing-size range (Guillet et al., 1985a). Products
with large particles lost efficacy in turbid
water during the rainy season or after floods
90 Formulation of bacteria, viruses and protozoa to control insects
due to competition with natural particles for
capture and ingestion (Guillet et al., 1985b).
Products with fine particles suffered from
these problems much less.
Incorporation of wet fermentation solids as
a water-in-oil emulsion has the advantage of
combining the slow sinking rate of fine parti-
cles with the buoyancy of oil. However, an
early emulsion of B. thuringiensis ssp. israelen-
sis inhibited feeding of Simulium larvae above
1 p.p.m. for 15 min; a mineral oil, kerosene,
had a similar effect (Molloy et al., 1981). Dou-
ble-strength Teknar (Teknar 2X) flowable aqu-
eous concentrate improved blackfly control in
USA streams. Less satisfactory results were
achieved with a flowable of equal potency,
based on dry solids in oil to try to keep parti-
cles afloat to improve carry (Table 3.10 in sec-
tion 3.3.3a, using dry solids in ssp. israelensis
and modified method) (Lacey and Heitzman,
1985). The reaction of mosquito larvae was not
affected adversely by vegetable oil added to
an inert carrier (Aly and Mulla, 1986).
A difficulty lies in the assessment of viscos-
ity. Viscous products have poor dispersibility
and a high rate of settling, as well as creating
pumping problems when refilling aircraft and
spraying, although sprays need not be fine as
with control of epigeal forest pests. An
improved method of measuring viscosity has
been developed in the West African onch-
ocerciasis programme, although a visual
assessment provides valuable first-hand
information - a product is suitable if the
drops break up when they hit the water and
disperse well into clouds of fine particles
(Guillet et al., 1990).
For most applications to water, suspension
concentrates have been the basic products of
choice, particularly for use by air with ULV
technology. They gave good control of con-
tainer-inhabiting mosquito species by ULV
treatment of piles of tyres (Lee et al., 1996).
They can be used to treat rice fields at the
position of water inlet during flooding
(McLaughlin and Vidrine, 1984). They are
particularly useful for point applications
against blackflies to rivers, being easy to
apply, e.g. typical streamside time for hand-
ling, mixing and application was ea x3 less
(5-10 min) than for powders (20-25 min), also
they were miscible and poured adequately at
freezing temperatures ((9C) (Molloy and
Struble, 1989). They are bettered by wettable
powders only when long shelf-life is at a
premium, although suspension concentrates
have been sufficiently stable in the heat of
West Africa if stored in the open (Guillet
et al., 1990). Suspension concentrates have
the commercial disadvantage of being
heavy to package and transport, as well as
needing preservatives to prevent bacterial
activity in storage (section 3.3.3b); however,
they are less expensive to apply (Knepper
et aI., 1991).
3.6.3 PENETRATION OF FOLIAGE FOR
MOSQUITO CONTROL
Although they are the products of choice for
most applications against blackfly, high- or
low-volume sprays of suspension concen-
trates are impracticable for treating many
mosquito habitats under foliage canopies
because sprays do not readily penetrate
dense foliage. However, successful mosquito
control under canopies has recently been
reported using aerial ULV sprays with dro-
plets with volume mean diameters of 150-
200 pm (Cyanamid, 1992); presumably
because enough spray drifts through the foli-
age into the water.
For the consistent penetration of foliar cano-
pies, e.g. forest, rice field or salt marsh, gran-
ule-sized products need to be heavy enough
to roll down leaves into the water. Successful
carriers include corn cob grits (Lacey and
Inman, 1985, 1 mm diameter; Lacey, 1986;
Lacey et aI., 1988, 12/14 mesh; Wilmot et aI.,
1993,5-8 mesh), clay (Lacey and Inman, 1985,
1 mm diameter), sand (Becker and Margalit,
1993; Table 3.20) and polymers (Table 3.22 in
section 3.6.4 below). Except for the polymers,
the bacteria are applied to the surfaces of the
 3.6.4 Floating and slow-release products 91
Table 3.20 Production of heavy, fast-release granules of Bacillus thuringiensis ssp. israelensis (Bti) suitable
for preparation by the user for control of mosquito larvae (Lisansky et al., 1993)

Ingredients Percentage (w/w) Function Cost ($/kg product)

Technical powder 3.88 Agent 1.09
Blasting sand 94.2 Carrier 0.03
Wessalon S 0.Q75 Free-flow agent 0.02
Energol WT-l 1.88 Sticker oil 0.18

Preparation
1 Mix technical Bti powder (80 000 IU/mg) and silica powder (e.g. Wessalon S) in the ratio 49:1 (w/w).
Market in separate container
2 Thoroughly mix 94% dry sand, 100-500 tLm, with sticker in rotary (e.g. cement) mixer 5-10 min. The
sand can be local to avoid bulky transport and the oil can be marketed with the Bti in a separate
container
3 Stop mixer, add 4% Bti-silica mixture (1), cover mouth of the machine, mix for 10 min
4 Use product immediately at 5 kg/ha to penetrate foliage canopy. The sticker is designed to release Bti
on entry into water so that the Bti floats. Many stickers, such as Golden Bear oil, do not readily release
the Bti. For open water, other carriers can be used, such as corn grits, which float and so should not be
used in windy weather

granules. Granules can be delivered by air or
ground equipment (Table 2.2).
3.6.4 FLOATING AND SLOW-RELEASE
PRODUCTS
Products can be made to float, suspend (Table
3.21) or sink (Table 3.20), according to the
feeding types of target mosquitoes (section
3.6.1). They can release the bacteria rapidly
or slowly. Gustatory stimulants can be
added to optimize larval feeding.
Rapid-release effervescent tablets have
been designed for easy transport and use in
small water masses. These float and dispense
particles into the water from the water sur-
face. To be economic, a high unit activity was
used (750 lTV / mg). From each tablet, the bac-
teria were spread over 1-3 m 2 of water if not
obstructed by vegetation, and gave nearly
100% control of susceptible Aedes (Most and
Quinlan, 1986; Skovmand and Eriksen, 1993).
Granules with rather slower release, for
protection of water masses of all sizes, have
been formed by adhering as much technical
powder as possible to the surface of inert
carriers. The high concentration minimizes
the amount of product to be applied and,
hence, the weight to be carried by aircraft.
The cost of transporting and packaging
heavy, bulky granules can be avoided by
keeping granule composition simple to enable
mixing with local materials near the site of use
(Table 3.20). Lacey et al. (1988) reported 5%
powder content by weight, and Lacey and
Inman (1985) 5-17%. As soon as they are
wetted, granules dehisce, releasing particles
of variable size. Crystals of B. thuringiensis
have a density of 1.40 and spores 1.32 (K.
Bernhard, Lorrach, Germany, personal com-
munication), and low density carriers such as
corn cob grits readily form floating granules;
these gave excellent control of Psorophora
columbiae in re-flooded rice fields when
applied by air before or after flooding
(Lacey, 1986). However, rice hulls treated
with B. thuringiensis spp. israelensis wettable
powder adhered with gelatin gave poor con-
trol compared with the wettable powder
applied as a spray, because the water carried
them away from mosquito larvae in rice fields
(McLaughlin and Billodeaux, 1983). In unob-
structed water bodies, wind bunched light,
floating products to windward. These
92 Formulation of bacteria, viruses and protozoa to control insects
Table 3.21 Manufacture of heavy, storable, quick-release granules of Bacillus thuringiensis ssp. israelensis
for control of mosquito larvae* (Sjogren, 1996)

Ingredient (Appendix I) Percentage (w/w) Function

Technical powder, 10 000 IU or more/mg 4.7 Larvicidal powder
Dicaperl HP 920 (perlite powder) 3.7 Flotation agent
Water 1.2 Solvent
Morwet EFW 0.01 Surfactant
Glycerol 1.4 Plasticizer
Fish gelatin (45%) 2.0 Adhesive
Blast sand, Texas 12/20, heat dried 86.7 Heavy core carrier
Sipernat 22 0.3 Free-flow drying agent

Manufacture of 1000 lb batch

1 Mix technical powder and Dicaperl thoroughly in a sealed powder blender (air, ribbon or rotary type).
Can be stored
2 Blend water, Morwet, glycerol and gelatin at 55-60 'C (do not exceed 65 'C) in heat-jacketed mixer with
a stirring paddle to form a viscous brown blend
3 Rotate sand in a rotary mixer (e.g. Munson or Continental), while lightly spraying in the blend, using a
flat-fan nozzle (e.g. Spray Systems 8001), and simultaneously adding the powder with a vibratory unit,
both additions timed to extend over 5-7 min
4 Rotate for 1 min
5 Add Sipernat over 1 min, still rotating. The granules should be free-flowing, 0.5-1.0 mm in diameter.
The amount of Sipernat can be varied according to ambient humidity and moisture content of the
granules. The final total moisture content after manufacture should not exceed 7% of the ingredients,
including sand to ensure good storage
6 Sieve to remove excess powder. Retain a sample in a water vapour-proof container for reference
7 Pack in bags with water vapour proof-liner
* Mortality of A~des ['<,xalls larvae was 92.4-100% in 17 out of 19 field tests treated at 5 kg granules/ha.

problems can be avoided by using heavy sand
granules (Table 3.20; Becker and Margalit,
1993). After impact, the oil helps to suspend
the bacteria in the water. A particulate flota-
tion additive can be used and a surfactant to
improve release of bacteria from the granule
(Table 3.21); these avoid any risk of sticker oils
(Table 3.20) inhibiting larval feeding. A com-
mercial product, LarvXSG, based on Table
3.21, releases the bacteria over O.l-72h, eco-
nomizes application to 5 kg/ha and is suitable
for storage and transport. Some additives
have an adverse effect, e.g. a cement (Guillet
et al., 1985a) and some oils.
Sustained-release granules can be made by
altering the ingredients in Table 3.21, viz:
technical powder, 15.0%; flotation agent, Pro-
pyltex (polypropylene powder), 12.7%; water,
10.8%; sticker, phagostimulant, Technical Pro-
tein Colloid 90014, 2.7%; cross-linking alde-
hyde to reduce water solubility of dry
colloid, 40% glyoxal, 0.1%; sand, 58.2%; free-
flow drying agent, Sipernat 22, 0.5%. The bact-
eria are released over a period of 10-30 days.
Floating slow-release granules containing
B. sphaericus doubled the control period of
Anopheles gambiae in clear water pools or of
Culex in sewage water containers and - at
the high dosage of 30 kg/ha - in cesspools.
The granules initially floated and spread on
the surface, then sank slowly while disinteg-
rating into small particles, 10-20% of which
still floated after several days. They were
compared with a flowable concentrate of
equivalent potency. Big granules, 1-2 mm in
diameter, were effective for much longer than
high doses of flowable concentrates (Skov-
mand and Bauduin, 1998).

Slow-or sustained-release pellets and bri-
quettes, for easy hand treatment of small
water masses, can be made by compounding
bacterial powder with fine inert additives and
dispersants (section 7.7.3c). These can be
made buoyant by choosing light-weight
additives. Cork, polypropylene powder
(Accurel, details in Appendix Table 1.4) and
moulding plaster have been used (Lacey et al.,
1984; Kase and Branton, 1986). Compounding
B. sphaericus in pellets with partially hydro-
genated vegetable oil, talc and a starch-based
super-absorbent polymer (Supersorb, details
in Appendix Table 1.4) extended residual
activity against Culex in large and small
plots, including poilufed water. When pellets
were applied to dry artificial larval habitats 5
days before flooding, Psoropl1Ora colurnbiae
hatching at flooding was eliminated, while
the technical powder was ineffective as a pre-
flood treatment, apparently due to solar in-
activation of the toxin (Lord, 1991). As a
dispersant, powdered sucrose has the dis-
advantage of encouraging growth of fungi
and microorganisms (Lacey et al., 1984).
Sucrose-polypropylene pellets provided lim-
ited control of Culex for 3 weeks with B. thur-
ingiensis ssp. israelensis (Ragoonanansingh et
al., 1992) and good control for 8 weeks with
B. sphaericus (Lacey et al., 1984). Briquettes of
both species gave effective control for 3 weeks
when the experiment had to be terminated
(Lacey et aI., 1988). A relatively large amount
of bacteria per unit area was used, but this is
balanced out by residual control and ease of
use. In an attempt to obtain season-long con-
trol of Aedes albopictus in domestic containers,
higher-than-Iabel dosages of Bactimos pellets
were used; complete control lasted only 60
days, and significant levels of control were
found up to 360 days (Nasci et aI., 1994).
Spread of inoculum from pellets and bri-
quettes is restricted, especially in environ-
ments with emergent vegetation in small
water bodies. A delay in the release of suffi-
cient toxin for immediate kill of older larval
instars is a disadvantage that can be remedied
3.6.4 Floating and slow-release products 93
by surface-coating with wettable powder that
would release more bacteria initially (Lacey et
al., 1988). Rose (1989) suggested that inclusion
of bread yeast or baking powder in a floating
matrix would increase flotation by producing
CO2 . One product containing 10% B. thurin-
giensis, the Bactimos Briquet, is available com-
mercially.
Floating controlled-release briquettes, pel-
lets and granules (Tables 3.22 and 3.23) were
made from Culigel polymers capable of
absorbing over x100 to ca x5000 their weight
of water (Levy, 1989). Rate of release of both
bacterial species from these super-absorbent
polymer granular matrices depended on the
inert ingredients admixed with a technical
bacterial powder during fabrication of the
insecticidal granules by aqueous microspong-
ing or entrapment techniques (Tables 3.22,
3.23). Bacterial powders and formulants were
shaken with preformed granules (2-3 or
4-5 mm diameter) in water for several hours,
strained, washed and air-dried at 27C on
screen trays for 1-4 days. The granules may
absorb over 50% by weight of powder and
additive. The rate and percentage of pesticide
loading within Culigel granules were related
to the super-absorbency, porosity and size of
the granules, as well as to the bacterial and
inert formulants added to the water (Tables
3.22 and 3.23). Culigel water-insoluble com-
ponents are non-toxic, biodegradable/erod-
ible, and protectant against oxidation and
sun (Levy et aI., 1992a, b). The rate and dura-
tion of the effective sustained release were
affected by the water quality of a mosquito
habitat. Release kinetics could be adjusted
for a specific water quality, or a range of
water qualities, by altering the type and/or
concentration of inert dispersant or dispersant
complex (Table 3.23). Bacteria can be applied
with other insecticides as oil- or water-based,
variable viscosity Culigel sprays. Pellets and
briquettes can be made by agglomeration
techniques (section 7.7.3a; Levy et al.,
1993a-c). Two proprietary delivery systems,
Matricap and Gelgrade, were based on similar
94 Formulation of bacteria, viruses and protozoa to control insects
Table 3.22 Slow-release Culigel granules with bacteria for application to water to control mosquito larvae

Granule
Culigel with Bacillus thuringiensis ssp. israelensis
(Bti) and B. sphaericus against Aedes and Culex
spp.
Culigel with Bti (34%) + 3.8% dispersant B or B.
sphaericus (16%) + 16% dispersant A (Table
3.23) at 14-16 kg/ha
Culigel3 with Bti (18%) + dispersant A (18%) or
an aliphatic alcohol + salts, or B. sphaericus
(11%) + dispersant B (11%) (Table 3.23)
Culigel cross-linked modified polyacrylamide
type and cross-linked acrylic types with Bti
and B. sphaericus
Data
90-100% control lasted 68-133 days: type II granules
= III > I > IV (Table 3.23) in water with 10% sea
water at 16-22 kg/ha, giving equal or better control
to chemical alternatives!
Granules (type IV, Table 3.23) control 6-7 larval
generations of Culex quinquefasciatus in fresh and
brackish water for 2-3 months. Shelf life> 124-224
d ays 2
Granules (type III) control C. quinquefasciatus for >4
months at 20 kg/ha in 10% seawater3
Granules, loaded by shaking with aqueous technical
powders in the presence of a non-ionic liquid
heterocyclic dispersant of ethoxylated fatty alcohol
surfactant, control Culex for 3-4 months. Ionic
dispersants produced poor release profiles4

References
1 Levy et a/., 1993a
2 Levy et a/., 1993b
3 Levy et a/., 1993c
4 Levy et a/., 1992b

Table 3.23 Dispersant complexes used in Culigel polymers (Levy et a/., 1993c)

Types of granular Culigel superabsorbent polymers evaluated as controlled-release matrices

Type Description
I Cross-linked copolymer of acrylamide and sodium acrylate
II Lightly cross-linked potassium polyacrylate
III Partial sodium salt of a lightly cross-linked polypropenoic acid
IV Cross-linked potassium polyacrylate/polyacrylamide copolymer
Types of inert ingredients used in dispersant complexes to regulate pesticide release from Culigel granules
Type Description
Alcohols Surfactants
Surfactants Binders
Emulsifiers Suspending agents
Solvents Compatibility agents
Salts Wetters
Diluents Oils
Inert dispersant complexes used to regulate pesticide release from Culigel type III granules
Dispersant complex Description
A 2-Ethyl hexanol + magnesium chloride
B 2-Ethyl hexanol + acrylic acid, copolymer
C Sulphated alkyl carboxylate and sulphonated alkyl napththalene,
sodium salt + acrylic acid, copolymer
D Sodium alkyl aryl sulphonate + acrylic acid, copolymer

materials. With proprietary coating adjuvants,
they provided controlled delivery composi-
tions of comparable performance, depending
on the incorporated formulants (Levy, 1997;
Levy et al., 1996, 1997).
Formulated bait can be used to increase
feeding and to attract mosquito larvae to a
product, whereas inert materials may depress
feeding. Anopheles albimanus larvae found bait
by random locomotion, not by directional
movement (Aly and Mulla, 1986). On contact
with floating food, larvae stopped swimming
and fed rapidly, resulting in aggregation of
larvae at the food source. Inert materials did
not influence swimming and diving in search
of food; filtering larvae expelled collected
inert particles. Aedes vexans gathered food par-
ticles (wheat flour, fishmeal or yeast) x 3 faster
than inert particles (kaolin, pumice or syn-
thetic cellulose). Aqueous fishmeal extract
accelerated ingestion of inert particles to
equal the ingestion rate of food particles,
demonstrating gustatory stimulation of larvae
(Aly, 1983). The presence of food in particles
probably also regulates feeding by larvae of
Culex pipiens (Dadd et aI., 1982). Aggregations
of mosquito larvae have been observed feed-
ing under decomposing carcasses of larger
animals (G. M. Roberts, Wolfson Mosquito
Control Project, University of Southampton,
personal communication). Aggregation of
mosquito larvae was induced by carbo-
hydrates, nucleotides and proteins, as well as
a variety of foods including corn cob flour. It
was not influenced by cellulose and a range of
inert materials. Vegetable oil added to an inert
material (kaolin) did not change larval reac-
tion (for review see Aly and Mulla, 1986), but
mosquito larvae aggregated around latex
beads treated with yeast extract (Aly, 1988).
Oils had an adverse effect on blackfly control
(section 3.6.2). Satiation caused mosquito lar-
vae given unlimited food to reduce feeding
and larvae stopped feeding in the presence
of unlimited inert materials. Larvae given
small amounts of food (dried flour) fed at a
steady rate. Becker et al. (1991) found tablets
3.6.5 Encapsulation 95
containing wettable bacterial powder, flour
and inert material (1:1:1) were no more
attractive than tablets without the food, and
suggested that spent fermentation ingredients
in the powder were already a strong attrac-
tant. Many technical powders contain addi-
tives such as lactose to facilitate harvest and
spray drying (e.g. Lacey et al., 1988; section
3.2). Low density and softness of particles
might be more important factors for ingestion
than absolute size ranges; flat, flocculent par-
ticles, a little above 100 pm, have been
observed being ingested by fourth instar lar-
vae (Dahl, 1988), but flocculent particles sink
rapidly.
The concentration of bacteria used in baited
products depends on whether maximum dis-
persion across the habitat for short-term con-
trol is intended. If so, the minimum to achieve
complete kill is 0.2% technical powder (Aly et
al., 1987). Otherwise much higher quantities
are desirable, e.g. 5% technical powder in corn
cob grit granules, 5-13% in briquettes and
30% in sustained-release pellets (Lacey et al.,
1988).
Formulated baits have been used in pellets
to attract bottom-feeding species. Fishmeal
increased effectiveness (Aly, 1983). Becker et
al. (1991) designed tablets to sink to the bottom
of domestic water reservoirs from which
amounts of water were continually taken and
the reservoirs periodically topped up. The
turbulence thus caused shortened the effective
life of the pellets. Incorporation of 33% flour
caused pellets to break up sooner, possibly by
encouraging growth of microorganisms.
3.6.5 ENCAPSULAnON
Formulated matrices (section 3.3.1) offer
organisms some protection from environ-
mental conditions, and the distinct skin
around capsules offers more, as well as
greater, opportunities of improving suspend-
ability in water. Margalit et al. (1984) made
two types of capsule: (1) by stirring a mixture
of B. thuringiensis ssp. israelensis and dried
96 Formulation of bacteria, viruses and protozoa to control insects
yeast or yeast extract in a solution of low
density polyethylene in cyclohexane, and (2)
by stirring the bacteria into a slowly cooling,
fine emulsion of a fatty acid (decanoic, palm-
itic or stearic). The capsules were filtered and
dried. Both types increased the flotation
coefficient and improved the insecticidal
activity against Culex and Aedes larvae in
glass containers with mud on the bottom.
Cheung and Hammock (1985) microencap-
sulated pure crystals in lipid droplets. These
were mostly 3-12 pm in diameter, ideal sizes
for larval feeding. After application to the sur-
face, over 50% of the capsules remained in the
top 0.2 inches of water for 9 h; ca 30% were
still there after 24 h, while the rest dispersed
evenly down to 12 inches. Efficacy improved
x2-3 against Culex and Aedes larvae and x20
against Anopheles. Lipid capsules of different
buoyancy can be formed.
Prill (Table 5.2) were formulated by thor-
oughly mixing B. sphaericus into aqueous alu-
minium carboxymethylcellulose (CMC), a
medium-viscosity polymer with a degree of
substitution of 0.7 (Sigma), and dropping
into aqueous 0.05-M Ah(S04)2 at pH 3.4 and
4 DC (El<;in et al., 1995). The best release profile,
over 14 days, was given by a CMC concentra-
tion of 1%. Compared with free bacterial
preparations in water, prill protected ger-
mination after exposure at 50 DC for 7 days,
at pH 3 to some extent for 45 days, and under
DVC radiation slightly for 24 h, while potency
in Culex spp. was well protected from these
extreme conditions for 45 and 60 days and
48 h, respectively.
A microencapsulated formulation of B.
thuringiensis had little advantage over the wet-
table powder product, Bactimos, for control of
the surface-feeding Anopheles stephensi, but
extended the effective period from 2 to 8
days for the bottom-feeding Aedes aegypti
(Vorgetts and Buescher, 1985).
Of all the above large-grained formulations,
only corn grit, clay and sand-and-oil granules,
as well as various pellets and briquettes, have
been marketed to date.
3.6.6 MONOMOLECULAR SURFACE FILMS
Monomolecular surface films (monolayers)
combine many of the objectives of formula-
tion for aquatic use. They float, spread, per-
sist, resist movement by wind (except on large
water areas, although larvae tend to move
too), and are easily used in both small and
large water bodies, while being innocuous to
most non-target insect groups and to verte-
brates (Levy et a/., 1984; Roberts and Burges,
1984; Roberts, 1989a, b; 1991).
Monolayers spread instantaneously on
application to a point on the surface of water
and distribute technical bacterial products
suspended in them. The bacteria are released
into the water, decreasing in quantity with
distance from the point of application for at
least 15 m (Roberts and Burges, 1984). When
the whole water surface has been covered by a
single layer of molecules, excess monolayer
remains as a reservoir globule at the surface
and retains many suspended particles. Mole-
cules stretched across the surface are slowly
degraded and are replaced from the globule,
carrying particles along with them. As mole-
cules spread across the water surface, some
particles are released immediately, others are
retained at the surface and released later
(Roberts, 1989a, b). In clear tropical waters,
the effective period of mosquito control was
extended from 9 to 15 days before adults
started to emerge again from the regenerated
population. The extension was less in pol-
luted waters and more in temperate condi-
tions (Roberts and Burges, 1984; Roberts,
1989a).
Monolayers interrupt the breathing of lar-
vae and pupae at the water surface and prev-
ent egg laying by adults of species that alight
on the water, often destroying them by
drowning (Reiter and McMullen, 1978). Gen-
erally the monolayer kills larvae more slowly
than B. thuringiensis ssp. israelensis, and also
kills pupae. The lethality of monolayers to
insect stages not attacked by the toxin
adds complementary mortality to its many

advantages as a formulation ingredient. The
toxin deteriorates during long-term storage in
monolayers, e.g. Monoxi, so the two ingredi-
ents are packaged separately. They are stable
in the short term and can be held together
briefly after mixing on site, potency being
maintained for 35 days at 5C, 21 at 25C
and 10 at 35 C, after which it slowly declines
and is lost in 60 days at 35C (Roberts, 1989a).
Good suspension can be maintained by an
insoluble gelling additive such as 5% fumed
silica (Cab-O-Sil; G. M. Roberts, personal com-
munication). Spreading of monolayers from
point sources enables coarse sprays to be
used or, in small water masses, drops from a
dropping bottle. Both ingredients are innocu-
ous and can be used safely by anyone, includ-
ing children in a community mosquito control
programme in the tropics (Roberts, 1989a, b).
Tested monolayer chemicals include egg
lecithin (Reiter and McMullen, 1978); Liparol
(soybean lecithin and C 12 -e J4 isoparaffins;
Becker and Ludwig, 1983) and Arosurf non-
ionic surfactant, the 2 M ethoxylate of iso-
stearyl alcohol (Levy et al., 1984). Monoxi, a
1:1 v Iv mix of oleyl alcohol monoethoxylate
suspended at 15% by high-speed mixing in
water, is an effective but bulky product.
This, together with two mixtures not requir-
ing pre-suspension in water, (1) oleyl alcohol
monoethoxylate mixed with 10% v Iv cetyl
stearyl diethoxylate, and (2) oleic acid mono-
ethoxylate mixed with 10% v Iv cetyl stearyl
diethoxylate, were superior to Arosurf, which
has the disadvantage of being more miscible
in water (Roberts, 1989a; 1991).
There are some conflicting data on the relat-
ive efficacies of bacteria and monolayers. The
LC so of B. thuringiensis ssp. israelensis over 1-2
days was greater in the presence of a mono-
layer (Nugud and White, 1982), while the
reverse was found with the monolayer,
Monoxi (Roberts, 1989a). Aly et al. (1987)
reported that mixtures of bacteria and Arosurf
in field tests did not kill Culex larvae at con-
centrations of bacteria effective without the
monolayer. They found that 1-5% Arosurf,
3.7.1 Pathogen production 97
mixed with bacteria and flour, impaired larval
feeding and hence ingestion of bacteria. This
is probably an effect of the monolayer wetting
the setae on the breathing siphons and redu-
cing larval activity, as well as holding bacteria
at the surface as a slow-release mechanism.
However, slow-release products typically
contain high concentrations of bacteria in
order to extend mortality over a protracted
period. It can be concluded that both control
methods are effective and that comparisons
depend on the monolayer in use. Over an
extended period, mixtures of Monoxi and
related compounds (Roberts, 1989a; 1991) are
most effective, giving a valuable extension of
the period between treatments, also with
overall reduced costs. Monolayers have been
marketed for use with bacteria on a small
scale.
3.7 FUTURE TRENDS AND RESEARCH IN
FORMULATION TECHNOLOGY
3.7.1 PATHOGEN PRODUCTION
Production of Bacillus thuringiensis worldwide
is mainly confined to deep liquid fermenta-
tion for maximum efficiency and ease of
handling and of scale-up. At harvest, water
is removed by centrifugation or filtration and
the liquid slurry stabilized by spray drying or
preservatives. For products marketed dry
(dusts, granules, capsules, wettable powders
and water-dispersible granules), universal
formulation requirements continue to need
attention, i.e. to avoid caking and improve
palatability as well as sunscreening. Thus,
avoidance of hygroscopic nutrients and stabil-
izers for spray-drying would prevent caking.
Use of multipurpose additives could be more
economical than a series of individual addit-
ives. For example, during spray-drying the
use of heat protectants with sticking, phagos-
timulant and sunscreening properties (section
3.7.2c) would reduce the need for additives
with these properties later during formula-
tion, particularly since the stickers among
98 Formulation of bacteria, viruses and protozoa to control insects
them are incorporated at relatively high per-
centages, e.g. skimmed milk powder and
starch products. Some of the newer additives,
e.g. starches and lignin (alone and in com-
binations), need investigation for a variety of
useful qualities including heat protection dur-
ing spray-drying and - before drying - pre-
servation (e.g. by acidity) and suspension in
aqueous concentrates. In addition, growth of
the vegetative B. thuringiensis cells remaining
at harvest and of possible small numbers of
contaminating bacteria and fungi must be
prevented, as well as enzymatic breakdown
of the protein toxin crystals. Residual protei-
nases remain at harvest and there are intersti-
tial proteinases in the crystal itself, with
marked variation between different strains.
There is little published work on preservat-
ives, an area that should reward further
research; a specialist study of enzymes and
their inhibitors would probably lead to valu-
able improvement in stability. An option is
pH manipulation, although different patho-
gens have different optimal pH requirements.
B. thuringiensis tolerates strongly acid condi-
tions to inhibit alkaline proteases that attack
the crystal toxin; B. sphaericus and viruses
store best at neutral pH, which inhibits alka-
line proteases in occlusion bodies (section
3.3.6).
Formulations of bacteria as aqueous sus-
pensions avoid the cost of drying and are
easier to apply, but have the continuous prob-
lem of preservation in the presence of water.
These key contrasting features should main-
tain a roughly equal market share for dry and
liquid bacterial products.
With the other two pathogen types, in vivo
production is at present the only method for
Protozoa and the most cost-effective method
for many wild-type viruses, particularly in
developing countries (Jones, 1988b). Virus is
harvested as a slurry and most commonly
stabilized by freeze-or spray-drying, the relat-
ive success of the two methods being likely to
depend on careful research and attention to
the physical and chemical conditions. Trends
are greater use of formulation additives, as
described for B. thuringiensis, with some
potential for dual function of additives as fil-
tration aids at harvest and as stabilizers dur-
ing drying.
There is an increasing move towards in vitro
production of viruses. This is largely driven
by the development of genetically engineered
products (Hawtin and Possee, 1993). Many of
these are producible only in vitro due to the
formation of transformed insect-specific tox-
ins during viral replication (Vlak, 1994). Com-
pared with in vivo products, the relative
absence of contaminants has the formulation
advantage of easier stabilization; however
there is no insect debris or melanin, etc.,
which are collectively effective sunscreens,
stickers a~d phagostimulants. Hence, there is
likely to be a greater trend towards use of
relevant additives, many with a potentially
dual action. At present, some viruses are
being engineered for short environmental
persistence to ease regulatory problems
(Vlak, 1994; Wood, 1994). However, as geneti-
cally engineered and formulated products
become more acceptable, it is likely that pro-
ducts will be engineered and formulated for
greater persistence. Already a number of stu-
dies have been directed toward selection of
more UV tolerant strains, with some - albeit
limited - success in the laboratory (e.g. Brassel
and Benz, 1979).
The trend in formulation and development
of peroral insect pathogens has been towards
sophistication. However, only limited sophis-
tication has been adopted by industry. This is
mainly due to the increased cost that it would
incur, because the biologicals are competing
with the less expensive chemicals. The early
microbiaIs had two important disadvantages
- difficulty of use and high cost. Formulation
research has greatly improved user friendli-
ness and is beginning to target the most cost-
efficient options / compromises. Basic studies
on the lethal pathogen content of individual
droplets/particles have helped, and need
more emphasis.

Shelf-life of dry products during storage is
good and comparable with that of chemicals.
This gives them a competitive edge over
liquid concentrates, which tend to store well
for only 18 months in ambient conditions for
B. thuringiensis, and for impracticably short
times for viruses and Protozoa.
Whilst there are some common aspects to
formulation for application to land and water,
the differences in these environments cause
different approaches to be taken.
3.7.2 TRENDS IN FORMULATION FOR USE ON
LAND
3.7.2a Products applied dry Corn borer con-
trol is representative of the specialist uses of
products applied dry, and indicates the most
likely direction of their future development.
In leafaxils of the corn plant where most of
the material lodges, the most important envir-
onmental factor is moisture, not sunlight. The
new starch-based granules show a promising
level of protection. Sucrose is incorporated to
make the starch easier to handle, but fungi
grow in moist leafaxils, normally curbed by
using a fungicide. Continuation of the present
very active research should be rewarding; an
alternative sugar not usable by fungi might be
worth investigating. When a phagostimulant,
Coax, is added to make the granules more
palatable they become more attractive than
fresh leaf and act as bait, so that dosage of B.
thuringiensis can be reduced by 75%. Because
information on cost of manufacturing the
granules is unavailable, it is unclear whether
it is economical to replace part of the bacteria
with Coax (McGuire and Shasha, 1995). How-
ever, land pests are mostly controlled by
sprays.
3.7.2b Products applied as sprays In the short
term, the types of spraying machines avail-
able on site are unlikely to be changed for
use with microbial insecticides, except poss-
ibly in forestry. Therefore, the main avenues
3.7.2 Trends in formulation for use on land 99
of progress are formulation and machinery
modifications, such as nozzle changes. The
two must go together because formulation
and delivery systems are inextricably interre-
lated. Research on sprays is being guided by
the environmentally friendly nature of insect
pathogens. The main considerations are the
biological effect on the insect pests and the
economy of active ingredients. There are vir-
tually no concerns about hazards to man, ver-
tebrates and non-target organisms. For
example, if there is spray drift - reduction of
which lowers crop protection efficiency (Tay-
lor et aI., 1993) - the real problem is loss of
pathogen reaching the feeding area of the
insect within the spray target zone, not toxic
hazard to man, animal and plant in the zone
reached by the drift.
The three prime requirements of pest con-
trol by peroral pathogens are to place them as
evenly as possible where they are most likely
to be eaten, to make them palatable and to
protect them once they are there. Thus, the
target is the insects' food, not the insects
themselves. The problem is complex, depend-
ing on the species of pathogen, insect and
food plant, as well as on the interactions of
their ecologies and of weather. Recently,
formulation research has been aided by
modelling (Taylor et al., 1993; Cooke and
Regniere, 1996). For example, modelling sug-
gested avoidance of B. thuringiensis by gypsy
moth larvae (Hall et al., 1995). Modelling opti-
mizes research effort and permits large num-
bers of theoretical simulations, but is not a
substitute for experiment and observation.
Notoriously, however, effects demonstrated
in the laboratory often do not show up in
field tests, but models are valuable in erecting
hypotheses for testing in the field, although
the models are only as good as their databases
and the assumptions made within them. The
field environment introduces another echelon
of variation and ecological imponderables.
For example, in a multi-instar larval popula-
tion in which instars I and II feed only on the
lower surface of the leaf without breaking
100 Formulation of bacteria, viruses and protozoa to control insects
though to the upper surface, while instar III
chews through the leaf, should a model con-
centrate on effects on the very susceptible
instars I and II, even though less deposit
reaches the lower leaf surface; or should atten-
tion be directed to the refractory instar III
because they are most likely to survive any-
way? Field tests quantify variation in pest
control levels, allowing calculation of dosage
levels needed to reduce the probability of fail-
ure to a reasonable proportion of treatments.
In response to the above considerations,
here have been three major formulation
trends, each with strengths and weakness.
Highly efficient suspension concentrates of
bacteria have been developed.
Wettable powders have been improved and
made into user-friendly water-dispersible
granules.
The effectiveness of products for low- to high-
volume sprays has been increased by the
use of capsules/granules. These trends
should continue to dominate use of these
pathogens and their research requirements.
The main motive for the trend towards sus-
pension concentrates has been to obtain good
coverage of inaccessible environments, such as
forest canopies with aerial ULV, CDA sprays
(section 3.3.3a). B. thuringiensis ready-to-use
oil-in-water emulsions have been effectively
formulated directly from newly harvested
fermenter slurries. This may not always be
industrially convenient and some suspension
concentrates are formulated from spray- or
freeze-dried technical powders. These pow-
ders have the advantage that they can be bulk
stored in a very stable state until required for
formulation, which facilitates formulation of
specialist products for relatively small mar-
kets. It also gives the option of formulating
the concentrate using oil as the carrier. It has
the twin disadvantages of (1) the cost of spray-
drying, plus the small decrease in activity that
is often incurred, and (2) the cbst of grinding
the powder without producing a damaging
degree of heat and, if a ULV product is to be
made, the need to grind very fine. Recent
developments with and without anti-evapor-
ants provide another option - the formulation
of products for ULV application in water as the
carrier (Parnell et al., 1996). This is a probable
area for future studies. The formulation of var-
ious liquid products from technical powders is
likely to continue.
The most concentrated B. thuringiensis
products contain in the order of 2-4% crystals
per g or per ml of technical material. Increases
obtainable by improvement of fermentation
and strain would be useful, but use of a crys-
tal extraction technique to increase concentra-
tion would be too expensive. However, the
interaction of a larva of some insect species
with B. thuringiensis requires yet more concen-
tration of products to obtain the highest mor-
tality from the first larval meal after spraying
(section 3.3.3a). Spray bulk can be reduced by
fermenting spore-free mutants, thus eliminat-
ing the spore. These mutants are unlikely to
be used for strains applied against land pests,
because many of these pest species are less
susceptible in the absence of spores. CellCap
products have no spores in order to benefit
from the protective action of the cellular
capsule (section 3.3.5), which appears to com-
pensate for the absence of the action of spores,
probably because the infective and destruct-
ive septicaemic roles of vegetative B. thurin-
giensis cells are partly taken over by insect gut
flora, which originates from phylloplane epi-
phytes (Li et al., 1987; Dubois and Dean, 1995).
However, further research may show that this
compensation may be inadequate in some
insect species for which some spore-contain-
ing technical concentrate could be formulated
into specialist products. The addition of only a
small proportion of spores may be effective
(Li et al., 1987). In contrast, spore-free prod-
ucts are widely used against target aquatic
insects, which do not have reduced suscept-
ibility in the absence of spores.
Development of liquid products for viruses
is in its infancy. If stabilization of contamin-
ants can be mastered without harming the

occlusion bodies, an aqueous product would
utilize the good storage qualities of some
viruses in water. Oil-based flow ables, which
facilitate ULV and CDA application, have
been produced (section 3.3.3.b), but improve-
ment of shelf-life beyond 15 months is desir-
able. Because the lethal dose of a baculovirus
by weight is usually very low, the first larval
meal should be lethal.
The second of the three trends, the
improvement of B. thuringiensis wettable pow-
ders, flourished because they have the best
stability in storage. This advantage is
balanced by the cost of spray-drying. Grind-
ing can be minimized by ensuring that a fri-
able technical product is produced by
fermentation and harvest. Long shelf-life will
always ensure a market place for wettable
powders, even though they are more difficult
and time-consuming to tank mix than suspen-
sion concentrates, sometimes requiring extra
mixing machinery. High potency water-dis-
persible granules are just coming onto the
market. They are easier to handle and tank
mix than powders. Unlike granules of chemi-
cal pesticides, production of less atmospheric
dust during their production and use has little
atoxic safety advantage, because the patho-
gens themselves are not hazardous, although
additives may cause irritation. Whether or not
granules supersede wettable powders is likely
to depend not only on the value placed on
decreased operator time against the extra
cost of processing, plus the cost of binder
and dispersant, but also on whatever devel-
ops as convention in the pesticides industry.
The third trend, development of capsules/
granules for low- and high-volume sprays,
arises from the need to use additives at these
volumes. When applied to tank mixes, wet-
ters, stickers, sunscreens, phagostimulants
and allelochemical masks become increas-
ingly uneconomic the greater the application
volume. For example, the cost of a sunscreen
at 5% in 500l/ha may be prohibitive. Most
additives, particularly sunscreens, function
best when in close juxtaposition to pathogen
3.7.2 Trends in formulation for use on land 101
particles. One answer to these problems is to
microencapsulate the additives with the
pathogens, making the product independent
of spray volume, minimizing the quantity of
additives and maximizing their efficiency
(section 3.3.5). Recent studies have produced
starch and lignin microcapsules which screen
organisms effectively from the sun but are not
rainfast (Shasha et al., 1995; Tamez-Guerra et
al., 1996). More work is needed to produce
capsules that stick to leaves and resist wash-
off (e.g. Morales Ramos et al., 1998). With B.
thuringiensis, each microcapsule is more than
large enough to contain sufficient toxin to kill
a young larva before feeding is depressed, as
already discussed in section 3.3.3a for
droplets of CDA and ULV sprays. Empty cap-
sules can be avoided by choosing appropriate
production methodology. Success of the cap-
sule system has been proven by the commer-
cially profitable CellCap products: obstacles
for fabricated capsules/ granules are probably
economic.
The economics of incorporating additives is
largely determined by the ratio of the expens-
ive technical pathogen products to the addit-
ives chosen for their low cost. This ratio
affects efficiency of the additives, but bulk
may necessitate compromise. For example,
Tamez-Guerra et al. (1996) varied the ratio of
the B. tllllringiensis technical powder to the
carrier / sunscreen, flour+cornstarch+sucrose
powder, by 100:0, 3:97, 10:90, 25:75 and
50:50, then exposed the microcapsules to sun-
light and obtained 19,96,86,64 and 42')'0 kills,
respectively, with test larvae. Thus the greater
the proportion of carrier, the greater the pro-
tection from sunlight. Applying 0.5 kg of B.
t/llIringiensis/ha at a ratio of 3:97 in a spray
to obtain a nearly complete kill would need
16.6 kg of formulated product. The cost of the
carrier is relatively low, en US$O.5/kg, and
would not push up product cost extensively,
but it would increase the cost of making the
capsules. To obtain a similar kill, only 2 kg/ha
of product would be needed at 1:1, which
is more representative of a commercial
102 Formulation of bacteria, viruses and protozoa to control insects
formulation. Further knowledge of manufact-
uring costs would permit a benefit analysis of
the extra protection of juxtaposition against
the cost of encapsulating ingredients and pro-
cessing, plus the slight loss of potency that
occurs during some processing.
S. H. Bpk (Korea Research Institute of
Bioscience' & Biotechnology, Kist, Yusong,
Taejon, South Korea, personal communica-
tion) made rice-soybean-based capsules with
added nutrients and ca 10% of technical B.
thuringiensis. Sprayed at rates similar to wett-
able powders, insect activity was greatly
extended, postulated as being due to demon-
strated bacterial replication in the field. How-
ever, any effect due to replication is not likely
to be reliable as it would occur only in condi-
tions humid enough to increase the water
activity sufficiently in the capsules.
Formation of formulated microcapsules by
spray-drying might be a breakthrough in their
economical manufacture, if the normal drying
process at fermentation harvest can be used.
This would depend on the stabilization and
handling qualities of the encapsulating mater-
ial and formulants in commercial driers.
Hopefully, they might replace the usual
stabilizers, so that the only extra cost of encap-
sulation would be the cost of its inert compon-
ents. Whole or part fermenter loads might be
used for batches of different products merely
by changing the ingredients.
3.7.2c Additives Molasses is one of the most
useful additives, and one of the few that
almost always shows positive benefits in
both laboratory and field. This is probably
because it is multifunctional as a sunscreen
(as effective as five other good screens; Mar-
tignoni and lwai, 1985), a thickener, a phagos-
timulant and a mask for adverse foliage
factors. In one experimental product, it also
functions as one of two preservatives present
during storage. It is also widely available and
cheap, making it ideal for use in developing
countries. However, it has the disadvantages
of being bulky and messy as a spray tank
additive, and sometimes causes problems
with filtration equipment e.g. clogging during
spraying. Attempts to replace it by a combina-
tion of more user-friendly materials illustrate
an increasingly common trend in formulation
development. It is desirable to find more
multifunctional additives. Examples are
starch products (encapsulation matrix, sunsc-
reen, buffer and sticker) and Tinopal LPW
(powerful synergist for virus and sunscreen).
A watchful eye should be kept for bonus ben-
efits, such as the enhancement by Tinopal of
natural virus already causing cyclic disease in
forest insect populations or of partially in-
activated virus, and also for adverse effects,
such as slight repulsion of some larvae by
Tinopal LPW.
Provided that they do not impair release of
peroral pathogens in the insect gut, stickers
cannot overstick this type of pathogen to
plants, in contrast to the contact-acting fungi
for which pick-up of spores from the plant
onto the external parts of the insect body is
important for infection (section 10.8).
Sunscreens protect peroral pathogens while
they are on leaves, but other possible actions,
both in storage and in the insect gut, should
also be considered. Some protectants against
adverse plant factors exert their effects while
on the leaf, but many also have important
oxidative or alkylative activity in the insect
gut. Attempts to neutralize adverse effects
of these foliage factors on pathogens in the
field have had limited or no success, even
when possible modes of action of factors
have been identified. Additives that mask
the effects of foliage, .e.g. phagostimulants,
have had more success. Possibly much benefit
could still be gained by identifying more
factors specific to particular crops and addit-
ives that might neutralize them. For example,
exceptionally high synergism multiples of
x15 to x1584 (part V of Table 3.17 in section
3.4.6, references 11 and 13) for Tinopal LPW
with NPVs in diet bioassays translate to
large significant field multiples of x2.1 to
x1l.7 for gypsy moth on oak (part V of

Table 3.17 in section 3.4.6, reference 12) but
only to xl-2.3 for armyworms (Pseudoletia
unipuncta, Table 3.4 in section 3.3.1) on cotton
and soybean (part V of Table 3.17 in section
3.4.6, reference 11) at concentrations between
0.1 and 1.0%. The industrially viable improve-
ment on oak compared with the modest
improvement on row crops may have been'
due to plant-specific allelochemicals and to
insect/plant interactions. More investigation
of masking/neutralizing plant factors on cot-
ton and oak may be rewarding.
Synergistic plant allelochemicals and
other known synergists need more investiga-
tion as potential formulation additives. For
instance, piperonyl butoxide - used with
pyrethrins and a known inhibitor of mixed-
function oxidase activity in insects - might
synergize certain of the allelochemicals
(Hedin et al., 1988) and, possibly, have direct
action on the pathogens as well. Whether
allelochemicals and synergists react with
spores, crystals or both, requires study.
Those reacting with crystals may be particu-
larly important in genetically engineered
plants that express this toxin, even to the
extent of selecting candidate plant varieties
with a low content of possible toxin inhibitors
such as tannin, or with a high content if the
action of an inhibitor in the insect supple-
ments that of the toxin. A continued search
for counteractors of pathogen inhibitors is
required; modes of action of synergists (sec-
tion 3.4.6, Table 3.17) need more study to try
to enhance this search.
Some synergists may have general activity
applicable to all the pest-plant systems for
which a product is recommended. Others, or
more particularly counteractors of pathogen
inhibitors, may be limited to certain systems.
These particular systems can be important,
e.g. tannins in oak impairing the activity of
B. thuringiensis on oak in stands of mixed tree
species (Appel and Schultz, 1994) or on tan-
nin-rich cotton. Such limited synergists/coun-
teractors would be best added to the tank
mixes only for the relevant plants.
3.7.2 Trends in formulation for use on land 103
Additives may also harm biocontrol agents.
Those inflicting severe harm are soon weeded
out, but those causing slight harm over an
extended period present a problem of recog-
nition. This problem becomes more difficult
with mixtures of additives and with propriet-
ary adjuvants. The best remedy is to build up
an assemblage of data, such as that presented
in the tables and in Appendix I.
Many of the so-called inert, non-insecticidal
additives formulated with chemical pesticides
are toxic or potentially dangerous to man and
environment. The US Environmental Protec-
tion Agency has listed 'inerts' in four cate-
gories: (1) high toxicological concern (57
compounds, no new approvals are being
granted for products containing these); (2)
high priority for testing (62); (3) to be re-
viewed later (800); (4) no concern. With micro-
bial pesticides, the high degree of safety and
the absence of phytotoxicity of the microbes
themselves should be backed up by use of
additives only in category 4, preferably of
food-grade quality. The use of xylol (category
2) has been discontinued in suspension con-
centrates of B. thuringiensis.
Trought (1989) lists companies specializing
in conventional encapsulation systems. Costs
and the limitations of developing for niche
markets are likely to determine the commer-
cial future of these systems for B. thuringiensis.
Lignin and starch are probably the materials
nearest to industrial use, particularly after the
recent economies and streamlining in manu-
facture (section 3.2 and 3.7.1). This technology
has been licensed by the US Department of
Agriculture to the Biotechnology Research
Development Corporation, Peoria, Illinois
(Anon., 1992). If this type of encapsulation
can be made competitive costwise, this
powerful research organisation stands a
good chance of success.
The insect pigment melanin, a very power-
ful photoprotectant (Table 3.16 in section
3.4.4), has been produced by Streptomyces
lividans transformed with a tyrosinase gene
(Uu et al., 1993). Similar transformation of
104 Formulation of bacteria, viruses and protozoa to control insects
B. thuringiensis cells may enable CellCap type
(section 3.3.5) capsules highly protected
from solar radiation to be produced (section
10.9).
More emphasis should be given to develop-
ing formulations for control of stored grain
pests, soil iI1sects and nematodes. A continual
stream of new additives and new chemical
groups, such as organosilicone super-wetters,
are being applied with chemical pesticides.
These are ready-made candidates for use
with pathogens. They must first be examined
for harmful effects on the pathogens, after
assessing clues to be gained from chemical
structure, general toxicity, phytotoxicity and
known environmental effects. This should be
followed by comparisons with formulants
already in common use, structured so as to
pinpoint effects specific to the test substances
and also to unexpected activities, such as
synergism and dual action. The search should
of course be continued for additives for patho-
gens de novo - another Tinopal LPW (section
3.4.6) may be around the corner.
Improvement of formulation is playing an
increasingly complex role in the development
of commercial products. Both research organ-
izations such as the Canadian Forest Service,
and primary producers of microbial insecti-
cides, are forging confidential links with com-
panies having formulation experience to gain
access to their skills and technology.
3.7.3 PRODUCTS FOR USE IN WATER
There is great scope for diversity of formula-
tions for application to water, which has
inspired research. Industry, to date, has incor-
porated only a limited selection of the find-
ings in marketed products.
3.7.3a Suspension concentrates and wettable
powders Because of the overriding require-
ment to use small particles that stay sus-
pended as long as possible, flowable
suspension concentrates should remain the
dominant products for treatment of both
large and small water bodies. Concentrates
can be manufactured ready-to-spray by ULV
without dilution to minimize loading time, a
critical feature for aerial application. Research
is important to increase potency and to reduce
the aircraft load, which can be accomplished,
among other ways, by using spore-free
mutants to dispense with the weight of the
spore (sections 3.7.1, 3.7.2b, 3.7.3e). Surfac-
tants are necessary as dispersants in the pro-
ducts to maintain suspension in storage and
to ensure good mixing when the product
reaches the water.
When limited shelf-life of suspensions is a
problem, the more stable wettable powders
can be used, despite their more rapid settling.
Light-weight materials added during or after
drying would increase suspendability or
make particles float. They would also increase
particle size and improve the efficiency with
which the target larvae filtered them from the
water. Particles of <45 f.lm diameter could be
made highly suspendable. Although these are
near optimum laboratory sizes for feeding,
they may be disadvantaged in flowing water
by more entrapment in vegetation. Oil is
probably the easiest material to add.
Although the balance of evidence shows that
at least vegetable oils are palatable to mos-
quito larvae, there is some evidence that
mineral oil inhibits the feeding of blackfly
larvae. Research on different oils should be
rewarding. An additive that produces gas in
water is an alternative, aiming to form on each
particle a very small bubble that remains
attached, although bubbles would probably
be dislodged by fast-flowing water.
3.7.3b Granules, pellets and briquettes The
practical problems encountered during mos-
quito larva control converge to suggest the
use of formulations of granule size in prefer-
ence to dry powders. Size and weight are
needed to make granules roll down foliage
cover into the water. Particles must float or
suspend in water to be maintained in the
various larval feeding zones (Table 3.19 in

section 3.6). Controlled release of these
particles from granules is needed to extend
the short life typical of suspension concen-
trates and wettable powders in the relatively
static water bodies where mosquito larvae
live. Blowing of floating granules by wind
into limited surface areas must be avoided.
Perhaps the ideal is a 2 mm granule, heavy
enough to roll down and penetrate the water
surface, yet buoyant enough to bob up and
float just below it; initially, sufficient filterable
particles should be released to kill all larvae
including the least-susceptible late instars,
then particles should be dispensed at a steady
rate to kill the highly susceptible larvae emer-
ging over time from eggs; however, particles
must not be too heavy to increase, due to high
weight, the cost of transporting them to the
site of use and applying them by air. Techni-
cal powder, coated on to the granule surface,
could release particles rapidly; it could be
combined with a phagostimulant to entice
larvae to accumulate below the granules and
hasten their eating a lethal meal. A spate of
research in the 1980s produced much data
addressing these factors individually. This
led to products on the market based on corn
grits, clay and sand, each designed primarily
for a specific purpose, but subject to either the
problem of wind drift or that of sinking too
fast. However, a start was made towards a
multipurpose formula by adding 25% heavy
particles to a buoyant corn grit granule
(Sutherland, 1990). Manufacture was limited
to the simplest procedures of spraying a tech-
nical product on to the granules or mixing it
with them. Progress has been curbed by the
cost of more sophisticated processing and
materials, as well as lack of assurance that
improved efficiency and less frequent ap-
plication will repay any extra cost (section
10.4). Encouragingly, a patent for a complex
granule (LarvXSG) has recently been filed
(Sjogren, 1996), but more research on these
granules is required. More urgently, com-
parative field trials with granules of known
composition are needed to assess the advan-
3.7.3 Products for use in water 105
tages of sophisticated granules, varying the
composition to examine the value of each
formulation aspect, since most of these values
are still based mainly on theory. Or, will non-
target fauna eat up such tasty phagostimulant
morsels before the full benefit of prolonged
controlled-release can be obtained?
A multipurpose formulation may not
always be needed, for example, for single-
species populations. Great flexibility could
be obtained by packaging different gran-
ules/ capsules separately with the intention
of using them either singly or roughly mixed
on site for multispecies populations. Granules
intended to float could incorporate a sun-
screen, which would also be valuable for
granules marketed for application to dry
ground prior to expected flooding (section
3.6.4). Possible advantages of capsules over
granules might be better control of buoyancy
and better protection of the toxin crystals from
deterioration, but the comparison is essent-
ially that of comparing carrier materials and
manufacturing processes.
Zaritsky et al. (1991) have bioencapsulated B.
thuringiensis ssp. israelensis by feeding it to the
ciliate Tetrahymena pyriformis, which in turn is
eaten by mosquito larvae. Although such tran-
sient entrapment does not seem a practical
method of biocontrol, it does demonstrate
that filter-feeding organisms trap B. thuringien-
sis ssp. israelensis applied for mosquito control.
Manasherob et al. (1996) obtained higher mor-
tality of larvae with bioencapsulated bacteria
than with the bacteria alone.
Pellets and briquettes are used mainly in
small water masses for convenience and for
minimum frequency of application. Their
technical requisites are much the same as
those for granules. Their use may increase in
the domestic market, but putrefaction may be
a problem in domestic water supplies.
Although application to water is currently
restricted to bacteria, future trends may
include other microorganisms. Williams et al.
(1992) conducted an initial study on the
impact of an iridescent virus on blackflies,
106 Formulation of bacteria, viruses and protozoa to control insects
which has the advantage of being able to
spread through the population.
3.7.3c Monomolecular layers Monolayers
combine so many of the objectives of formula-
tion for aquatic use, as well as complementing
the action of the bacterial toxins on larvae,
that they surely are prime subjects for more
development for the formulation of bacteria.
Compounds are available that persist longer
than Arosurf (section 3.6.6; Roberts, 1989a). A
method is required to prevent long-term dete-
rioration of toxin when formulated in mono-
layer on the shelf, possibly by lowering the
moisture content. The cost needs reducing
and marketing needs improvement.
3.7.3d Bacillus sphaericus B. sphaericus (Table
3.1 in section 3.1) is more persistent than B.
thuringiensis ssp. israelensis; control can
remain substantial up to 4 weeks, although
reasons for the difference are not understood
(for review see Lacey and Vndeen, 1986).
Because of its greater durability, it may bene-
fit more from formulation, and thus may be
the better candidate for use in slow-release
products. While being more active against
species of Anopheles and Culex, but less against
Aedes, it has the slight disadvantage of being
more susceptible to solar radiation (section
3.6.1). The spore and crystal of B. sphaericus
do not separate and the complex settles in
water less rapidly than free crystals of B.
thuringiensis ssp. israelensis, possibly because
the less dense spore acts as a float (Karch and
Hougard, 1986). Perhaps production of an oil
globule beside the crystal could be obtained
by genetic engineering to improve flotation.
However, greater flotation would increase the
importance of sunlight, which could be count-
ered by an insoluble sunscreen or by inserting
a gene for production of melanin, a very
powerful natural screen (section 3.7.2c).
3.7.3e Products without live spores When a
formulated product free from live spores is
needed to treat water, spores can be killed
by VVC or gamma radiation. 'Dead' products
are as effective as 'live' products in controlling
mosquito and blackfly larvae (sections 3.7.2b,
3.7.3a; Lacey et al., 1978; Engler et al., 1980;
Krieg et al., 1980a; Burke et al., 1983). There
are, however, a few reports of reduced
potency after radiation: a small reduction
after VVC treatment (Li et al., 1987) and after
gamma radiation from 172 to 137 lTV (Beck
and Becker, 1992) with B. thuringiensis ssp.
israelensis, also small reductions after dosages
of gamma radiation up to 1800 kR, statistically
significant with B. thuringiensis ssp. israelensis
but not with B. sphaericus (Lacey and Smittle,
1985). It can be concluded that the radiation
has only an unimportant effect on the crystal
and that infection caused by the spore exerts
virtually no effect on the control of target
aquatic insect larvae.
Better products are obtained from spore-
free mutants. Spore weight is absent and
there is no risk of radiation damage to the
crystal. Fermentation could be made even
more efficient if some of the cell resources
normally used to produce a spore could be
diverted to production of larger crystals.
3.7.4 SYSTEMIC EXPRESSION OF MICROBIAL
FACTORS
The challenge of maintaining the presence of
B. thuringiensis endotoxin in new, rapidly
growing foliage has been met in two ways:
crop plants are transformed directly with
toxin genes;
toxin genes are transformed into endophytic
microorganisms that can be applied as
microbial insecticides.
Both have the key advantage of being self
replicating.
Transgenic plants have greater commercial
potential. However, crop varieties have to be
transformed individually, a considerable
commercial limitation. Also, in near-monocul-
ture situations, there is the spectre of intense
selection pressure causing insects to develop
 3.7.4 Systemic expression of microbial factors
resistance to the toxin. This can be countered
by various strategies (Marrone and MacIn-
tosh, 1993), the present favourite being the
planting of refugia not involving B. thuringien-
sis (mandatory for cotton in the USA)
combined with crops producing high concen-
trations of toxin, e.g. >25 times the LC 99 for
the target insect (Gelernter, 1997). Some
suppliers sell seed conditional on certain
anti-resistance steps being used as a part of
cultural practice. Plant varieties resistant to
diseases and/or pests, and tailored by tradi-
tional genetic techniques, are collectively an
outstanding success in control programmes.
When the disease organisms and pests resist-
ant to these genetic varieties have appeared,
either the operational scope of the varieties
has been reduced, or the varieties have been
superseded by new re-tailored resistant vari-
eties. There appears to be no reason why plant
varieties genetically engineered with B. thur-
ingiensis toxins should not become as success-
ful as, or even more widely used than,
microbial insecticides. Registration require-
ments for transgenic plants have been eased
in the USA and should not inhibit commer-
cialization. Use during 1996 against Heliothis
spp. met some problems (Gelernter, 1997).
Predictably, damage was sometimes caused
by pests secondary to H. virescens, such as H.
zea and Spodoptera exigua. This could be coun-
tered in future by transforming more toxins
into the plants, a ploy that would also reduce
the likelihood of toxin-resistant insects. No
induced resistance was found in 1996 in H.
virescens due to commercial growing of trans-
genic plants (Gelernter, 1997). Naturally
resistant wild strains of Plodia interpunctella
occur in food stores. Outdoors, resistant
strains of Plutella maculipennis have occurred
in response to the long-term use of B. thurin-
giensis spp. kurstaki insecticides, including
cross-resistance to abamectin (Wright et al.,
1995).
Despite the potential of transgenic plants, in
many situations, particularly in developing
countries, spray application of microbes is
107
likely to be the method of choice due to the
cost and unavailability of seed supply; even
the supply of normal certified seed is prob-
lematic in many developing countries.
In water, algae transformed to express the
B. thuringiensis ssp. israelensis toxin will be
limited by the range of water masses favour-
able to growth of the species of alga used. Rice
areas may be good targets. Additional fea-
tures in the alga, such as nitrogen fixation
(Boussiba et al., 1992) would increase the
chances of industrial success.
The use of endophytes, which are delicate
organisms vulnerable both during storage
and when infecting the plants, will need
sophisticated formulation required for organ-
isms that do not form tough spores. Epiphytic
organisms are also relatively delicate and will
face shelf-life problems. The commensal
microorganisms will have the problems of
genetically manipulated organisms in obtain-
ing regulatory permission for release into the
environment. Use of commensals has two
advantages: (1) they would be applied only
once to a crop; and (2) they would function as
living microcapsules protecting the toxin from
deteriorative factors. However, in tests on
maize to date, crop damage reduction has
been variable and obtainable only in maize
hybrids compatible with the endophyte
(Anon., 1991). This suggests that future use
will depend on further technical improve-
ments.
Although transgenic genes from insect
pathogens used to protect organisms are cur-
rently limited to B. thuringiensis endotoxin
genes, a wider range may be used in future.
For example, Hanzlik et al. (1995) produced
infectious particles of H. armigera stunt virus
in non-insect cells, and report on the possibil-
ity of developing transgenic plants or other
non-insect hosts that produce this virus.
Genes from sources other than insect patho-
gens can also be included.
The genetic engineering of baculoviruses is
now well established. It is aimed mainly at
increasing the speed of kill or stopping insects
108 FOYIIllllatiol/ of bacteria, viruses and protozoa to col/trol insects
feeding by the insertion into the virus of
genetic sequences that express specific toxins,
or by the deletion of some sequences from the
baculovirus genome (Hawtin and Possee,
1993; Vlak, 1994). Transformed virus products
should be commercially available in the near
future, with an increasing variety and func-
tion.
In the future, from the formulation view-
point, the outstanding success should con-
tinue to be the insertion of B. tllllringiensis
toxin genes into plants, subject to the curbs
of resistance development in insects and lim-
itation to only some pests in a pest complex.
Resistance can be delayed by anti-resistance
strategies and use of different combinations of
toxin genes. A wider range of pests can be
controlled by co-insertion of other types of
genes.
3.7.5 NOVEL STRATEGIES FOR CONTROL
A wider range of control strategies, involving
novel approaches with different formulations,
is likely to be employed in future. For ex-
ample, traps may be used to attract insects
and infect them with a pathogen (e.g. Tatchell,
1(81). A sunscreen would not be needed in
the formulation, because the pathogen is in a
protected environment. Also, insects may be
reared for release and infection with a patho-
gen to induce an epizootic, as has been done
with the rhinoceros beetle infected with its
non-occluded baculovirus; the virus is likely
to be formulated only for storage because
healthy beetles are immersed in a simple
virus suspension to infect them (Bedford,
19tH).
There should be an increasing range of
prod ucts available for a larger range of pests.
For example, the range of B. thuringiensis tox-
ins being isolated is rapidly expanding. Crick-
more ct Ill. (1995) list 50 different sequenced
crystal protein genes. Bravo et al. (1995) report
that toxins are now known with activity
against Lepidoptera, Diptera, Coleoptera,
Hymenoptera and Acarina, as well as Nema-
toda, Trematoda and Protozoa. As a result
targets such as nematodes and blowflies will
be included in product development research.
These new targets will involve new environ-
ments, e.g. dried fish for blowflies (Turner et
al., 1994), which present new challenges to the
form ulator.
Probably more sophisticated models of the
interaction of spray volume, droplet size,
product type, persistence and effect of addit-
ives will be developed to facilitate assessment
of the effect of changes in formulation on
product action. This should be very important
for predicting the action of genetically altered
products where the cost of safety testing prior
to field experimentation is high.
3.7.6 RESEARCH PRIORITIES
From the summary above, a number of prior-
ities become clear.
A stage has been reached when the true pract-
ical field value of recent formulation inno-
vations needs better comparative study.
Sufficient innovations have been taken up
by industry to encourage work to be limited
to marketed products, which is largely
research in the dark because product com-
positions are proprietary. It is so frustrating
to read of significant trial differences
between proprietary products of equal
potency and not be able to find reasons for
these differences due to absence of formula-
tion data. More comparative work is needed
with formulations of published composi-
tion, so that the value of known material
combinations can be assessed and combina-
tions improved. Inclusion of some propriet-
ary products would help to show the
consequences of unknown industrial com-
promises that presumably were thought
necessary, for instance to limit costs.
Costs are crucial. They largely determine the
degree of use made of microbial insecticides
and the opportunity for economies of scale.
Cost reductions and streamlining in manu-

facture, distribution and user strategy need
priority integrated study. Aspects of the
outstanding example of manufacturer-user
cooperation in the development and use of
ULV application of pathogens in North
America (Fig. 3.1; section 3.3.3a) could
well be applied to other fields.
Research should be concentrated on some
obvious weak points. One weak point is
limited shelf-life of B. t/wringiensis flow-
abIes and even of dry virus products;
some outstanding improvements in harvest
and storage of fungal pathogens (Chapter 4)
may be applicable to viruses. Another weak
point is the limited post-application life of
the pathogens; this- mainly translates into
putting and preserving pathogens where
they are eaten most on land foliage, to
maintaining them in the feeding zones of
mosquito larvae, and to improvement of
carry in running water for blackfly larvae.
Scope for formulation improvement has
recently been most obvious in the design
of experimental granules for use both on
land and in water. This momentum must
be maintained and coupled with studies of
how far granule complexity and cost can be
increased before being negated by
uncontrollable natural forces, such as dilu-
tion of cover on new foliage by plant
growth, and the removal or masking of
products in water by physical forces (e.g.
trapment) and by non-target biota.
The search for new additives must continue.
The rapid increase in our knowledge-base
should enable this search to be better
guided.
To achieve viable market size, manufacturers
must often make products broad spectrum.
User flexibility can be improved by research
on tank mix technology for locally available
types of spray machine, and by manufac-
turer research on making products more
compatible with the requirements of differ-
ent tank mixes for different situations.
More attention should be given to optimizing
concentration and particle/droplet size for
References 109
individual pest scenarios. These vary
according to whether only young larvae
are to be targeted because older ones have
poor susceptibilities, or whether it is feas-
ible to kill all larvae. With B. thuringiensis,
the objective is rapid acquisition of a lethal
dose before the toxin stops the insect feed-
ing, a matter of good cover near the initial
feeding sites of neonate or instar II larvae
and sufficiently concentrated cover to leth-
ally poison older larvae during their diur-
nal feeding bouts.
There is one area of unfulfilled promise in
mosquito larviciding: the combination of
B. thuringiensis with monolayers. Early
experience with Arosurf has been dis-
appointing. Some of the newer materials
need testing, with thought being given to
the most compatible bacterial products and
to combined marketing of bacteria and
monolayer. Experiments should be
designed to illustrate advantages to be
gained by reduced application frequency.
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Yendo!, W. G., Heldung, R. C. and Lewis, F. B.
(1977) Field investigation of a baculovirus of the
gypsy moth. J. Econ. Entomol. 70, 598-602.
Young, S. Y (1989) Problems associated with the
production and use of viral pesticides. Mem.
lnst. Oswaldo Cruz., Rio de Janeiro, 84 (suppl.
III), 67-73.
Young, S. Y. and Yearian, W. C. (1976) Influence of
buffers on pH of cotton leaf activity of a Heliothis
nuclear polyhedrosis virus. J. Ga. Entomol. Soc.
11,277-82.
Young, S. Y and Yearian, W. C. (1986) Formulation
and application of baculoviruses, in The Biology
of Baculoviruses, Vol. II, Practical Application
for Insect Control (eds R. R. Granados and B. A.
Federici), CRC Press, Boca Raton, pp. 157-79.
Zaritsky, A., Zalkinder, V., Ben-Dov, E. and Barak,
Z. (1991) Bioencapsulation and delivery to mos-
quito larvae of Bacillus thuringiensis H14 toxi-
city by Tetrahymena pyriformis. J. Invertebr,
Pathol. 58, 455-7.
Ziniu, Y, Songya, L. Jingyuan et al. (1996) Cloning
and expression of Bacillus thuringiensis Cry IIA
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Zou, Y and Young, S. (1996) Use of fluorescent
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Entomol. 89,92-6.
PART THREE

ORGANISMS WITH A CONTACT MODE

OF ACTION

Microorganisms that penetrate or associate with the outer surfaces of insects,

growing plants and plant-disease organisms target at application the outer
surface of insect and plant, also the environment. They can attack sucking
insects as well as chewing insects and rarely attack through the insect gut. They
are formulated to survive and grow in these positions.
FORMULATION OF MYCOINSECTICIDES 4
H. Denis Burges

CONTENTS
4.1 Introduction 132
4.2 Water relationships of fungi 135
4.3 Application of fungal sprays 137
4.3.1 Formulation of sprays for dry climates 137
4.3.2 Formulation of sprays for wet climates 139
4.3.3 Sunlight and sunscreens 142
4.3.4 Humidity, humectants and pH 146
4.3.5 Rain and stickers 146
4.3.6 Wetters and emulsions 147
4.3.7 Formulation of fungi with chemical pesticides 147
4.3.8 Endophytic growth of Beauveria bassiana in corn 148
4.4 Application of fungi to soil 149
4.4.1 Drenches and sprays 149
4.4.2 Granules 149
4.4.3 Baits and dusts 151
4.5 Application of fungi to water 152
4.6 Storage 154
4.6.1 Storage temperature 154
4.6.2 Survival during drying and improvement by formulation 155
4.6.3 Unformulated dry conidia 156
4.6.4 Formulated dusts, granules and wettable powders 156
4.6.5 Storage of conidia in oil 159
4.6.6 Storage in water 162
4.6.7 Effects of storage on speed of germination and virulence 165
4.7 Production 166
4.7.1 Production on solid substrates 166
4.7.2 Production by liquid fermentation 170
4.7.3 Optimizing spore vigour 171
4.8 Future development and research 172
4.8.1 Application 172
4.8.2 Storage 174
4.8.3 Production and quality of spores 175
References 176

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
132 Formulation of mycoinsecticides
4.1 INTRODUCTION
The range of insects controllable by fungi is
wide. Fungi kill insects primarily by infection
through the external insect cuticle, arising
from spores reaching the insect surface by
chance, infection rarely occurring through
the gut. Since spores need not be eaten, they
attack sucking as well as chewing insects. The
host insects most commonly Inentioned in this
chapter are aphids, whiteflies (scale insects),
locusts and grasshoppers, lepidopterous lar-
vae, ants, termites and ground beetles in foliar
and soil habitats.
In the simplest life cycle, an airborne distri-
butive phase, a conidiospore (conidium; Fig.
4.1) has considerable tolerance of harsh envir-
onmental conditions (Thomas et aI., 1996). It
alights on the insect's cuticle and germinates,
producing a tube that penetrates into the body
cavity and grows into a mycelium. This forms
relatively thin-walled blastospores which

Figure 4.1 Scanning electron mi.c~ograph of conidiogenesis of BemllJeril1 bassianl1 on solid medium; a,
characteristic ampliform conidiogenous cell; c, conidium; h, hypha; bar = 5 {tm. (Source: G. D. Inglis;
reproduced with permission of Agriculture and Agri-Food Canada.

circulate within the insect, eventually forming
a lethal mycelial biomass. This mycelium
grows back out again through the cuticle,
swathes the body surface and forms distribu-
tive conidia in vast numbers (Fig. 4.1). Later,
when adverse conditions end the insect's
reproductive season, the fungus survives as
mycelial biomass (Fig. 4.1) and conidia in
cadavers. This simple cycle is universal in
the Hyphomycetes, which contain most of
the genera used for insect control - listed in
Table 4.1, together with important features of
each genus. The other major group of fungi
considered for practical use, the Ento-
mophthorales (Table 4.1), form additional
very tough, specialized resting spores (oos-
pores), with copious oil food reserves, for
long-term survival and slow dissemination
of the fungus.
4.1 Introduction 133
For pest control, the natural cycle is mod-
ified to reduce the elements of chance and
attack the insects before they reach damaging
numbers. The fungal spores are mass pro-
duced on an industrial scale, stored until
required, then applied to the insects as effect-
ively as practicable. It is important to mimic
natural conditions as far as possible to pro-
duce spores in prime condition in vitro and
apply them effectively. The function of formu-
lation is to improve spore harvesting, survival
in store, application and post-application sur-
vival.
The structure of the mycoinsecticide opera-
tion is illustrated in Fig. 4.2 The most critical
decisions about formulation are made at the
end point of the operation, i.e. the application
of spores to insects in different habitats and
the need to ensure their survival in field

Table 4.1 Fungus pathogens on insects and mites, roughly in order of importance

Fungal genus Important features

Hyphomycetes (= Mitosporic Fungi; Hawksworth et aT., 1996)

Beauveria, Hosts: many pests of crop, pasture,
white muscadine; plantation, orchard and forest. Host range:
Metarhiziwn, wide for fungus species, narrow for strains.
green muscadine; Dusty spores (conidia) easily produced on
Paecilo111yces solids, blastospores in liquids. Temperature range usually lower for
Beauveria species than for Metarhiziu111
Verticilliul1l Hosts: aphids, scale insects and a few other groups. Production as
above, but conidia formed in sticky globular heads. Used in
glasshouses
Hirsutella Hosts: mites, particularly in orchards. Production less easy than
above genera, conidia not formed in liquid culture
No 111 urea Hosts: mainly Lepidoptera on field crops.
(Spica ria) Production, conidia on solid media. Blastospores not infective to
Heliothis zea larvae
Aschersonia Hosts: scale insects. Considered for control in glasshouses
Tolypocladium, Culicinomyces Hosts: mosquitoes. Production, conidia on solids, blastospores in
liquids. Considered for control of larvae

Entomophthorales
Entomophthora, Hosts: aphids, Lepidoptera and many other groups. Delicate conidia
Erynia and related genera formed on solid media. Very durable resting spores formed in
special conditions. Difficult to use for insect control
134 Formulation of mycoinsecticides
Line 1 Line 2 Line 3

Insect, Foliage Soil

foliage

i
Dry climate Wet climate

i
ULV spray
i
HV spray Granule

I
applicator

Formulate
i
Store
t
Store ....- - - - - - - - - ,
in field
with oil

i
Store with
r
Formulate as
r
Media-free
desiccant wettable granules

I
powder

t
Harvest and Dry Fluid bed
dry drier

i
Mycelium
1
Centrifuge:
i
Screen
produces technical
aerial slurry

i
conidia

i
Aerated bags Mycelium Mycelial Sterilized
of sterilized producing balls granular

/
granular blastospores solids in
medium end-user

i ~
containers

Inoculate .....1 --------- Liquid fermenter ---------I~~

t
Inoculate

t
Flasks

t
Plates

t
Slant

Figure 4.2 Optimal industrial flow lines in the use, storage and production of insect pathogenic fungi.

conditions. The implications of these deci-
sions are illustrated along three flow lines
working back to the starting point, spore pro-
duction. This chapter is ordered in that direc-
tion to ensure that full account is taken of
nature's requirements and their interactions
with the constraints of the chosen methods
of application and production.
The performance of mass-produced fungus
material will be studied in its unformulated
state, as a base line against which improve-
ments obtainable by formulation will be
assessed. The very nature of these fungal life
stages limits the possible improvements to an
unknown extent. An attempt will be made to
estimate these limits as ultimate objectives for
improvement.
The production system most easily auto-
mated and handled is deep liquid fermenta-
tion, which usually produces blastospores.
Ensuring survival and persistence of these
relatively delicate spores, or alternatively of
fragmented mycelium, is one of the greatest
challenges of mycoinsecticide formulation.
The tougher conidia are normally produced
only on discrete surfaces of liquid media or
solid substrates. Solids are more difficult to
handle and scale up than liquid systems. The
great durability of oospores is an advantage
offset by the difficulty of synchronous pro-
duction and, later, synchronous germination.
Consequently, most production and formula-
tion technology has been developed for blas-
tospores and conidia. Technology has to be
optimized for individual fungus species or
even strains. Finally, this chapter offers
suggestions for further research and develop-
ment.
Despite the wide host range of the fungi,
the number of commercial products has been
small. Most of these products have tended to
be ephemeral and so it is difficult to specify
the products available for sale at anyone date.
Those on sale at present include a few wett-
able powders that have been available since
the 1980s, e.g. Vertalec against aphids and
Mycotal against whiteflies, sporadically used
4.2 Water relationships offllngi 135
in European greenhouses and containing blas-
tospores of different strains of Verticillill/l1 leca-
nii. Most other currently available products
are wettable powders containing conidia.
Beallveria bassialla is used on a large scale on
various outdoor pests in Russia (Boverin),
China and more recently Brazil (Metaquino).
New products include Bio-Blast (Metarhizilll1l
anisopliae) against termites in the USA, Natur-
alis (B. bassinna) in France, Bairoisa-kamikiri
(Benllveria brongniartii) in Japan, Mycotrol
(non-dusty wettable powder and easily
mixed emulsifiable suspension; B. bnssiann)
in the USA and products containing Paecilo-
myces fumosorosells, e.g. Pfr 97 water dispers-
ible granule in the USA and Europe. Many
central and south American countries have
named products containing Beallverin and
Metarhizilll11 with some Pnecilol1lyces and a
few Nomllrnen and Verticillillm (R. M. Pereira,
University of Tennessee, Knoxville, personal
communication). Most of these products are
wettable powders or granules requiring refri-
gerated storage, and some have low quality.
Many co-operatives produce fungi for their
own use. Thus Brazil has wettable powders
formulated in clay or diatomaceous earth, a
pure conidia product and a rice-based gran-
ule. A few new products are approaching
commercialization, e.g. Green Muscle against
grasshoppers and locusts. Some products
have been withdrawn over the years. One of
the main reasons for product failure has been
poor and erratic pest control performance.
Formulation is a key factor in improving per-
formance.
4.2 WATER RELATIONSHIPS OF FUNGI
Water is critical for a fungus at all stages of its
life cycle; water availability can be influenced
by formulation.
The various units defined in the glossary
(Appendix III) and used to express water rela-
tionships are difficult to interrelate, so some
comparisons are made in Table 4.2. The equi-
librium relative humidity (RH) is the most
136 Formulation of mycoinsecticides
Table 4.2 Key points of water relationships in soil (Brady, 1984; Studdert et al., 1990)

Water content (percentage of dry weight)

Water tension (bar)* RH (%) Sandy loamt Peaty muckt Key points
-0.1 > 99 40 163 Saturation = field capacity
-0.1 to -0.3 > 99 40-15 163-88 Greatest bacterial activity
-3 to-6 99 11-10 68-67 Bacterial activity declines
rapidly
-0.1 to -15 99 40-9 163-58 Good growth of plants;
fungi grow
-15 98.9 9 58 Permanent wilting point t
-31 97 :::;9 :::; 58 No liquid water, only
vapour
-6 to -50 99-96 10 to :::; 9 68to:::;58 Fungal activity declines
rapidly
-50 to -400 96-75 :::;9 :::; 58 A few fungi can grow,
activity very low
-200 86 :::;9 :::; 58 Fungi cannot grow
-1500 33 :::;9 :::; 58
* 0 to -15 bars = matric tension: ::; -15 bars = matric tension + osmotic tension. Soil appears wet at 0 to -2 bars;
moderately dry at -10 to -15 and dry at < -15.
t Percentage saturation at -15 bar is 20% for a carefully defined sandy loam and 31% for a specific peat.

universally useful. It indicates the ambient
hydrating and evaporative power of air on
an inert surface such as glass; on a surface
with a partial barrier to moisture movement
such as the cuticle of an insect or plant with an
associated microclimate; on a solid growing
medium; or in soil. Its powers are largely
independent of temperature (> OC) because
it is a vapour pressure ratio (Appendix III).
Water activity (a w ), another useful tempera-
ture-independent pressure ratio (Appendix
III), is functionally interchangeable with relat-
ive humidity.
Equilibrium relative humidity is also the
most useful expression of water relationships
in soil. The critical physical and biological
points of soil are given in Table 4.2 alongside
water tension (= water suction). This is based
on matric potentials (negative bar values),
controlled by the forces of adhesion and
capillarity holding water to solid matter
(matrix) in soil. In soil with a high solute
content, and in dry soil, the osmotic potential
becomes significant and so is combined
with the matric potential to form another
unit, soil water potential. In soil that is
moister than the permanent wilting point
(-15 bar, 98.9% RH) water is available to
plant roots in liquid form. The moisture con-
tents of different types of soil differ consider-
ably at each critical point due to variation
in the content of sand, clay and humus
(Table 4.2).
There are three features of outstanding
importance for insect pathogenic fungi deter-
mined at constant humidities. Spore ger-
mination and mycelial growth are optimal
between 95 and 100% RH and cease at 92%
(Walstad et al., 1970; Ferron, 1977; Gillespie,
1984; Magan and Lacey, 1984; Inch and
Trind, 1987; Jimenez, 1989). Spore production
(e.g. blastospores of Paecilomyces farinosus)
is increased by a reduction of water activity
to an optimum of 0.958 (Inch and Trind,
1987). Survival of conidia (e.g. Metarhizium
anisopliae) in storage is best at extreme humid-
ities approaching 100 and 0% RH (section
4.6.3).
 4.3.1
4.3 APPLICATION OF FUNGAL SPRAYS
The impressive success of oil-formulated ultra
low-volume (ULV) sprays on locusts in dry
climates (Fig. 4.2, flow line 1) confounds the
concept that fungi infect insects only at high
humidity, and establishes new principles,
explored in section 4.3.1. The small droplet
size required in ULV (Appendix Table 11.2
and Fig. 11.1) restricts the use of additives,
thus limiting formulation options valuable in
the higher range of spray volumes used in wet
climates (Fig. 4.2, flow line 2). These will be
elaborated in section 4.3.2.
4.3.1 FORMULATION OF SPRAYS FOR DRY
CLIMATES
Application of ULV sprays with oil as carrier
has many advantages (section 2.2.2b). The
high work rate enables large areas to be trea-
ted during periods of suitable meteorological
conditions, which often occur only briefly
early in the morning. Droplet distribution is
relatively efficient and is most even with con-
trolled droplet application (CDA) of droplets
in a narrow size range (Table 2.4 in section
2.2.2). A relatively viscous product, suitable
for appliances with a feed pump, can be
diluted with a light oil (Appendix Table 11.4)
for those with gravity feed to give droplets
that do not evaporate excessively, such that
impaction is prevented (Bateman, 1994a). Oil
readily wets the hydrophobic, lipophilic sur-
faces of insects and leaves, as demonstrated
with the scanning electron microscope by
L. Ibrahim, T. M. Butt, A. Beckett and S. J.
Clark (IACR Rothamsted, Harpenden, perso-
nal communication). Oil also wets the dry,
dusty type of conidia, allowing conidia to
suspend easily in oil.
On impact, oil sprays spread rapidly over
the hydrophobic surfaces of leaves and
insects, before being absorbed into the cuti-
cles. A key advantage is the consequent
broad distribution of conidia over an insect,
carrying many conidia to protected spaces
Formulation of sprays for dry climates 137
between the in-folded intersegmental mem-
branes. Here the soft, thin cuticle and the
relatively humid microenvironment, even in
dry climates, favour rapid germination and
fungal penetration in the race to beat shed-
ding of conidia with the old cuticle at the
next insect moult (Fargues and Vey, 1974).
Carriage of spores by oil into humid micro-
environments probably explains the success
of infection by oil formulations in dry clim-
ates, because conidia of Metarhizium anisopliae
formulated in water or in oil needed aw ;;:'0.98
in nutrient-free agar to germinate; the pre-
sence of nutrients or insect extracts increased
the proportion of germinating conidia but not
the germination-permissive range of aw
(L. Ibrahim, T. M. Butt, A. Beckett and S. J.
Clark, personal communication).
There is considerable physical evidence of
efficient adhesion to the cuticles of insects and
plants of hydrophobic conidia formulated in
oil. Boucias et al. (1988) reported passive and
non-specific adhesion of conidia to insect cuti-
cle. This adhesion is thought to be a property
of the rodlet layer on the conidium surface
(Boucias and Pendland, 1991). Scanning elec-
tron microscopy of conidia applied to locust
cuticle showed germ tubes, appressoria and
penetration structures in the presence of an oil
film; thus oil does not appear to impede infec-
tion, and may assist it by disrupting the waxy
layer on the insect cuticle, as well as protect-
ing conidia from evaporation (Bateman et al.,
1993; L. Ibrahim, T. M. Butt, A. Beckett and
S. J. Clark, personal communication). Inglis et
al. (1993) found that Beauveria bassiana conidia
dispersed better in oil (Mycotech 9209) than in
0.05% aqueous Tween 80, while substantial
clumping occurred in a 5% emulsion of the
oil in water, even after homogenization. How-
ever, in three formulations, oil at ULV, water,
and emulsion at high volume (HV), conidia
formed aggregations on alfalfa (Fig. 4.3) and
crested wheatgrass Agropyron eristatum, and
stuck on the hairs of alfalfa. Tracer dye
showed 62-80% grasshoppers hit by wind-
dispersed ULV oil droplets of ea 70 mm
138 Formulation of myeoinseetieides
Figure 4.3 Scanning electron micrograph of circle of Bcoul'eria bassiana conidia left on alfalfa leaf by a
spray droplet of water with 0.05(X, Tween 80; bar = 10 pill. (Source: G. D. Inglis; reproduced with
permission of Agriculture and Agri-Food Canada.)
volume mean diameter (VMD) sprayed at 11/
ha (Lomer et Ill., 1993), although hits in the
field may range from 40 to > 90'70 of hoppers.
Laboratory work on Metllrhizium and Beau-
vaia conidia formulated in oil has provided
much evidence of infection at low humidities
and the importance of penetration at interseg-
mental membranes. Conidial germination on
cinch bugs (Bliss/ls leueopterus) tended to be
localized at the articulation between coxa
and trochanter (Ramoska, 1984). Conidia
infected grasshoppers at 12-30'Yo RH (Marcan-
dier and Khachatourians, 1987). Topical appli-
cation of conidia in oil was more effective
than in water plus wetting agent on the
mouth parts of the cocoa weevil Pantorhytes
plutllS by x36 (Prior et al., 1988) and under the
pronotal shield of locusts by x 146 at ea 35%
RH (Bateman et al., 1993). The LT50 (lethal
time to kill half the insects) was lower if bee-
tIes were inoculated under the elytra than on
the elytra where conditions were shown to be
more fungistatic (Butt et al., 1995). Conidia in
oil were more active than in water when
applied to grasshopper baits (section 4.4.3)
and in safety tests on honey bees (Ball et al.,
1994). Efficient killing in the presence of
oil was indicated by steep dose-mortality
curves more akin to those for chemicals
than for microbial agents (Burges and
Thompson, 1971; Lai et al., 1982; Bateman et
al., 1993).
Such laboratory results have been matched
by those in the field. CDA oil sprays of con-
idia from hand-held centrifugal disc sprayers
(Appendix Fig. ILl) can cause> 90% locust
mortality in < 10 days in both arid (15-80'~{,
RH day-night) and humid (mean 70% RH)
conditions (Bateman, 1992; Bateman et al.,
1992). In contrast, HV aqueous sprays of
 4.3.2
B. bassiana (oil emulsion plus 4% clay) against
grasshoppers in hot, dry, sunny conditions
failed to significantly reduce populations
due to field environmental conditions (Inglis
et al., 1997c). In dry conditions (45-100% RH
day-night), Paecilomyces fumosoroseus conidia
sprayed in oil (ShellsoIT:rape seed oil, 7:3)
and in two emulsions (1 and 10% Codacide
emulsifiable oil in water) killed 80-100%
whitefly (Bemisia tabaci) scales, but killed
none when sprayed in 0.1% aqueous Tween
80 (all sprays applied at 2 ml per cucumber
leaf on potted plants; Smith, 1994). B. bassiana
conidia in oil (Mycotech No. 9209) at ULV
gave distribution and penetration through
crested wheatgrass and alfalfa canopies simi-
lar to that both in water with Tween 80 and in
No. 9209 oil-in-water emulsion at HV; the
formulations had no obvious effect on persist-
ence (Inglis et al., 1993). Metarhizium flavoviride
conidia in sprays of oil and oil-in-water
emulsions survived longer on foliage than
those in water, presumably because the oil
component gave greater protection from
environmental stresses (Jenkins and Thomas,
1996). Six different vegetable oils were com-
patible with Nomuraea rileyi conidia in sprays
on detached leaves (Vimala Devi and Prasad,
1996). ULV oil sprays of B. bassiana gave better
control of tent caterpillars (Dendrolimus spp.)
by air and ground in China than aqueous
sprays made from wettable powder at xO.25
the comprehensive cost by air (Yin, 1981).
Post-spray effects of different oils have not
been studied.
Insects pick up spores from foliage, as well
as acquiring them from direct hits by droplets
(Bateman, 1997). Scanning electron micro-
scopy revealed conidia of B. bassiana exposed
on leaf cuticle and hairs, many aggregated in
intracellular depressions and some protrud-
ing from stomata (Inglis et al., 1993). In north
Benin, 39.8% of untreated grasshoppers
exposed to fresh M. flavoviride conidia resi-
dues for 2 days picked up a lethal infection,
the proportion in later exposures falling
progressively to 2.2% of hoppers exposed
Formulation of sprays for wet climates 139
after 10 days (Lomer et al., 1997; Sawyer
et al., 1997). Other workers reported spore
survival and pick-up (Prior et al., 1988;
Lomer et al., 1993; Bateman et al., 1994; Jenkins
and Thomas, 1996; Thomas et al., 1996; Lange-
wald et al., 1997), although Lobo Lima et al.
(1992) and Johnson et al. (1992) found that
application of conidia to vegetation had little
effect. Spores are also acquired by grasshop-
pers eating foliage, causing infection probably
via the buccal cavity rather than the gut
(Inglis et al., 1993, 1996a).
During the dry season, low humidity pre-
vents germination of conidia and growth of
fungus on treated plants, also conidiation
over the surface of cadavers. Thus the fungus
does not spread through insect populations to
improve control soon after application, as it
does in the wet season and in more amenable
climates. However, M. flavoviride can persist
in fragments of infected grasshopper, particu-
larly the heavily sclerotized, and survive the
Sahelian dry season (Sawyer et al., 1997).
Thomas et al. (1996) demonstrated that the
severity of natural infestations, when wet con-
ditions favourable for fungus spread returned
again, was related to the density of infected
cadavers surviving in the intervening dry sea-
son habitat from the previous wet season.
Increasing the concentration of conidia in
Sunspray Oil (Appendix Table 1.1) caused
intermittent blockage of the restrictor in a
Micro-Ulva sprayer. Application was satis-
factory at 100 gil, giving droplets of 30-
300 mm diameter and typically 20-40 spores
per droplet (Johnson et al., 1992). The desired
viscosity was obtained by adjusting the blend
of heavy and light oils (Lomer et al., 1993).
4.3.2 FORMULAnON OF SPRAYS FOR WET
CLIMATES
Treatment is more effective in humid than in
dry conditions because the fungus is spread
by conidia formed on insect cadavers. Even
better pest control results when the fungus
can grow and sporulate on the plant on
140 Formulation of mycoinsecticides
nutrient additives included in the larger dro-
plets formed by very low-volume (VLV), low-
volume (LV) and HV sprays (Table 2.4 in
section 2.2.2), as in flow line 2 (Fig. 4.2). By
absorbing water in the moist night time
and slowly losing it in the dry daytime, nutri-
ents act as water-availability buffers for the
fungi.
Nutrients such as cereal flour can be formu-
lated as the carrier in a dust or wettable pow-
der (e.g. Table 4.8 in section 4.6.4). The best
known example is Verticillium lecanii in the
products Vertalec and Mycotal, used against
aphid and whitefly intermittently since 1980
(Burges, 1981; Hall, 1981, 1982a; Ravensberg et
al., 1990).
Pioneered in controlled, warm, moist, low-
UV conditions under glass, flour-based V leca-
nii products gave better pest control than
sprays of spores alone (Hall, 1982a, b; Kana-
garatnam et al., 1982). On drying, droplets
leave little islands of nutrients, invisible to
the naked eye, as with sunscreens (section
4.3.3c). In 4-5 days fungal growth appears,
chiefly on the under sides of leaves; fortun-
ately this is where most of the pests are sited,
so upwardly directed nozzles should be used
(Hall, 1982a; Milner and Lutton, 1986).
Ca x 40 more spores may be produced by
growth on leaves than are applied (Hall and
Papierok, 1982). Gillespie et al. (1982), Hall
(1982a) and Milner and Lutton (1986) showed
that some aphids were infected by direct hits,
others by spores picked up from the leaf
surface and later some by spores growing on
the leaves. In an apparatus that mimicked the
greenhouse, but with constant humidity,
growth of V lecanii was optimal at 100% RH
with free water present, inhibited at 93%, and
almost nil at 80% (Milner and Lutton, 1986).
Control failures have been caused by dry
conditions (Kanagaratnam et al., 1982) and
the presence of relatively immobile pest
species (Hall and Burges, 1979).
After spraying there is a lag period before
the first diseased insects appear, e.g. 6 days at
20C with V lecanii on the aphid Myzus
persicae (G. A. Gill, Horticulture Research
International, Wellesbourne, personal com-
munication). Meanwhile, pests continue to
breed and damage crops; spraying early in
the infestation is therefore vital. This period
can be reduced by pre-soaking spores (see
below) and by formulation to speed germina-
tion (section 4.6.7) and fungal growth. Also,
solid nutrients in wettable powders can be
supplemented by additives to the spray tank
(Table 4.3). Glycerol consistently speeded
aphid death presumably by its humectant,
nutrient and adhesive qualities, or possibly
(section 4.8.3) by supplementing the spore
polyol content. Cutinol, an emulsifiable oil,
had less effect, probably acting as a wetter!
spreader carrying spores between interseg-
mental membranes (section 4.3.1). Other addi-
tives, including oils, had too small an effect to
be significant, while two, SAS 90 and Output,
inhibited the fungus on the tentative evidence
of a single assay, possibly by toxic action
(Table 4.3). Without a solid flour carrier, add-
ing 2% glycerol or 1% gelatin to aqueous con-
idial suspensions improved powdery mildew
control with V lecanii (Spencer and Ebben,
1983).
Under glass, it may be essential on some
crops to artificially raise humidity to achieve
lasting pest control. When nutrient formula-
tions are used this enables the fungus to grow
and sporulate on the leaves, as well as increas-
ing sporulation on cadavers, to continually
infect invading pests. Sheets covering crops
for other purposes improved aphid control.
For example, modern thermal screens not
only raised temperature but also humidity
(N. L. Helyer and G. A. Gill, Horticulture
Research International, personal communica-
tion), as did polythene blackouts to alter day-
length (Hall and Burges, 1979; Helyer et al.,
1992). Control was also improved by water
fogs (Helyer et al., 1992).
Water-based ULV and VLV applications
(Table 2.3 in section 2.2.2), preferably with
nutrients, controlled insects effectively in
greenhouses. This was partly because the
 4.3.2 Formulation of sprays for wet climates 141
Table 4.3 Effect of additives in a tank-mix of cereal flour-based Verticilium lecanii wettable powder* on the
speed and extent of mortality of the aphid Myzus persicae, in order of performance (N. L. Helyer, G. A. Gill
and M. de Courcy Williams, personal communication)

Additive (detail in Constituents and Laboratory assays! Greenhouse

Appendix 1) mode of action test!
Single assay Four assays

Glycerol, 2% Humectant/nutrient + +++sig +++sig

Cutinol, 0.1% Rapeseed oil + emulsifier + +sig
Emultex, 0.5% Sticker gum ++sig +
Potato dextrose agar Nutrient/humectant +
Gelatin, 0.2% Humectant/nutrient +
Skimmed milk, 0.2% Nutrient/humectant + <l
Codacide, 0.1 % Rapeseed oil + emulsifier. + =, +
Spreader
Actipron, 0.1% Mineral oil + emulsifier.
Spreader
Sabouraud dextrose agar Nutrient/humectant
Esterol 123, 0.2% Ethyl oleate. Penetrating
spreader
Mowiol Polyvinyl alcohol. Sticker
Cutinol V7, 0.1% Rapeseed oil + emulsifier.
Spreader
NuFilm, 0.2 % Terpene. Spreader
Vicchem EOP, 0.2% Ethyl oleate. Cuticle-
penetrating spreader
SAS 90,0.2% Surfactant. Spreader
Output, 0.4% Mineral oil + emulsifier.
Spreader

* Vertalec.
t Symbols express effect of additives on LT50, ranging from shortening of en 2 days (+++), to virtually no change (=), to
lengthening of ca 2 days (-). The LT 50 was typically 6 days with Vertalec alone (0.5 ml of 0.2% suspension) sprayed on
8-cm discs of Chinese cabbage kept at 25 'c, ca 100% RH.
1: Symbols express control equivalent to Vertalec alone (=), ranging to a x4 improvement (+++) on Chinese cabbage,
uncovered or under fleece covers in 11 days.
Aphid counts significantly different from Vertalec alone.
.- Codacide (5%) improved aphid control on cucumber in a greenhouse trial at 18-25 'c, 60-85% RH, mean 78%
(Helyer, 1993).

relatively high in-house humidity decreased
evaporation from droplets en route from
sprayer to leaf, giving good settling and crop
cover over the short distances involved. A
variety of sprayers were used, including an
electrostatically charged rotary atomizer
(Helyer and Wardlow, 1987; Sopp et al.,
1989). Wright and Kennedy (1996) reported
a similar versatility for a commercial B. bassi-
ana flowable product, and control equal to or
better than that achieved with conventional
chemical products.
One feared potential side effect did not
occur: nutrients used with V lecanii did not
increase white rust disease of chrysanthe-
mums (Helyer and Whipps, 1993).
Outdoors, a variety of nutrients promoted
growth of other entomopathogenic species on
foliage. In Mycar, fragmented mycelium of
Hirsutella thomsonii was formulated in a solid
nutrient carrier with variable results (Burges
and Hall, 1982). For control of brown plant
hopper (Nilaparvata lugens) on rice, skimmed
milk has been added to M. anisopliae conidia
142 Formulation of mycoinsecticides
(Gillespie, 1984). Yeast extract enhanced viru-
lence against the beetle Phaedon cochleriae on
oilseed rape, as well as stimulating germina-
tion of conidia in the laboratory (David-
Henriet et al., 1996). A wettable powder of
Pandora delphacis in a carrier of 20% sorghum
powder with 5% clay as a non-nutrient dis-
persant gave better results than a standard
chemical; lignite fly ash from thermal coal
mines as dispersant was less effective (P. Nar-
ayanasamy, Annamalainagar, India, personal
communication). B. bassiana conidia in dusts
and granules applied dry to leaf whorls gave
better control of European corn borer (Ostrinia
nubilalis) than aqueous sprays with or without
detergent. In granules, corn meal was super-
ior to the non-nutrient attapulgite clay, and
mycelium was observed growing in whorls
on the dust. Growth in the laboratory was
better on flour than on corn starch (Bartlett
and Lefebvre, 1934; York, 1958).
Outdoors, it is often necessary to formulate
for unpredictable weather conditions. Sprays
containing water initially support spore ger-
mination, which is an advantage during
weather that maintains high humidity to
allow germination to continue after water
from spray droplets evaporates. At low hum-
idities, however, germination will be arrested
after evaporation; water in the spray may be a
disadvantage, sensitizing spores by allowing
germination to start and increasing the like-
lihood of spore death if the spore dries. In dry
conditions, waterless formulations such as
dusts and oil flowables may be better to
avoid germination being initiated until
conditions are humid enough to support it.
Protection from dryness may result from
encapsulating pellets of mycelium in alginate
(Beauveria, with added wheat bran, Knudsen
et al., 1991; Pandora gloeospora, Keil, 1994).
Conidiation from the Beauveria pellets was
speeded after application to the target if the
pellets had been soaked in the osmotic regu-
lator polyethylene glycol (PEG) part way
through the drying process at the end of
production.
Pre-soaking of wettable powders of some
fungi in water speeded germination and
reduced infection time, hence the period
spores were exposed to adverse conditions,
thereby increasing pest mortality. Pre-soaking
with water was beneficial with blastospores of
V lecanii in wettable powders (Hall and
Papierok, 1982) and with fresh conidia of
M. anisopliae (Hassan et al., 1989). Soaking
conidia of Tolypocladium cylindrosporum (Mate-
wele, 1986) and blastospores of P. farinosus
with broth was also beneficial (Prenerova,
1995), but was not successful with conidia of
Hirslltella thompsonii because these start to ger-
minate only 2-3 min after wetting (McCoy
and Couch, 1978), nor with M. fiavoviride in
which the ability to germinate is impaired
(Moore et al., 1997; section 4.6.7).
Application of a low dose (ca LC lO ) of feed-
ing inhibitor, cypermethrin, with M. fiavoviride
advanced the onset of locust mortality by 48 h.
This occurred at a dose of 13 000 conidia per
adult but not at doses as low as 375 conidia.
Cypermethrin was not toxic to the fungus in in
vitro tests (Sanyang and van Emden, 1996).
4.3.3 SUNLIGHT AND SUNSCREENS
The lethal effect of sunlight on spores of B.
bassiana was recognised at the dawn of know-
ledge about microbial infection by Bassi, who
is accredited with the first proof that a micro-
organism caused disease. He recommended
disinfection of leaves fed to silkworms by
exposure to sun as a cure for muscardine dis-
ease (Bassi, 1836). Sunlight is one of the most
damaging factors faced by fungi on foliage.
4.3.3a Susceptibility of fungi to sunlight Sun-
light begins to kill fungi within hours after
exposure begins (Daoust and Pereira, 1986;
Moore et al., 1993; Inglis et al., 1995; Morley-
Davies et al., 1995; Moore et al., 1996b;
Fargues et al., 1996). In the field, negative cor-
relation of persistence of conidia with cumu-
lative total solar radiation was strong (Inglis et
al., 1995).

When considering how to protect fungi
against harmful light, it is important to know
the effects of the constituent wavelengths.
Harmful wavelengths reaching plants out-
doors (UVB, 280-320 nm and UV A, 320-400
nm) and in glasshouses (UVA) are described
in section 2.2.3a. Of various broad light bands,
near-UV (320-450 nm, peak 370) was the most
harmful in tests with different wavebands at
approximately constant energy delivery in
identical experimental conditions (Osman
and Valadon, 1981). Within the UV range the
most damaging wavelengths reaching foliage
are UVB (280-320 nm) and, to a lesser extent,
UVA (320-400 nm). Moore et al. (1996b) quant-
ified energy levels; UVB damaged conidia of
M. flavoviride but UVA did so only slightly
(Moore et al., 1993), even though its energy
level was x8 greater. Comparisons within
each system used by different authors are
valid and will be used to unravel the princi-
ples involved in the effects of UV light. Com-
parisons between different studies are difficult
and must involve consideration of energy
levels and temperatures at the point of contact.
The developmental stages of fungi vary in
susceptibility to UVB light, which in turn is
influenced by environmental conditions and
fungal species. Dry mycelium of M. anisopliae
was comparatively resistant (Jimenez, 1989),
but this advantage is lost because the myce-
lium must conidiate after application and the
conidia are then still exposed to sun (Pereira
and Roberts, 1991). Aerial conidia of B. bassi-
ana were more susceptible to UVC (254 nm;
screened out by the atmosphere) than conidia
produced in submerged culture, which in
turn were much more susceptible than hyphal
bodies (Jimenez, 1989). Conidia of M. flavovir-
ide from agar cultures 30-40 days old at 25C
were less susceptible to UVB than conidia
from 14-20 day old cultures (Moore et al.,
1993), probably due to increased pigmenta-
tion. Damage was proportional to the radia-
tion received, and periods of darkness
between exposures did not reverse the loss
of viability; a delay in germinating conidia
4.3.3 Sunlight and sunscreens 143
after exposure further reduced viability
(Hunt et al., 1994). Before a lethal exposure
was reached, UV light delayed the germina-
tion of many conidia from 24 to 48 h or more
at 25C (Zimmerman, 1982; Moore et al., 1993;
Hunt et al., 1994; Moore et al., 1996b). The
effects of UV light increased drastically with
increasing temperature from 5 to 55 T (Moore
and Morley-Davies, 1994; Moore et al., 1996b).
UV damage to fungal spores is due to muta-
genesis and photoreactions (Inglis et al., 1995).
There are large differences in susceptibility to
simulated sunlight between fungal species,
only partially related to spore pigmentation,
e.g. AspergillUS niger cinnamomeus (black con-
idia) is less susceptible than M. flavoviride
(heavily pigmented green conidia, 92% of iso-
lates exhibited 50% survival after 1 h of irra-
diation) < B. bassiana (unpigmented, 61%) <
M. anisopliae (paler green, 26%) < P. fumosor-
oseus (lightly pigmented, 3%). There were
wide variations in susceptibility between dif-
ferent strains of the same species (Fargues et
al., 1996; section 10.9).
4.3.3b Protection against sunlight by carriers
Carrier liquids in sprays can partially protect
conidia against UV light (Moore et al., 1993;
Inglis et al., 1995). On glass surfaces, simu-
lated sunlight or UVB light rapidly reduced
survival of conidia in 15-60 min in water,
which probably offered no protection, where-
as in oil there was 25-74% less reduction,
indicating considerable protection by the oil.
However, survival of conidia in oil on leaf
was substantially less relative to that on
glass, attributed to absorption of the oil away
from the conidia into mesophyll cells (Moore
et al. 1993; Inglis et al., 1995). Rehydration of
conidia in water may increase susceptibly.
4.3.3c Sunscreen additives Table 4.4 sum-
marizes available data and compares the
protection given to some fungal spores by
different sunscreens. Those used with B. bassi-
ana were tested for toxicity: none harmed
spores, including Blankophor optical
144 Formulation of mycoinsecticides
Table 4.4 Protection of conidia from UV light by sunscreens, expressed as ratio of percentage survival
with and without the screen. B, Beauveria bassiana; M, Metarhizium flavoviride; 5, protection significant; N5
not significant; (5) survival significantly less than control. Each significance entry represents one set of
experiments

Protectant Laboratory Field

Percentage Simulated sun (h)* Protection Sun (h) Protection

Water carrier
Water control 3 B: 0.22%t 8 0.24%t
Congo Red 5 3 B: x 454,5 8 x 2.46, N5
optical brightener P167 5 3 B: x 436,5
(OB)
Blankphor B5U (OB) 5 3 B: x 436,5 8 x 0.12+,5, N5
Blankophor BBH OB 0.25 3 B: x 286,5
(= Tinopal LPW)
Tinopal LPW (Caicofluor 5 3 B: x 218,5 8 x 6.6,5
White)
Clay~ 12*1 3 B: x 195,5 8 x 8.0,5
LPG-Blankophor OB 0.25 3 B: x 82, N5
HR5-Blankophor OB 2 3 B: x 50, N5
RKH-Blankophor OB 0.25 3 B: x 32, N5
DML-Blankophor OB 0.25 3 B: x 25, N5
5% paraffinic oil emulsion 5 3 B: x 0.06, (5)
Graessorb 0 5 3 B: 0.044, (5)

Oil carrier
Paraffinic oil control 3 B: 0.14%t 8 0.89%t
3 M: 28
Parsol MCX 5 3 B: x 93,5
1 2 M:5,N5
2,2-Hydroxy-4- 4 3 B: x 71,5
octobenzophenone
2,2-Dihydroxy-4- 2 3 B: x 17, N5
methoxybenzophenone
Eusolex 8020 5 3 B: x 14, N5
Eusolex 4360 1 2 M: N5,N5
Eusolex 6300 1 2 M:5,N5
Eusolex 8021 (= 61 % 8020 + 2,4 4 M:N5
39% 6300) 1 2 M: 5,5
Oxybenzone 5 3 B: x 7.8, N5 8 x 5.4, N5
2,4 4 M:N5
1 2 M: N5,N5
Ethyl trans-cinnamate 5 3 B: x 4.6, N5 8 x 5.0, N5
1 3 M: 2.1,5
Ethyl cinnamate 2,4 4 M:N5
1 2 M:5,N5
Benzyl cinnamate 1 2 M:5,N5
Florisil 60-100 mesh 2 3 B: x 4.6, N5
Graessorb 5 5 3 B: 2.2,5 8 x 5.0, N5
1 3 M: 1.5,5
1 2 M: N5,N5
 4.3.3 Sunlight and sunscreens 145
Parsol1789 5 3 B: 3.1, NS
1 2 M:NS,NS
Mexenone 1 2 M: NS,NS
Tinuvin 328 1 2 M:S,NS
Tinuvin P 1 2 M:S,NS
Cyasorb UV 531 1 2 M:S,NS
Chimmasorb 81 1 2 M:S,NS
Univul049 1 2 M: NS,NS
* M. flavoviride: 2 h exposures of two strains from Hunt et al. (1994); 4 h exposures from Moore and Morley-Davies
(1994); 3 h exposures of B. bassiana from Inglis et al., 1995 and of M. flavoviride from Moore et aI., 1993.
t Conidial survival (%).
t Mean of two trials, one similar survival to water and the other significantly less than in water.
Solutions of these additives saturated at these concentrations.
"if 12% clay (w Iv), 15% oil (v Iv) in water.

brightener (BBH-OB), despite its high pH of
9.5. Modes of action of screens are reviewed in
sections 2.2.3a and 3.4.4e. Their absorptance
spectra were favourable, peaking in the UVB
band and extending a little into UVA and
UVC (Appendix Tables 1.9 to I.12). The pH
and the solvent or carrier alter the spectra
(Shaath, 1990; Moore et al., 1993). I have been
unable to detect any correlation between
recorded spectra and protection afforded by
different screens. The exposure periods in
Table 4.4 were chosen to show differences
between screens, rather than to protect
enough conidia to kill insect pests. The
expression of spore survival as the ratio of
percentage survival with and without a screen
partially eliminates the effects of variation
between different spore batches, strains and
species and of using different radiation
dosages in different studies. Even so, the pro-
tection given by different screens spanned a
wide range. They are conveniently assessed in
two groups, those compatible with water, and
those with oil.
Of the water-compatible sunscreens, five
absorbers and a UV blocker, clay, gave the
best protection (Table 4.4). The absorbers
comprised a dye, Congo Red, and four stil-
bene optical brighteners: PI67-0B, BSU-OB,
BBH-OB and Tinopal LPW, although poor
solubility in water limited the concentration
of BBH-OB to only 0.25% (Appendix Table
I.10). Of four screens selected for field tests,
only clay and Tinopal LPW gave good field
protection - these did not perform the best in
the laboratory.
Oil-soluble sunscreens usable in ULV
sprays on leaf gave less protection in the
laboratory than the water-compatible screens
(Table 4.4). This may be explained, at least in
part, by the physical behaviour of the carriers.
Inglis et al. (1995) state that
"on both leaves and [glass] coverslips, drop-
lets of water were localized and evaporated
within 15 min of placement. On coverslips, oil
droplets covered a larger area (4 to 5 mm dia-
meter) than did water droplets (1.5 to 2.0 mm
diameter). On wheatgrass leaves, oil spread
rapidly across the lamina and an oil sheen
was usually observed".
Since oil spread more than water on both glass
and leaf, less screen would be sited over
spores to stop UV light in oil than in water.
Also, since water evaporates rapidly, it would
drag the screen into an ever-decreasing
volume and deposit it in concentric rings, as
in the familiar rings around a drying puddle
on a path; finally the last remaining liquid
would retreat around solid particles such as
spores, depositing most screen around them.
Because oil evaporates slowly, less screen
would be expected to aggregate around spores
in oil. Inglis et al. (1993) observed that conidia
of B. bassiana aggregated in rings around both
impacted water and oil droplets (Fig. 4.3).
146 Formulation of mycoinsecticides
Absorption of oil into the leaf may reduce the
amount of screen sited over the spores, parti-
cularly if the solvent oil carried the screen into
the leaf. In the laboratory (Table 4.4), two
sunscreens, Parsol MCX and 2,2-dihydroxy-
4-octobenzophenone, gave significantly better
protection compared with oil alone and with
the other screens; possibly these two screens
penetrated into the leaf less than the others. In
the field, although their effects were not sig-
nificant, the three screens applied in oil, oxy-
benzone, ethyl cinnamate and Graesorb S
were superior to oil alone, and nearly as
good as the two best screens applied in
water, Tinopal LPW and clay (Table 4.4).
The value of sunscreens will depend on
whether they can improve pest control, but
few field trials on fungi with insects have yet
been conducted. Field mortality of the grass-
hopper Krausella amabile was not increased by
adding 2% oxybenzone to a ULV oil forma-
tion of M. flavoviride in Mali (Shah et al., 1998).
A wider range of screens for the protection of
bacteria and viruses is described and dis-
cussed more comprehensively in sections
3.4.4a-e, and screens are listed in approximate
order of merit in the Note at the foot of Table
3.16 in section 3.4.4. Some of the best screens
with fungi were also among the best with
bacteria and viruses. From the environmental
viewpoint, UV blockers such as clay, starch
and carbon are preferable because they are
either environmentally innocuous or easily
decomposed. Some, e.g. clay, are useful
additives to improve harvest and storage (sec-
tion 4.6).
4.3.4 HUMIDITY, HUMECTANTS AND PH
Available evidence suggests that the fluctuat-
ing wet and dry conditions on foliage impair
spore survival (section 4.3.2). Once germina-
tion on a leaf has started, dryness prevents
fungal development and sporulation on
foliage, effects that can be a'meliorated by
humectants. These delay evaporation from
sprays, absorb water diurnally at peak high
night humidities and lose it at low day
humidities, buffering the fluctuation of water
activity and pH experienced by fungi.
Whether fungi gain water depends on
whether the average diurnal water availabil-
ity exceeds their absorptive power.
Many additives are hygroscopic (section
4.3.2; Table 4.3 in that section). Some of these
are powerful humectants with possible nutri-
ent qualities, e.g. glycerol; others are primar-
ily nutrients with some humectant action, e,g.
Sabouraud dextrose agar, skimmed milk and
solid nutrients such as flour. Some are also
stickers, or wetters and/or pH buffers. As a
further complication, some of these additives
have important effects on droplet size spectra
of sprays. It is impossible to ascribe accurately
the relativ.e importance of these various qual-
ities, but glycerol may act primarily as a
humectant. The effect of leaf surface pH on
survival and germination of fungal spores is
poorly understood.
4.3.5 RAIN AND STICKERS
Adverse effects of rain washing spores off
leaves have not been demonstrated in the
field, possibly due to counteracting benefits
of humidification. Rainfall had no overriding
effect on the survival of B. bassiana conidia
sprayed on stands of wheatgrass and the car-
rier (oil, oil-in-water emulsion in 0.05% aqu-
eous Tween 80) had little or no effect on field
persistence (Inglis et al., 1993). Rain is unlikely
to wash influential numbers of conidia off the
insect body; even violent agitation such as
vortex washing in 0.05% aqueous Triton X-
100 removed only half the conidia from vine
weevil larvae previously dipped in a suspen-
sion of M, anisopliae conidia for 150 s (Moor-
house, 1990).
Obviously rain must wash some spores off
leaves, so stickers bring the benefit of a degree
of rainfastness. This benefit may be supple-
mented by other properties of some stickers.
For example, skimmed milk (0.5-2.5%) in-
creased the deposit of M. anisopliae conidia,

presumably by beneficial action on the beha-
viour of the spray. In the longer term, after 7
days, the number of spores was increased by
x 12 due to fungal growth on leaves, compared
with sprays in 0.025% Triton X-100 on rice
plants kept in perspex cages (25C; ca 100%
RH) under fluorescent lights, i.e. without
exposure to UV (Gillespie, 1984). Keller
(1992) used skimmed milk as sticker-sun-
screen in sprays of Beauveria brongniartii to
start infection in swarming cockchafers, Melo-
Iantha me/olontha. Stickers can both stimulate
and harm fungal weed pathogens (section 6.5).
4.3.6 WETTERS AND EMULSIONS
A wetter (Appendix Table 1.5) is essential to
allow aqueous sprays to wet and spread over
the hydrophobic surfaces of insect and leaf
cuticles. It also helps to rehydrate spores
stored dry (sections 4.6.4, 4.6.7) and to dis-
perse spore clumps. The wetters used most
frequently have been 0.01-0.5% Triton X-lOO
and Tween 80 (see also section 3.4.1). 5hort-
term exposure to these surfactants in sprays
probably does not harm the spores (Prior et
al., 1988), although there is limited evidence of
harm to fungal spores caused by Tween 80 in
long-term storage (section 4.6.6b). In assays,
0.02-0.05% Triton X-lOO slightly reduced the
infectivity of conidia of M. anisopliae (Moor-
house, 1990) and of V lecanii, and reduced
sporulation on aphid cadavers (Hall, 1982b).
Possibly, the wetting agent progressively neu-
tralized hydrophobic binding sites on the
insect cuticle (Boucias and Pendland, 1991).
Even at 1%, Triton X-100 did not harm con-
idial germination of V lecanii on agar (Hall,
1982b), although there is one report of possi-
ble harm to vegetative bacteria (section 3.4.1).
M. anisopliae conidia were not wetted by water
and some germinated in storage at room tem-
perature; premature germination was pre-
vented and excellent spore wetting and
suspension of 1-10% (wjv) conidial suspen-
sions was obtained with 0.5-1% dioctyl sul-
phosuccinate (055), while germination was
4.3.6 Wetters and emulsions 147
not impaired after 4 months' storage in
water with or without oil; drying with the
OSS then storage in water impaired germina-
tion, but not if the conidia were stored dry,
although then speed of germination was
reduced; premature germination was not
prevented by alkoxylated alcohol (Johal and
Marold, 1996). Measurement of wetting
effectiveness is usually by qualitative visual
assessment of wetting on leaf surfaces and
suspendability of spores seen under the
microscope, e.g. Kanagaratnam (1980),
although leaf water repellency can be quanti-
fied by contact angle measurements (Appen-
dix II, Fig II.6). Tween 20 at 0.05% increased
the infection rate of Coccus viridis by V lecanii
in a field trial (Easwaramoorthy and Jayaraj,
1978). More Aschersonia placenta conidia ger-
minated in 0.01-0.05% Tween 70 than in pure
water or in 0.01-0.05% aqueous Welgro, a rich
nutrient medium (Appendix Table 1.5), prob-
ably because the surfactant provided the best
balance of nutrients for germination as well as
the best wetting of spores (Ibrahim et al.,
1993). Tween 20 and 5ilwet L77 can be toxic
to fungal weed pathogens, and Tween 80
stimulatory (section 6.5).
Lipophilic surfactants are widely used as
emulsifiers (Chapter 2). The oil carries hydro-
phobic spores, and large droplets of oil-in-
water emulsion splash off the leaf mainly
leaving the spores behind on the leaf surface.
Conidia of B. bassiana delivered in an emul-
sion persisted live on foliage for a similar time
to those in oil (Inglis et al., 1993). Miller
Nufilm, Chevron X-77, Plyac and Spray Oil
435 had no effect on germination of H. thomp-
sonii at field-use rates (Couch and Ignoffo,
1981).
4.3.7 FORMULAnON OF FUNGI WITH
CHEMICAL PESTICIDES
Pathogenic fungi may be used and even for-
mulated with chemicals to obtain a synergistic
or additive action. They may also be included
with chemicals as components of integrated
148 Formulation of mycoinsecticides
control packages, applied together if compat-
ible, or for application convenience. Such
usage involves risk of damage to the fungal
pathogens, especially by fungicides, and to a
lesser extent by insecticides and carriers or
other formulants with the chemicals. This
risk can be assessed reliably only in field
tests with the fungal pathogen in question,
together with the actual chemical products.
Laboratory tests with the chemicals, applied
to agar cultures of the pathogen or to bio-
assays with the target insects, give results
that are of limited use and sometimes mislead-
ing, as with microbial herbicides (section 6.5).
Even so, many chemicals have been tested ~n
the laboratory, e.g. eight fungicides against B.
bassiana by Feng and Chiang (1994), but only
some have been tested in the field, e.g. Kana-
garatnam (1980), Anderson and Roberts
(1983), Moorhouse (1990), Majchrowicz and
Poprawski (1993), Li and Holdom (1994),
Ravensberg et al. (1994) and industrial litera-
ture available from the Mycotech Corporation,
Butte, Montana. Pathogenic fungi can be reli-
ably formulated for storage with other micro-
bials, but not necessarily with chemicals.
Some chemicals can be used in the same tank
mix with certain pathogens. Precautions to
minimize risk include use of selective chemi-
cals, encapsulation of either pathogen or che-
mical and separation of application of the two
in time. Thus teflubenzuron, an insecticide
which selectively interrupts chitin synthesis
of insects - but not fungi - synergized mortal-
ity of third-instar desert locusts, erratically
with M. anisopliae and consistently with the
more specific pathogen, M. flavoviride (Joshi
et al., 1992). Also the fungicide dimethirimol,
used against cucumber mildew some time
before V lecanii sprays, did not impair white-
fly control by this fungus under glass (Kana-
garatnam, 1980; Kanagaratnam et al., 1982).
Consistent synergism of the well estab-
lished organochlorine, carbamate and organ-
ophosphate insecticides has not'been recorded
with fungal entomopathogens. However,
among the newer insecticides, in addition to
the action of teflubenzuron described above, a
sublethal concentration (100-1000p.p.m.) of
the systemic neurotoxic imidacloprid slowed
the development and movement of first-ins tar
citrus root weevil larvae, preventing contact
with the substrate which removes M. anisopliae
and B. bassiana conidia before germination and
penetration of the cuticle (Quintela and
McCoy, 1997b). Also, a confidential formulant
of the imidacloprid speeded and increased
conidial germination on both agar and insect
cuticle (Quintela and McCoy, 1997a). In field
tests, additive and synergistic effects were
detected at 8 days after treatment, but not
after 16 days (Krueger and McCoy, 1997).
4.3.8 ENDOPHYTIC GROWTH OF B. BASSIANA
IN CORN PLANTS
Growth of an entomopathogen in corn offers
an intriguing, but so far variable, method of
controlling an insect pest. B. bassiana applied
in corn grit granules to foliage of corn, or
injected directly into the plants, colonized
the plants, particularly the pith, and/or
moved in the xylem vessels. Yield data indi-
cate that it is not a plant pathogen (Lewis et al.,
1995). However, only some corn cultivars
were colonized. In a hot, dry year, there
were just a few successful colonizations last-
ing until harvest, mainly among injected
plants. In a wet year, most plants were colo-
nized (93-95%), resulting in ca 50% (foliage-
treated) and ca 20% (injected plants) reduction
in corn borer (Ostrinia nubilalis) damage,
while in the 2 years 0 and 33%, respectively,
of the untreated control plants were colonized
(Bing and Lewis, 1991, 1992). Possibly in the
hot year, high ambient temperature killed the
Beauveria as it can in insects (section 4.8.1b).
When corn borer mortality was supplemented
by the specific borer pathogen Bacillus thurin-
giensis, or the compatible chemical insecticide
carbofuran, yields were sometimes increased,
variability being introduced by other factors
that influenced yield, besides insects (Lewis et
al., 1995).

4.4 APPLICATION OF FUNGI TO SOIL
The moist nature of the soil environment
favours fungal survival and growth, so the
formulation strategies for application of pro-
ducts to soil follow industrial lines 3 and 2
(Fig. 4.2). Aqueous drench is used to penetrate
soil from the surface, but not oil spray because
the lesser quantity of oil is absorbed by soil
before it can carry spores over the insect cuti-
cle. Granules incorporated into soil imbibe
moisture, allowing the fungi to grow and pro-
duce more spores. Also, the slopes of bioas-
says with soil pests are low indicating wide
variation in the response of the insects (Moor-
house, 1990). Their dosage is less important
than in other environments, hence consider-
able deterioration in storage (section 4.6) can
be tolerated. UV light is not important because
the soil protects spores below its surface.
Soil-dwelling insects burrow and nest in
soil and many scavenge on the surface.
Usually they are long-lived, and development
may span a year or more. These features dic-
tate fungal application strategies, including
drenches for rapid soil penetration, baits and
sprays for surface scavengers, dusts for nests,
granules for soil incorporation (particularly at
potting, seed sowing and pasture renovation).
4.4.1 DRENCHES AND SPRAYS
Fungal spores applied to the soil surface in
drenches, with or without minimal wetter,
are mainly trapped in the upper 1-5cm by
the filtration and absorptive powers of soil
(Verticillium lecanii, Hirte et al., 1994; Metarhi-
zium anisopliae, Moorhouse, 1990). Here they
may be damaged by high temperatures
caused by long-wave sunlight. However,
enough M. anisopliae spores penetrate deeper
to control pests. In pots with established
plants, penetration depended on soil compo-
sition. The concentration of conidia increased
with depth in potted field soil but, in peat or a
peat-sand mix, only conidia in the highest of
three drench volumes penetrated extensively
4.4.2 Granules 149
to the lowest levels, a difference possibly
partly related to the different water-holding
capacities of these soils. Control of vine weevil
larvae was good and was not improved by
mixing conidia into soil at planting, so spore
penetration was not limiting. In fields, Beau-
veria bassiana conidia from an aqueous com-
mercial formulation sprayed on to the surface
of six well-drained, settled field soils were
mostly (92 to > 97%) trapped during the first
day in the upper 5 cm layer. In loams, the
degree of spore percolation was not correlated
with the clay/sand ratio. Enough conidia
penetrated 5-15 cm to kill test insects (No111ur-
aea rileyi, Ignoffo et al., 1977; B. bassiana, Storey
and Gardner, 1987, 1988; Storey et al., 1989;
V lecanii, Hirte et al., 1994). Prospects of
improving drenches by formulation are prob-
ably limited to experiments with more wetter.
4.4.2 GRANULES
Baseline data on survival of insect pathogenic
fungi in soil are very variable due to a variety
of abiotic and biotic factors. Temperature is
overriding, depending on the temperature
range of the different fungi. Water availability
(Table 4.2 in section 4.2) tends to be favour-
able, because fungi survive and grow best
above the permanent wilting point of plants
(Metarhiziu111 and Beauveria, Lingg and
Donaldson, 1981; Daoust and Pereira, 1986;
Studdert et al., 1990; Moorhouse, 1990; Li and
Holdom, 1993: V. lecanii, Hirte et al., 1994).
Survival may extend over several years, e.g.
105 colony-forming units (c.f.u.) of M. aniso-
pliae/g dry soil in field plots for 2 years (Mil-
ner and Lutton, 1976) and 104 /g for 3 years
(Rath, 1992).
Spore numbers fluctuate with time. Milner
and Lutton (1976) provided evidence that
M. anisopliae increased in summer. Conidia
of B. bassiana penetrating into the top 5.5 em
of soil declined by 86-95% in the first 12 days,
then increased two-fold apparently as a result
of fungal growth in the soil; a slow, erratic
decline followed until day 200 when 0-8200
150 Formulation of mycoinsecticides
d.u./ cm 3 dry soil were detected (Storey et al.,
1989). Survival varied greatly in different
strains of the same species (Fargues and
Robert, 1985; Moorhouse, 1990; Studdert et
al., 1990). There is some evidence for mycor-
rhizal status of B. brongniarti (Tillemans et al.,
1993).
Some entomopathogenic fungi are spread
naturally in soil by growth from infected
cadavers. M. anisopliae infecting larvae of
pecan weevils and vine weevils in soil was
confined to the cadavers, sporulating on the
cadaver surface; spread to other insects was
not observed. In contrast, B. bassiana grew
through the soil into colonies up to 8 cm
across, infecting other weevils and arthropods
(Gottwald and Tedders, 1984; Moorhouse,
1990). Similar ramifying mycelium of B. brong-
niartii formed compact pellets within which
conidia formed (Callot et al., 1996).
Spread from cadavers can be mimicked by
formulation of the fungus into granules.
Counts of B. bassiana d.u. in the top Scm of
soil from a commercial wettable powder
spray (formulation details not given) declined
to ca 4% in the first 21 days, then slowly to
zero over 202 days. In contrast, counts from
granules scattered over the soil surface fell
less to 37% in 21 days, x 10 higher than counts
from the aqueous suspension; 111 c.f.u./cm 3
soil were recorded after 202 days (Storey et al.,
1989). Of three natural nitrogenous fertilizers,
fresh cow manure depressed persistence of
B. bassiana at 25C and high rates of compost
were beneficial (Rosin et aI., 1996). Thus com-
bination with soil manuring in compost pel-
lets could be beneficial.
Grain on which the fungus is produced can
be used as formulated granules. Keller (1992)
drilled barley granules with sporulating B.
brongniartii into soil, 3-5 cm deep. Sporulating
fungus was clearly visible on the grain for 2-5
months after drilling. One year later, infection
of grubs was 98% in a meadow and 84% in an
orchard. Pest control in the orchard was suffi-
cient, a result clearly visible in tree health,
which was better than that achieved with che-
micals. Moorhouse (1990) obtained vine wee-
vil control effective for 16 weeks and partially
effective for 52 weeks with M. anisopliae on
millet. Unsporulated mycelium grown on ver-
miculite granules (lncitec Ltd, nutrient status
not given) had an inevitable delay until spor-
ulation made it readily infective. Sporulation
was best at -15 bar and very poor at 0 to
- 2 bar, but conidia survived in the soil no
better than conidia applied unformulated, the
best of ten combinations of soil conditions giv-
ing 62% survival after 100 days (Li and Hol-
dom, 1993). The fungistatic action of the
natural soil biota is important in limiting insect
pathogens in soil, as are temperature and
nutrients. Amendment of a sterilized palouse
silt loam with carbon and nitrogen, or com-
binations of both, led to vigorous growth of
B. bassiana, showing that sterile soil was not
inhibitory. However, the same amendments to
the same soil - not sterilized - decreased
survival time of conidia, probably due to
enhancement of the growth of fungistatic
organisms, notably the common Penicillium
urticae (Lingg and Donaldson, 1981). Formula-
tion of the juxtaposed amending additives in
granules with the insect pathogen may over-
come this problem by allowing concentrated
growth of the pathogen to fight the soil organ-
isms around the granules.
Formulation with clays protects insect
pathogens against fungistasis, as with other
beneficial organisms (sections 5.3.4 and
7.6.1). Milled attapulgite gave good survival
of M. anisopliae in sugarcane soil, resulting in
elevated conidial numbers above background
levels for 3 years, but which failed to control
larvae of the sugarcane soldier fly, Inopus
rubriceps (Samson et al., 1994). Studdert et al.
(1990) coated dry B. bassiana conidia with a
bentonite clay by mixing 1:3 by weight, moist-
ening a thin layer lightly with a water mist,
then drying. The clay increased the half-life of
conidia in both a sandy loam and a peat from
2-7 weeks to 7-12 weeks at 30C and from 12-
44 weeks to 20-64 weeks at 10 DC, at different
combinations of soil and moisture content.

Fargues et al. (1983) prolonged survival of
B. bassiana blastospores by clay coating. Rei-
singer et al. (1977) coated blastospores with an
equal weight of clay by mixing in water, cen-
trifuging and enclosing in a fine mesh bag. On
incubation in a greenhouse soil, the clay
reduced the conidial death rate by protecting
conidia from attack by bacteria and Amoeba.
Clarsol and the calcium form of montmorillo-
nite gave the best results among the clays
tested.
The above means of benefitting insect
pathogens can be combined in fabricated
granules; protective clays can be formulated
with other protective additives and with
nutrients. Prophylactic treatment should con-
trol the long-lived soil insects, which pick up
infection as they burrow, so more - but sma-
ller - granules should be the most effective.
Protection is difficult when fungistasis is most
severe, i.e. in waterlogged soil (Obar), when
an insect pathogen competes with fast-grow-
ing bacteria in conditions unfavourable to its
own growth. If possible, application should be
timed to avoid waterlogged conditions and
heavy rain, since the fungal pathogens grow
best in moderately dry soil (Studdert et al.,
1990; Li and Holdom, 1993), where they com-
pete mainly with other fungi and with preda-
tors of fungi.
Fabricated granules can also be designed to
improve pathogen survival during another
period of stress, drought. This is because sur-
vival of conidia decreases as soil dries above
-15 bar (Studdert et aI., 1990), when spore
death is probably due to physiological causes.
Granules containing the most drought-resist-
ant fungal life stage can be applied to dry soil
to await rain or, as soil dries, a granule can be
a haven in which the fungus can develop its
own drought resistance. Alginate pellets
enable many additives to be fabricated
together (Abd-el-Moity, 1986; sections 5.3.3
and 7.7.3d). Presoaking in PEG during
production speeds germination after water
imbibition (Knudsen et al., 1991) following
application to soil.
4.4.3 Baits and dusts 151
4.4.3 BAITS AND DUSTS
The above granules can be made more effec-
tive against insects that feed at the soil surface
by using attractive substrates to convert the
granules into baits. Either the fungus can be
grown on the substrate or spores can be
applied to the bait (pathway 3, Fig. 4.2). In
the laboratory, B. bassiana conidia on bran
killed grasshoppers at a similar rate to that
on fresh lettuce, but oil was superior to
water as the carrier (Inglis et al., 1996a). Bran
bait, with 4.4% conidia (2.0 x 1013 /ha) and 2%
molasses applied with a motorized blower to
fallow wheat fields with sparse clover
cover, infected 70% of the grasshopper
population, possibly aided by rain soon after
application. Prepared on site, the bait cont-
ained 21% water and was unsuitable for sto-
rage. Conidia did not adhere to the bran and -
in practice - were applied largely as a dust,
which may have infected many insects by
direct hits. A sticker might improve adhesion
to the bran and increase infection by oral con-
tact (Johnson and Goettel, 1993). Oil and / or
conidia did not alter palatability or effective-
ness of oat flake bait (Goettel et al., 1995). In
field cages on pasture grass, bran baited with
V. lecanii caused mortality similar to that
caused by aqueous conidial sprays on grass-
hoppers (Johnson et al., 1988). Extruded
maize-starch pesta pellets (Tables 6.8 and 6.9
in section 6.8) were highly palatable to locusts
(Schistocerca gregaria; Caudwell and Gate-
house, 1996).
Against foraging, nesting insects, baits were
in general inferior to dusts. The main route of
infection of fire ants with B. bassiana conidia is
oral and tarsal (Siebeneicher et al., 1992), but
Pereira and Stimac (1992) concluded from
infection experiments that reliance on trans-
mission within the nest is not a viable strat-
egy. Following work with laboratory colonies,
Stimac et al., 1993a, band Oi et al., 1994 found
that boiled rice bearing conidiating mycelium
scattered over the surface of nests in pastures
gave moderate control, as did a 10% conidial
152 Formulation of mycoinsecticides
powder in diatomaceous earth (Appendix
Table 1.2), blown by CO2 under pressure
through a tube pushed into the nests. To
reduce loading of nests, 90-95% diatomaceous
earth dusts were used, an important function
of the clay being to prevent spores sticking to
application iffiachinery. Use of a hydrophobic
carrier, fumed silica (TS-720, Appendix Table
1.2) greatly improved dispersion in the nest
and adherence to ant bodies, their mouth
parts frequently being clogged. These changes
progressively improved ant control, particu-
larly the silica, which itself gave considerable
control in a silica-only treatment, although no
treatment gave control adequate for practical
purposes. Deaths resulted from contact with
dust. The fungus did not spread in space or
time, mortality being limited by hygiene prac-
tices of the ants.
With leaf-cutting ants, incorporation of an
attractant base - dried orange - with M. ani-
sopliae conidia showed some potential in
masking a repellent action of the foreign fun-
gus (Blowers et al., 1992).
M. anisopliae conidial bait infected many
Macrotermes michaelseni workers and did not
cause avoidance in choice tests. Inoculation of
7 g dry conidia into mature mounds caused
80% mortality, 10 g mycelial fragments in
sawdust bait caused 60% mortality, or in 20 g
rice bait 58% mortality, 7 weeks after treat-
ment (Gitonga et al., 1996). Cornitermes cumu-
lans was controlled by applying 8 g of a
formulation of 25% spores of B. bassiana
(100% kill) and M. anisopliae (80%) in talc to
nests in the field. Control was no better with
formulations containing 20% spores in rice,
termite-nest soil or clay (Fernandes et al.,
1991). Microgranules would be easier to han-
dle than dusts and would reduce risk of inha-
lation by operators.
4.5 APPLICAnON OF FUNGI TO WATER
The only aquatic targets to date have been
mosquito larvae. All species breathe at the
water surface and many feed there, so formu-
lations of pathogens that infect on contact
should be designed to float.
B. bassiana conidia dusted onto the water
surface infected the perispiracular lobes at
the apex of the larval siphon, probably be-
cause the spores remained fixed in the oily
coating of the stigmatal plate and were pro-
tected from wash-off by folding of the lobes as
larvae submerged. There were enough nutri-
ents in the water to stimulate germination,
which made spores less infective. Formula-
tion in vegetable oil has the advantages of
preventing germination during storage and
floating after application to water. Oil
droplets accumulate on the stigmatal plates,
then enter the stigmatal trunks, where spores
germinate and penetrate the tracheal walls.
Formulation in a silicone oil (Dow Corning
550 Fluid) changed the infection site to the
cuticle of the mouthparts (Clark et al., 1968).
Formulation of M. anisopliae conidia in clays
or mineral and botanical carriers (listed in
Table 4.6) reduced virulence, probably by
impairing attachment of conidia around the
siphon. Two exceptions, Bentonite GO-II
and Thixcin R, improved virulence. Thixcin
R, being a dried modified castor oil derivat-
ive, probably has the lipophilic quality of an
oil, improving spread over the insect cuticle
(Daoust et al., 1982).
Culicinomyces clavosporus sporulates at
the water surface so spores must be allowed
to float. Unlike most other fungal insect
pathogens, its spores infect on ingestion.
Formulation by drying a mycelial pad and
grinding produced particles that floated and
sporulated in the surface-feeding zone of
Anopheles larvae. Drying in an antidesiccant
sucrose solution produced viable particles,
but a glucose solution did not (McDonald
et al., 1989). This inoculum is more virulent
than aqueous spore suspensions (Sweeney,
1989). Blastospores formulated in oil were
x4 more virulent than in water, probably
because oil carried them to the more suscept-
ible sites on the larval cuticle (Matewele,
1986).
 4.5 Application offungi to water 153
Table 4.5 Longest half-lives (LTso ) in six temperature bands taken from 58 sets of data in 39 research
papers on entomogenous Hyphomycete fungi

Range eC) LTso * Species, spore type, formulation, condition

Deep-freeze
-20 2.66 years VerticiIlium lecanii conidia in water + best additive, 10% skimmed
milk 1
-20 2.00 years Metarhizium anisopliae conidia unformulated at best humidity, 97%
RH 2
-70 0.50 years Culicinomyces clavosporus conidia in water3
Cold store
4 8.50 years Beauveria bassiana conidia in best dry clay carrier, attapulgite, at 0%
RH 4
4-10 7 years B. bassiana conidia pellets and powders
8 much> 2.44 years M. jlavoviride conidia in oil + silica gel, 48 h germination 6
8 much> 1.74 years B. bassiana conidia powder over CaCI/
Temperate room
17 much> 2.44 years M. flavoviride conidia in oil + silica gel, 48 h germination 6
19 > 2 years M. anisopliae conidia unformulated at best humidity, 97% RH 2
16,23 1.53 years M. anisopliae conidia in soil, moisture 25-50%, equilibrium ca 99%
RH 8
20 1.43 years B. bassiana conidia over CaCh powder 9

Tropical room or store

30 0.77 years B. bassiana conidia powder over CaCl/
30 0.684 years M. anisopliae conidia in soil moisture 25-50%, equilibrium, ca 99%
RH 8
25-37 0.50 years M. flavoviride conidia pre-dried to 5% moisture, stored with silica
gel lO
38 0.33 year M. flavoviride conidia pre-dried to 5% moisture stored in undried
oil, no silica gel l l
Tropical field storage
50 ca 90 days M. flavoviride most tolerant strain, dried, stored with silica gel 12
40 > 60 days M. flavoviride conidia in oil with silica gel 13
55 15 days M. flavoviride pre-dried to 5% moisture, stored in undried oil, no
silica gel ll
60 2h M. flavoviride conidia in oil with silica gel 13
70 1.5 h M. flavoviride conidia in oil with silica gel 13
80 1.0 h M. flavoviride conidia in oil with silica gel 13
Spray drying
50 ca 20 s Aschersonia aleyrodis pycnidiospores in water 14
55 10 s A. aleyrodis pycnidiospores in 0.05% aqueous Kelzan l4

References
1 Kanagaratnam, 1980 8 Milner and Lutton, 1976
2 Daoust and Roberts, 1983 9 Sandhu et aI., 1993
3 Sweeney, 1981 10 Moore et aI., 1996a
4 Ward,1984 11 Hedgecock et aI., 1995
5 Starcova and Weiser, 1992 12 Morley-Davies et al., 1995
6 Moore et aI., 1995 13 McClatchie et al., 1994
7 Clerk and Madelin, 1965 14 Fransen, 1991
* For spore batches with germination at day a (Go) substantially less than 100%, germination percentages after storage
(G s) were corrected by WaGs/Go.
154 Formulation of mycoinsecticides
Encapsulation of presporangia of Lageni-
dium giganteum in calcium alginate (sections
5.3.3,6.8, 7.7.3d) dramatically increased reten-
tion of infectivity to 75 days in storage at 15C
plus 25 days after application to water. Field
results with encapsulated oospores were
excellent (Axtell and Guzman, 1987).
either very dry (near 0% RH) or very moist
(section 4.6.3). Dry storage is the best option
for products to apply to foliage (Fig. 4.2,
industrial flow lines 1 and 2), and moist sto-
rage is good for products intended for soil
(line 3). Shelf-life is universally dependent
on temperature.

4.6 STORAGE 4.6.1 STORAGE TEMPERATURE

Storage options are second only to mode of
application in decision-making about indus-
trial flow patterns for fungal products (Fig.
4.2). To be competitive with other pest-control
products, a fungus needs a shelf-life of at least
1.5 years. Above OC, fungi survive longest
As well as depending on temperature, shelf-
life varies widely in response to many factors,
such as fungal species and life stage, water
availability, production and harvest method.
Heckley (1978) has reviewed extensive data
on non-entomogenous fungi. The longest

Table 4.6 Effect of dry formulation diluents at 0% RH on the survival (%) over 1 year of two batches of
Metarhizium al1isopliae conidia, one grown on rice and the other on YpSs agar, compared with
unformulated dry conidia (denominator) (Daoust et al., 1983)

Temperature
Type Diluent', t 20C

Attapulgite Attaclay 24/48 63/71, 73/53 32/36,5/0

Attaclay X-250 66/71,92/53 55/36,42/0
Montmorillonite Bentonite GO-ll (Bentone 38) 79/71,70/53 16/36,0/0
Pyrophyllite Pyrax ABB 76/71,69/53 38/36, 1/0
Kaolinite Continental clay 86/71,71/53 29/36,3/0
Barnet clay 87/71,62/53 42/36,3/0
Tenn K clay 84/71,73/53 40/36, 18/0
Rex clay 84/71, 72/53 25/36,3/0
Jordan clay 91/71 32/36
Diatomaceous earth Celite 209 77/71,84/53 50/36,8/0
Talc Vertal-5 87/71 26/36
Vertal-15 51/71 7/36
Illite Vermiculite 35/60 65/71,54/53 20/36,4/0
Vermiculite 8/35 57/71 19/36
Organic powder Thixcin R+ 74/71 31/36
Botanical Corn cob 14/40 (not dried) 67/71,8/53 15/36,0/0
Corn cob (dried) 62/71 1/36
Wood chips 64/71 2/36
Desiccants CaCb 41/66 2/3
CaCI 2 + kaolinite clay 49/66 9/3
Silica 45/66 2/3
Silica + kaolinite clay 62/66 12/3
Concentration of conidia was 10% for granules and powders.
t Numbers show particle size, passing US standard sieve No. 24, retained by No. 48.
:I: Modified, dried, castor oil derivative.
60 C. 2 h.
 4.6.2 Survival during drying and improvement by formulation
recorded survival times of insect pathogenic
species are summarized in Table 4.5. Of these,
Beauveria and Metarhizium conidia appeared
to survive longest at most temperatures, but
this may be an artefact of the focus of work on
these two genera. It should be possible to
improve their products by further study of
storage conditions; current achievements
with them could serve as targets for develop-
ment of other genera. For example, a shelf-life
of 2 years at temperate room temperature has
been claimed for the M. anisopliae product Bio-
Blast (A. C. Rath, EcoScience Corporation,
Worcester, Massachusetts, personal commun-
ication, 1995).
Shelf-life of a product also depends on the
carrier medium and on formulation, which
will be described in the following sections.
One of the best options is to store conidia in
a dry state; however, the spores have first to
survive the drying process.
4.6.2 SURVIVAL DURING DRYING AND
IMPROVEMENT BY FORMULAnON
In nature, Hyphomycete fungi survive dry
periods as mycelium, blastospores (= hyphal
bodies) and conidia, either encased in insect
cadavers or as aerial conidia. The dormancy
of conidia is exogenous, not constitutive
(section 4.6.3), so the key to prolonging their
survival is to stop germination and, as with
the preservation of mycelium, to reduce
metabolism as much as possible. This is most
easily done by drying. In nature, aerial
conidia may dry rapidly, but drying of the
fungus in insect cadavers is obviously relat-
ively slow.
Conidia harvested dry by aspiration off
solid media survive well, as do conidia har-
vested in water and air-dried. Bartlett and
Jaronski (1988) and Bailey and Rath (1994)
consider that rapid drying of M. anisopliae
conidia at ~ 30 - 35C is critical to preserva-
tion of spore viability. However, some evi-
dence suggests that rapid drying at <30C is
better (D. Moore, International Institute for
155
Biological Control, Ascot, personal commun-
ication). End moisture content is critical and is
probably the cause of much variation ascribed
to storage conditions and formulation in sec-
tions 4.6.3 to 4.6.5. Limited data show that
moisture contents affect spores of non-ento-
mogenous organisms to different degrees,
and the balance of opinion is that with most
organisms the lowest moisture levels give best
survival (Rey, 1975; Heckley, 1978). This may
not be so with blastospores of Paecilomyces
fumosoroseus; spores at 2-5% moisture after
drying at > 50% RH and 22 DC survived in
store longer than those at 2.5-1.5% moisture
and dried at <50% RH Oackson et al., 1995).
Drying of conidia produced on solid media
and harvested wet can be accelerated by
freeze-drying, vacuum-drying and desiccants
(sections 4.6.3 to 4.6.5). Unformulated B. bassi-
ana and M. anisopliae survived freeze-drying
(48-95% survival) better than vacuum drying
for 66 hat 22C, but there was great variation
between strains, possibly due to variation in
final moisture content. However, few blastos-
pores and few conidia of B. bassiana produced
in submerged liquid culture survived vacuum
drying Gimenez, 1989).
Additives, notably sugar solutions, improve
survival during drying. They serve the addi-
tional formulation purpose of carriers in tech-
nical concentrates and wettable powders. They
act by reducing disruption of membranes (sec-
tion 7.6.2) and probably by modifying the final
moisture content of spores. Maltose, glucose
and sucrose protected a thin entomophthoran-
type mycelial mat during air-drying (McCabe
and Soper, 1985; Pereira and Roberts, 1990).
Blastospores of V. lecanii survived freeze-
drying best in additive mixtures containing
sugars, e.g. 10% honey (55% survival) and
7.5% glucose (54%) (among other additives
listed in Table 4.12 in section 4.6.6 below;
Kanagaratnam, 1980). Blastospores of P. fumo-
soroseus survived (79-86%) freeze-drying in
10% aqueous lactose + 1% bovine serum albu-
min as well as air-drying mixed with 5% (w Iv)
diatomaceous earth Oackson et al., 1995, 1997).
156 Formulation of mycoinsecticides
Lactose, other sugars and skimmed milk are
good for freezing-drying organisms other than
fungi (Heckley, 1978).
High temperatures in another rapid drying
method, spray-drying, may be prohibitive
(Table 4.5), although conidia of Metarhizium
flavoviride successfully pass through exhaust-
nozzle sprayers fitted to the exhausts of trucks.
Enzyme (protein) stabilizers (Kelzan, but not
Dextran) gave some protection to pycnidio-
spores of Aschersonia aleyrodis (Fransen, 1991).
4.6.3 UNFORMULATED DRY CONIDIA
Dry, dusty conidia, adapted for aerial distribu-
tion, also have the role of survival in nature.
Evidence of this is the survival of M. flavoviride
conidia in grasshopper cadavers (section
4.3.1). Conidia are ideal for long-term storage;
study in their unformulated state reveals the
best basic storage conditions.
Humidity in equilibrium with conidia is
critical. Longest survival amongst fungi in
general falls into four types: (1) at high
humidity; (2) intermediate humidity; (3) low;
or (4) at both high and low humidity. Insect
pathogens occur in types 3 and 4. In type 3,
Beauveria and Paecilomyces survive longest at
extreme low humidity (nominal 0% RH),
although the highest humidity studied with
Paecilomyces was 75% RH, so it is possible that
survival of this genus may be equally as long
at 100% RH. In type 4, M. anisopliae survives
much better at both 0 and 100% RH than at
intermediate humidities, while 100% RH is
somewhat better than 0% RH. Differences
can be great, of many months, at the same
temperature (Clerk and Madelin, 1965;
Daoust and Roberts, 1983; Sandhu et al.,
1993; Jin et al., 1993). The moisture content of
conidia depends on humidity, equilibria after
7 days for M. flavoviride being 5% moisture at
5% RH; 9.4% at 33 % RH; and 19.2% at 76%
RH (Hedgecock et al., 1995).
In dry conditions, conidia survive by redu-
cing metabolism: the greater the reduction, the
longer the survival. This explains the long
shelf-life of conidia at 0% RH. Pre-drying con-
idia at harvest increased survival time (Table
4.8), presumably because the earlier drying
reduced metabolism sooner. Mixing silica gel
pellets with the conidia also increased survi-
val (Table 4.8), presumably by maintaining the
dry condition during storage. In another
study, exclusion of Oz improved survival, pre-
sumably further reducing metabolism. In air
(ca 20% Oz), no M. anisopliae conidia survived
2 months, but with no Oz, 56% survived at
25C and 26% at 37 cC (Jin et al., 1993).
In moist air, in contrast, conidia continued to
use Oz while no germination was observed in
the storage tubes, even at 96-98% RH (Daoust
and Roberts, 1983). Dormancy is maintained
by the nutritional environment, possibly
enough nutrients being present to help sup-
port metabolism, without the nutrients and
stimulants required for germination. When
Oz was removed, the germination of M. aniso-
pliae conidia at high humidity was reduced to
7% in 42 days at 22 cC (Jin et al., 1993).
Exclusion of light during storage increased
survival (Clerk and Madelin, 1965; Sandhu et
al., 1993).
Data on survival of unformulated conidia in
different conditions provide a baseline against
which formulation to improve shelf-life can
be measured.
4.6.4 FORMULATED DUSTS, GRANULES AND
WETTABLE POWDERS
For storage at 0% RH, addition of sachets of
silica gel pellets can be regarded as a break-
through, producing a x 3.3 improvement in
Metarhizium conidial survival (Table 4.8).
Easy on-site removal of pellets gave a mini-
mal-weight technical conidia powder with no
additives for tank mixing in oil for CDA
spraying (section 4.3.1). Colourless pellets
were used because indicator pellets harmed
the spores, probably due to the added chemi-
cals (N. E. Jenkins, International Institute for
Biological Control, Ascot, personal communi-
cation). However, powdered desiccants
 4.6.4 Formulated dusts, granules and wettable powders 157

Table 4.7 Production of a wettable powder from blastospores (adapted from Blachere et aI., 1973;
Lisansky et aI., 1993)

Ingredient Percentage (w/w) Function

Centrifuged spores 22 Pathogen
Clarcel Flo 2.2 Free-flow agent
Liquid paraffin 19.9 Anti-oxidant
Labrafil 2.1 Anti-oxidant
Sucrose 22 Osmotic protectant
Na glutamate 2 Osmotic protectant
Cereal flour 25.8 Carrier, nutrient
Bevaloid 116 2 Dispersant
Surfynol 104S 2 Wetting agent

Method
1 Ferment for 5 days in aerated liquid medium
2 Chill to 5-1OC
3 Centrifuge c:: 10 000 g, < 35 DC) to a paste containing about 22% dry solids with agitation of feed
4 Sample for viable spore counts and other tests
5 Add 1:1 dry to wet weight silica powder (Clarcel Flo, Wessalon S) to the paste and coarsely mix in a
mixer (e.g. Z Blade)
6 Per kg powder add 250 ml liquid paraffin containing 10% Labrafil (polyoxyethylene glyceryl
monooleate) and 250 ml aqueous sucrose (80%) + sodium glutamate (2%) and remix to a friable
powder at < 35 DC
7 Dry in thin layers in a ventilated drying cabinet at S 25 DC to S 7% moisture
8 Sample for moisture content and viable spore count tests
9 Add sufficient fungal nutrient, e.g. cereal flour, to lower viable spore count to the level chosen for
marketing
10 Bevaloid 116 (2%) can be considered as dispersant and Surfynol 104S (2%) as wetting agent, but not
before testing their effect on shelf-life
11 Dispense into containers

mixed with conidia did not improve survival,
except possibly a mixture of powdered silica
and kaolinite clay (last group in Table 4.6),
although Gasil GM2 is a possible successful
alternative (section 4.6.5).
Clays generally improved survival at 0%
RH to a smaller extent (Table 4.6). Their
surface pH varied between 4.0 and 9.2, but
pH was not correlated with survival (Daoust
et al., 1983). Neither was pH between 5.0
and 7.7 correlated with survival of B. bassiana
conidia in sterile and non-sterile moist soil
at 25-75% saturation (Lingg and Donaldson,
1981) at an equilibrium humidity of < 98%
RH (Table 4.2 in section 4.2 above). Ward
(1984) reviewed the physico-chemical pro-
perties of clays, especially their buffering
capacities. He believed that their ability to
control the pH of a formulation, to buffer its
microclimate and to absorb suppressive meta-
bolites improved the survival of spores in
storage. No correlations with these factors
could be seen in storage at 0% RH (Table
4.6); correlations may occur at higher humid-
ities.
Corn cob did not improve survival, even
when pre-dried to bring the conidia rapidly
into equilibrium with 0% RH (Table 4.6).
At 0-10% RH, a beneficial effect of the
absence of Oz is achieved by storage under
vacuum or after flushing with N z. It is main-
tained by inclusion in the package of a sachet
of an Oz absorbent, Ageless type 2-300
(fine iron filings, Mitsubishi), which can be
158 Formulation of mycoinsecticides
used with a bag of Drierite (anhydrous cal-
cium sulphate), a desiccant with the advan-
tage of not releasing absorbed water at high
ambient temperatures. A suitable flexible
packaging material is a polyethylene-alumi-
nium foil-polyethylene laminate, 0.003-0.007
inches thick, which is impermeable to gases
(transmission < 0.005ml O 2 and H 2 0/
100 in 2 / day) Gin et al., 1993).
At the opposite end of the humidity scale,
fungi of types 1 and 4 (section 4.6.3) have long
shelf-lives at 100% RH. Resting azygospores
of Conidiobolus obscurus broke their constitut-
ive dormancy in 3 months at 4C and 100%
RH, and remained 80% viable until 10 months
in the clays Clarsol STF and BBC, demonstrat-
ing similar survival to unformulated spores.
Survival was reduced in Cecaperl AM and
Clarcel F (Perry and Latge, 1982). However,
sterility is broken at harvest and the risk of
contaminants makes routine storage at 100%
RH impractical.
With conidia in granules, sterility can be
maintained by production in end-use contain-
ers (section 4.7.1). These conidia are not con-
stitutively dormant and are grown on solid
media such as cereal grains, which serve as
carriers. Conidial viability of B. brongniartii
was maintained at 98% for 2 years at 2C
(Aregger, 1992). At 100% RH in the presence
of O 2 , M. anisopliae conidia retained 84%
germination at room temperature Gin et al.,
1993). Suitable heat-sealable, autoclavable
packaging should be relatively impermeable
to water loss and freely permeable to O 2 and
CO2, e.g. high-density polyethylene (0.3 g H 2 0
10ss/24 h/100 in 2 /mil thickness at 35C, 90%
RH; 600 ml O 2 and 4500 ml CO2/24 h/100 in 2/
mil at 25C, 50% RH; 1 mil = 0.001 inch).
Blastospores, produced by deep liquid fer-
mentation, are more delicate than aerially pro-
duced conidia. Blastospores survived well in
dry powders obtained by the sophisticated
Blachere process (Table 4.7). In this, the
sodium glutamate extended shelf-life rather
than protecting the spores during the drying
process. Survival of B. brongniartii (2-7%
moisture) was 15% in 4 weeks, 0 in 7 weeks
at 23C, but 16% in 8 months at 4C. Drying
at 4 C to 6% moisture then storage under
vacuum at both 23 and 4C gave excellent
survival for 8 months (Blachere et al., 1973),
also with M. anisopliae for 1 year at 4C
(Andersch, 1992). At 6% moisture in air, sur-
vival of T. cylindrosporum in powder produced
by this process (Table 4.7) was good: 47-80%
in 6-8 weeks (d. 71% in water) at 25C and
60% in 5 months at 4C (Matewele, 1986). At
25C, survival was improved from 3 to 59% in
24 weeks by a supplement of compatible salts
(0.1 MMgS04, KH2P04, CaCh and NH 4S04)
made to the ingredients shown in Table 4.7,

Table 4.8 Effect of pre-drying conidia of Metarhizium flavoviride on survival when stored as a powder with
and without dried silica gel pellets (Moore et aI., 1996a)

Survival (%)
Moisture Weeks in Temperature
Conidia (%) No silica gel With silica gel store* (0C)

Undried 44.8 59 11 10-14

Pre-dried 4.3 93 11 10-14
Pre-dried 5.0 95 97 18 10-12
Undried 44.8 15 11 28-32
Pre-dried 4.3 4 11 28-32
Undried 44.0 0 51 14 28-32
Pre-dried 7.5 23 76 14 28-32
Pre-dried 5.0 9 42 18 25-37
* Three different batches of conidia were used as indicated by the storage times.

but reduced by supplementation or replace-
ment with peat, clay, powdered cellulose, talc
(magnesium silicate), sodium ascorbate (anti-
oxidant; see also sections 4.6.5 and 6), milk
powder, proline, malt extract, glucose or
molasses. Few blastospores of P fumosoroseus
survived drying on silica gel, sand or diato-
maceous earth and storage at 20-23 C for 24 h
(Cliquet and Jackson, 1997). However, modi-
fication of the production method gave blas-
tospores tolerant of drying (section 4.7.2). At
4C after air-drying, <25% survived 7 months,
improved to 60% by freeze-drying and stor-
age under vacuum. The spores were still
potent in bioassays (Jackson et al., 1997).
4.6.5 STORAGE OF CONIDIA IN OIL
Because of their lipophilic surfaces, dry, dusty
conidia mix readily in oil. However, conidia
of M. flavoviride freshly harvested by suction
and not pre-dried did not survive as well in
undried oil as in an unformulated powder.
Survivals of 1, 1 and 0% in oil compare with
59, 15 and 0%, respectively, taken from com-
parable batches of conidial powder (Table 4.8
in section 4.6.4).
Data from three studies on conidia stored in
oil have been assembled in Table 4.9. They
span temperatures from deep-freeze to tropi-
cal storage. Survivals of undried spores are
compared in consecutive lines of the table
with survivals of pre-dried spores from the
same batches at the same temperatures. As
with conidia stored as unformulated powder
(Table 4.8), pre-drying dramatically improved
survival in oil at all temperatures above freez-
ing in all but one spore batch (Table 4.9). Silica
gel pellets added to undried conidia in oil also
dramatically improved storage in one of three
batches. The combined effect of pre-drying
and storing the same batch with desiccant
pellets sometimes exceeded the added effects
of either treatment alone (Table 4.9).
More pre-dried conidia germinated after
storage as a dry powder with silica gel pellets
than as an oil suspension (Morley-Davies et
4.6.5 Storage of conidia in oil 159
al., 1995). For example, after 13 weeks' storage
at six temperatures between -10 and 50C,
the difference ranged between 3 and 12%
germination. The opposite effect was found
by Moore et al. (1996a) in their only batch of
pre-dried conidia stored both as a powder
(76% germination, Table 4.8) and in oil (91%
germination, Table 4.9). This effect was prob-
ably due to uptake of moisture from air by the
conidial powder because the containers used
were inadequately airtight (D. Moore, perso-
nal communication, 1996). Crude petroleum
extracts were successful as tank mixes but
not for storing B. bassiana conidia (Yin, 1981).
Thus the best procedure for oil sprays is to
store conidia as dry powder with silica gel,
then mix with the oil as near as possible to
spray time. Conidia readily absorb moisture
on reaching locust cuticle (Moore and Caud-
well, 1997). While pre-drying conidia and
keeping them dry is the key to long shelf-life
of M. flavoviride, their life as powder can be
further extended by low temperature, from
42% germination after 4.5 months at 25-37C
to 97% in cool store at 10-12 C (Table 4.8), or
for non-pre-dried conidia in oil with silica gel,
from 79% after 37 months at 17C to 89% at
8C (Moore et al., 1995). Surviving conidia
retained full virulence in insects (Moore et
al., 1995, 1996a). A silica zerogel powder,
Gasil GM2, is a promising alternative desic-
cant that could remain in the formulation dur-
ing ULV spraying (N. E. Jenkins, personal
communication).
Silica gel acts not only by drying, but prob-
ably also by absorbing metabolites. It should
also prevent growth of contaminants, such as
those found in some undried oils by Stathers
et al. (1993), and the germination of T. cylin-
drosporum conidia, oil being an important car-
bon source for growth of this species
(Matewele, 1986).
Very few oils have been tested in the pre-
sence of dried silica gel. With M. flavoviride,
there were no consistent differences between
diesel oil, odourless kerosene, Shellsol K,
aviation fuel, groundnut oil or soya oil.
160 Formulation of mycoinsecticides
Table 4.9 Effect of pre-drying conidia of Metarhizium flavoviride on survival when stored in oil formulated
with and without dried silica gel pellets

Survival (%)
Moisture Weeks in Temperature
Conidia (%) No Si gel With Si gel store (0C) Reference

Undried 19 75 * 18 -15 1
Pre-dried 5.0 73 18 -15 1
Undried 4 93 51 8 2
Undried 3.0 1.0 10 10-12 3
Pre-dried 89 87 10 10-12 3
Undried 44.8 1 41 11 10-14 3
Pre-dried 4.3 94 93 11 10-14 3
Undried 19.2 80 18 12 1
Pre-dried 5.0 75 18 12 1
Undried 0 84 51 17 2
Undried 19.2 50 18 25 1
Pre-dried 5.0 78 18 25 1
Undried 0 1 10 26-30 3
Pre-dried 53 72 10 26-30 3
Undried 44.0 0 9 14 28-32 3
Pre-dried 7.5 67 91 14 28-32 3
Undried 44.8 1 14 11 28-32 3
Pre-dried 4.3 85 84 11 28-32 3
Undried 19.2 23 2 38 1
Pre-dried 5.0 85 2 38 1
Undried 19.2 2 2 48 1
Pre-dried 5.0 60 2 48 1
Undried 19.2 1 2 55 1
Pre-dried 5.0 50 2 55 1

References
1 Hedgecock et al., 1995
2 Moore et a!., 1995
3 Moore et al., 1996a; light mineral oil or 1:1 mixtures with soya or groundnut oils were used, 1 g dried
non- indicating silica gel in 5 or 15 ml oil and (when specified) 2 x 109 conidia/ml or 1 g conidia/15 ml
* Gaps show where data were not determined.

Anti-oxidants (0.01 % butylated hydro- Without pre-drying conidia and adding

xyanisole+0.01% butylated hydroxytoluene)
improved conidial survival in groundnut oil
and in soya oil by 23-26% (Moore et al., 1995,
1996a). Moore and Caudwell (1997) discuss
oxidation in oils and anti-oxidants, and sug-
gest precautions such as avoiding high tem-
perature, exposure to light and contact with
air and O 2 , as well as maintaining strict clean-
liness by removal of traces of copper, iron and
oxidized fats, which act as catalysts. See also
sections 4.6.4 and 4.6.6.
silica gel, oil was not a good storage
medium, survival being low even during 2-9
weeks (Table 4.10) and much inferior to that
of dry, unformulated conidia (Table 4.8).
However, data on undried oils may indicate
the best oils to use in a fully desiccated
regime because adverse factors may not have
been curbed by desiccation. For ULV sprays
of chemical insecticides, oils with a high
aromatic content are not used so as to avoid
phytoxicity (Wrigley, 1973)-a property that
 4.6.5 Storage of conidia in oil 161

Table 4.10 Effect of storage in undried oil without silica gel on germination (%)* of spores of Metarhizium
spp. and Beauveria bassiana

Weeks at 4-8C Weeks at 17-25C

Oil 6-9 18 2 6-9 12-16 18
Vegetable
Almond t 87 0
Coconut t 10 4
Coconut! 98 0
Coconut: cottonseed 1:1 t 44 0
Corn t 100 0
Cottonseed t 79 0
Cottonseed (Sigma) 28 12 2 4
Cottonseed (Zimbabwe) 1 0 0 0
Linseed t 93 4
Neem 0 0 0
Olivet 53 31
Olive (Sigma) 5 7 5 4
Palm 0 0 0 0
Groundnutt 69 18
Groundnut (Benin, local market) 60 20 4 6
Rapeseed 70 0 0 0
Codacide (Rape + 5% emulsifier) 71 0 0 0
Soya t 44 0
Soya (Sigma, expt 1) 75 34 47 28
Soya (Sigma, expt 2) 100 ca 68
Sunflower' 46 22
Sunflowe~ 24--64 14-50 1-12 0-3
Wheatgerm t 57 37
Wheatgerm 0 0 0 0
Animal
Cod liver t 0 0
Cod liver 12 2 6 2
Mineral
Kerosene (Benin, experiment 1) 84 44 32 12
Kerosene (Benin, experiment 2) ea 79 ea 79
Shellsol 2 0 0 0
Shellsol K** 81 33
Liquid paraffin' tt 2 0 0 0
Mineral t 65 11
Naphthol, heavy aromatic t. II 69
Paraffin Oil~ 13-85 3-22 6-18 0-12
T-400-100 t." 0 0
Other
I'-Butyrolactone t 27 0
Glycerol (100%)t 0 0
Lactic acid t 0 0
Lactic acid~ 12-15 3-18 3-18 0
Linoleic acid t 93 0
Molasses 0 0 0
Propylene glycol t 6 0
* Germination after storage (G,) corrected for germination on day 0 (Go) in the different conidia batches by 100 G,jG o,
I M, anisopliae conidia (Daoust el al., 1983).
1 B. bassiana conidia (Prior el al., 1988).
M. flavoviride conidia (Slathers el al., 1993).
~ B. bassiana blastospores (Kleespies and Zimmermann, 1994).
** M.flavoviride conidia (Moore el al., 1995).
II Mixture of alkanes with> 12 carbon atoms/molecule.
11 Petroleum-based oil.
162 Formulation of mycoinsecticides
may also damage conidia. For conidial storage,
mineral oils collectively were similar to vege-
table oils, some of which have the disadvant-
ages of rancidity and solidification over long
periods at high ambient temperatures. No oil
was consistently best in all studies (Table 4.10).
Three of the best were included in a thorough
study: kerosene was slightly better than soya
oil and both were much better than groundnut
oil (Stathers et al., 1993). Certain fatty acids
from rancid oils inhibit germination of M.
fiavoviride conidia (Barnes and Moore, 1997).
Vegetable oils vary, being extracted from dif-
ferent plants, which in turn vary according to
variety, agronomy, climate and processing.
Thus in general, mineral oils are preferable to
vegetable oils.
The reasons for poor survival in undried
oil, apart from insufficient desiccation, are
usually unknown. Neem oil is pesticidal and
contains fungicidal materials. Cotton seed oils
may contain gossypol, which may be harmful.
Different specimens of other oils sometimes
gave very different results, e.g. wheatgerm
oil (Table 4.10), suggesting the presence of a
toxic substance in one specimen. Viscosity per
se in the range of 45-150 centipoises (Daoust et
al., 1983) and the degree of purification
(Stathers et al., 1993) had no effect. Addition
of an emulsifier did not reduce survival over 2
weeks (Table 4.10).
4.6.6 STORAGE IN WATER
Unlike beneficial organisms for application to
soil (Chapter 7), storage of entomopathogenic
fungi in water is not a preferred method for
industrial products because of the severity of
problems incurred. These include contamina-
tion, spore germination, dehabilitating meta-
bolism of spores, release of metabolites,
leaching of nutrients, an adverse effect of
high concentration of spores, and the need to
wet hydrophobic conidia. Such problems
caused most storage experiments to be termi-
nated after 4 months or less (Tables 4.11, 4.12
and 4.13).
4.6.6a Storage in pure water Survival in water
varies widely with fungal species. Beauveria
and Metarhizium conidia are the longest lived
with or without a little wetting agent, e.g. 80-
96% germination after 16-52 weeks at 2-10 C,
and 50-96% after 12-18 weeks at 15-25C
(Walstad et al., 1970; Gillespie, 1984; Jimenez,
1989; Moorhouse, 1990). Germination of T.
cylindrosporum conidia fell to 70% after 20
weeks at 25C (Table 4.11), and of V lecanii
blastospores to 50% after only 8 days at 2C
(Table 4.12). Thus survival of unformulated
spores in water is poorer than in the dry con-
dition (Table 4.5 in section 4.5 above).
Survival of spores in water is probably
linked to metabolic rate, germination and pos-
sibly anaerobiosis. The reduction in survival
of conidia with increasing temperature is
probably related to increased respiration and
metabolic activity, with consequent depletion
of endogenous reserves. Dillon and Charnley
(1986) reported that M. anisopliae conidia
would not swell or produce germ tubes in
distilled water 40 h) unless an exogenous
nutrient source was available. Conidial germi-
nation was, however, noted by Daoust et al.
(1983) and of T. cylindrosporum conidia by
Matewele (1986), during prolonged storage
of conidia in water. This may have been
induced by endogenous nutrients leaching
from some of the conidia. The limited nutrient
levels prevent further growth, but death and
lysis of the germ tubes could release nutrients
and this might support additional germina-
tion.
At 100% RH, 25C, depletion of O 2 killed
M. anisopliae conidia in 2 months (Jin et al.,
1993), and presumably conidia would also
die in water. In some studies, bottle caps
were screwed down hard (e.g. Daoust et al.,
1983) and in others access of atmospheric O 2
was not described, so possibly some survival
times reported for long storage may have
been shortened by anaerobiosis.
4.6.6b Effect of additives on survival in water
Apart from depressing spore germination
 4.6.6 Storage in water 163
Table 4.11 Effect of additives in water on conidia of Tolypocladium cylindrosporum at 25C at an unstated
spore concentration. At 4C, only 60% glycerol was substantially better than at 25C (Matewele, 1986)

Germination (%)

Additives in water 24h 4 weeks 6 weeks 10 weeks 20 weeks 24 weeks

Water 93 89 88 74 70 germ
Buffer, pH 5 (tris) 77 62 56 56
KCl5-20% 85 77 64 29
Glycerol 60%, 25C 95 58 55 51 19
Glycerol 60%, 4C 91 83 82 80 58 28
Glycerol 30%, 25C 89 65* 64* 54* cont t cont t
Glycerol 10%, 25C 73 69* 67* cant cant cant
KCl 20% + glycerol 10% 85 72 68 58
KCl 20% + sucrose 10% 93 85 54 24
NaCl5% 47 42 21
NaCl20% 37 8
NaC120% + sucrose 10% 68 22
NaCl 20% + glycerol 10% 62 18
Sodium silicate 5% 62 32 18
Sodium silicate 20% 32 16 7
Sucrose 20-40% 77 71* 68* 69* cont cont
Vegetable oil, neat 78 50 germ+ germ germ germ+
Mineral oil, neat 75 18 germ germ germ germ
Linoleic acid 35 5 6
Tributyl citrate 26 6 5
Dimethyl sulphoxide 10% 64 58* 43* cont cont cont
Dimethyl sulphoxide 20% 19 10
* Low to moderate bacterial contamination.
t cont - contaminated.
t germ - germinated.

Table 4.12 Effect of additives in water on survival (LT50) of blastospores of Verticillium lecanii at a dilution
of ca 50:1 spore pellet: additive solution (Kanagaratnam, 1980)

LT so days (fiducial limits)

Additives in water rc
Water (distilled) 8 (7, 8) 382 (216, 676)
Sodium glutamate 5% 39 (30,51) 50 (16, 155)
Glucose 7.5% in nutrient broth 32 (21, 46) 64 (43,96)
Honey 10% 29 (12,74) 26 (9,75)
Lactose 5% in 10% glycerol 26 (20, 34) 82 (61, 111)
Gelatin 3% + sucrose 3% 25 (20,31) 428 (367, 500)
Peptone 7% + sucrose 7% 21 (16,28) 27 (20, 36)
Glycerol 10% 21 (11,38) 16 (7, 36)
Hank's salt solution 15 (6,36) 1 (0,36)
Sabouraud broth, fresh 13 (5,39) 29 (19,42)
Skimmed milk 10% 13 (10, 16) 970 (668, 1409)
KH2 P04 , pH 7.2, 42 mg/l 12 (5,32) 107 (85, 134)
Inositol 5% 12 (9, 17) 36 (26, 50)
Sabouraud broth, spent 9 (8, 11) 6 (2, 16)
Horse serum 7 (1, 16) 129 (111, 149)
164 Formulation of mycoinsecticides
Table 4.13 Effect of additives in water on survival and virulence of blastospores of Metarhizium anisopliae
(Kleespies and Zimmermann, 1994)

Survival (%) after

Additive 18 weeks at 6 weeks at 4 days at Virulence t

40C 20C* 35C

Deionized water 19 44
Used medium 8 0
Ringer's solution 25-100% 64 14 10
Glycerol 10-25% 51 3 37
Sorbitol 100% (Karion F) 12 9
PEG 200, 10-100% 15 14 0 +
Maltose 10% 3 1
Hydroxyethyl starch 10% 81 33 0
Hydroxyethyl starch 10% + lecithin 0.05% 14 0 4
Tween 80 10% 9 0
Sodium alginate 1% (Manucol DH) 29 0 4
Lecithin IV-S and II-S, 0.05-0.10% 0-2 0 3-8
L- and L(+)-Ascorbic acid 0.05-0.10% 0-4 0 7
Q- Tocopherol 0.1 % 5 0 19
Paraffin oil 32 0 18
* All values differ significantly from those in deionized water, except PEG, starch + lecithin and sorbitol at 4 'C and
Ringer's at 20 'c.
t Locust LTso significantly higher than that with fresh blastospores, -; lower, +; difference not significant, =.

and contaminants, improvement of spore Various materials have been added in

survival by additives - in comparison with
deionized water - depended on fungus
species and temperature. Certain additives
improved survival of V lecanii and M. aniso-
pliae at 2-4C, but not at -20 or +20C
(Tables 4.12 and 4.13), nor with T. cylindros-
porum at any temperature (Table 4.11).
Germination and contamination of T. cylin-
drosporum were prevented by acidifying to
pH 5 or adding a high concentration of gly-
cerol (60%) or KCl (5-20%) (Table 4.11). In
comparison with pure water, glycerol also
improved survival of V lecanii and M. aniso-
pliae at 2-4 C (Tables 4.12 and 4.13). It may act
by stabilizing proteins, but an attempt to stab-
ilize enzymes by binding 'inert' proteins in
horse serum did not improve survival of V
lecanii (Table 4.12). Dilution of the stored sus-
pensions for spray application lowers the con-
centrations of additives to levels that would
not curb spore germination (see also sections
3.3.3b, 7.6.3).
attempts to improve the stability of spores.
Sodium glutamate was the most beneficial
with blastospores of V lecanii, extending the
LTso at 2C from 8 to 39 days in water
(Table 4.12). It also extended the half-life of
dry fungal spores in wettable powder (section
4.6.4). In water, sugars alone or in natural
products, or in combination with materials
such as glycerol, were nearly as beneficial to
V lecanii (Table 4.12) but not to T. cylindros-
porum, with which sucrose encouraged
bacterial contamination unless checked by
combination with KCI (Table 4.11). With M.
anisopliae, although maltose, sorbitol and PEG
were not beneficial, starch was the best pro-
tectant. Starch, Ringer's solution and glycerol
improved survival at 4C in comparison with
deionized water, but not at 20C (Table 4.13).
This species has a relatively high temperature
range, so it is possible that these additives
were giving some protection against cold
damage.
 4.6.7 Effect of storage on speed of germination and on virulence 165
A cell-wall protectant, the phospholipid of concentration that would be necessary for
lecithin, plus two anti-oxidants, ascorbic acid economic commercial storage.
and ex-tocopherol, were not beneficial, but
may have been deleterious (Table 4.13), as 4.6.6d Survival of frozen spores Below OC,
was sodium ascorbate in the preparation of a the critical factor is survival of the freeze-
wettable powder (section 4.6.4), in contrast to thaw process. Most entomopathogenic fungi
the two anti-oxidants tried with vegetable oils survive, except for some Entomophthorales.
(section 4.6.5). . Survival time spans years (Table 4.5 in section
A little surfactant is essential to wet and 4.5 above). The LT50 of V lecanii blastospores
disperse hydrophobic spores. M. anisopliae at -20C was 382 days in distilled water,
survived well in Triton X-100. In a 0.01 % solu- although two formulation additives, 10%
tion, 95-96% conidia and 82-92% blastospores skimmed milk (970 days) and 7.5% glucose
survived for 130 days at 10-25C (Gillespie, in horse serum, made some improvement
1984). Recently, Tween 80 has replaced it in (Table 4.12). Survival was less with other
fashion, despite some evidence of harm. At additives and least in Hank's salt solution (1
0.05%, compared with Tween 80, Triton day) and spent Sabouraud broth (6 days),
X-100 consistently gave better survival of suggesting that at the freezing point ice cryst-
conidia of six strains at 5-25C by margins als may have separated around the spores,
varying, for example, by 3-98% after 8 increasing the concentration of solutes to
weeks' storage (Moorhouse, 1990). A higher harmful levels. The performance of additives
concentration of Tween 80 (0.5%) gave only at -20C showed little relationship to those at
16% germination after 4 months at 4C com- 2C (Table 4.12).
pared with 42% in distilled water (Daoust et
al., 1983). An attempt to use it as a preserva-
4.6.7 EFFECT OF STORAGE ON SPEED OF
tive at a very high concentration (10%) was
GERMINATION AND ON VIRULENCE
harmful (Table 4.13). Thus the concentration
is best limited to the minimum that gives Prolonged storage sometimes slows sub-
efficient wetting. The beneficial effect of sequent spore germination. In germination
surfactants in sprays is described in section tests on agar early in storage, germination of
4.3.6. Metarhizium spp. was complete in 24 h, but
after longer storage it extended over 48 or
4.6.6c Effect of spore concentration on survival even 72h (Daoust et al., 1983; Moorhouse,
Increasing conidial concentration of M. aniso- 1990; Stathers et al., 1993; Moore et al., 1995).
pliae from 106 to 108 /ml, or an increase above Germination was slowed after storage of
1:1 w /w conidia:water (80% water content), conidia in powder form, in oil or in water. In
drastically reduced their survival time (Moor- powder or oil, long survival of Metarhizium
house, 1990; Bailey and Rath, 1994). About 106 conidia depends on thorough drying (sections
spores/ml were used in survival studies with 4.6.3, 4.6.4 and 4.6.5), which minimizes meta-
V lecanii blastospores (Table 4.12) and M. ani- bolism; the delay in germination may be due
sopliae conidia (Gillespie, 1984; Moorhouse, to the extra time taken for recovery from phy-
1990), while numbers were not given for T. siological dehabilitation in storage. In water,
cylindrosporum (Table 4.11), M. anisopliae conidia are not dehydrated and the operative
(Table 4.13) and B. bassiana (Jimenez, 1989). factors are probably recovery from faster de-
Possibly toxic levels of exudates or metabol- habilitation due to greater metabolism in
ites build up at high concentrations. Thus, water, and from possible leaching of nutrients
significantly, some of these studies are not from spore into water. In some studies on
applicable to the storage of slurries, a degree spore survival, germination on agar was
166 Formulation of mycoinsecticides
recorded over only 24 h and some spores not
germinated by then would be falsely included
in the death rate. A lag period while organ-
isms recovered from a period in storage is
important for organisms applied to soil for
the benefit of plants (sections 7.4, 7.5.1c).
While M. flavoviride conidia can be dried to
< 5% moisture content without harm, rehy-
drating the dried conidia directly in water
reduced germination from 92 to 64% on agar
in one batch and from 76 to 24% in another.
This reduction was avoided by first rehydrat-
ing for 1-13 h in humid air. Rehydration of
conidia in oil, or even pre-germination, before
spraying in oil onto locusts did not decrease
time to locust death, suggesting that dry con-
idia easily take up moisture on the locust
cuticle. In contrast, before spraying in water
- to avoid inbibition damage - spore packs
should be opened and the spores exposed to
moist air to allow gentle rehydration, which
creates logistical difficulties in the field
(Moore et al., 1977). Unlike M. flavoviride, P.
fumosoroseus conidia rehydrated in water were
slightly more active than dry conidia on Spo-
doptera frugiperda (Fargues et al., 1994), sug-
gesting that there may be advantage in
rehydrating this species before spraying in oil.
Apparent loss of spore virulence - as
opposed to germination - during storage
may be partly due to delayed germination of
some spores increasing the time taken to kill
test insects. Moore et al. (1995) found no sig-
nificant loss of virulence of M. flavoviride con-
idia after storage in oil, and Daoust and
Roberts (1983) found virulence of M. anisopliae
impaired after storage as an unformulated
powder, only when the spore death rate accel-
erated, i.e. when many of the spores were near
death. After storage at 0-10% RH, wetting
conidia powder with 0.01-0.001% surfactant
with a high hydrophile-lipophile balance
(HLB 10), in this case ethoxylated tridecyl
alcohol, improved germination from 21 % in
water alone to 85-88% (Jin et al., 1993). In most
studies, such wetting occurs anyway because,
when suspending conidia for germination
enumeration and for spraying, a surfactant is
normally added to ensure efficient mixing.
Dehydration may reduce germination by
increasing surface tension on conidia, while
the surfactant reduces the tension and facil-
itates rehydration. A model was developed by
Hong et al. (1997) to quantify the effect of
temperature and moisture content on the
longevity of conidia of M. flavoviride.
Stored in water, B. bassiana conidia and
blastospores did not lose virulence over 27
weeks at 5 or 20C (Jimenez, 1989). Storage
in PEG solution increased virulence of M. ani-
sopliae blastospores a little, while sorbitol and
sodium alginate caused a decrease, showing
no relationship to spore survival, i.e. to the
proportion of spores likely to be near death
(Table 4.13). Survival of T. cylindrosporum con-
idia fell to only 10% during 4 weeks' storage
in water or 20% KCl, but virulence fell rapidly
in water (x 100) and only slightly (x 1.2) in
KCl. This was probably due to gradual leach-
ing of nutrients in water and retention of
nutrients by the conidia in KCl.
4.7 PRODUCTION
Mass production of insect pathogenic fungi
will be reviewed from the viewpoint of facil-
itating formulation and application. The suit-
ability of a system will depend on the
intended use of the products, formulation
and method of application (Fig. 4.2). The sys-
tems can be classified according to the type of
medium, solid or liquid.
4.7.1 PRODUCTION ON SOLID SUBSTRATES
The most convenient and durable develop-
mental stage of the dusty spored Hyphomy-
cetes for application and storage is the
conidium, the natural distributive stage. In
nature, this forms on the surface of the insect
or internally in dry situations. For mass pro-
duction, nutrient granules provide the maxi-
mum surface area on which conidia can be
produced (Fig. 4.1 in section 4.1). Bartlett
 4.7.1 Production on solid substrates 167

and Jaronski (1988) reviewed in detail the
production requirements of nine species of
entomopathogenic fungi. They described
media, machinery and yields of conidia, as
well as industrial production of fungal
enzymes and Japanese fermented foods.
What appear to be optimal features of a num-
ber of systems have been assembled in Table
4.14 in a protocol that has never been tried as
such, but could be used as a starting point for
research into a large-scale, highly mechanized
industrial manufacturing system.
The simplest, most suitable low-cost gran-
ule is the cereal grain (Table 4.14). If neces-
sary, it can be broken to maximize surface
area, while maintaining a friable open texture.
It is soaked or boiled to achieve the necessary
moisture content, as well as to enlarge the
grain to increase available surface area and
intergranular air content, then sterilized by

Table 4.14 Production of debris-free, dry, technical conidial powder*

Ingredient Amounts for 4 kg batch Function
Yeast-sucrose (each 30 gil) broth 500 ml Seed broth
Buckwheat 4 kg Solid nutrient medium
Mushroom spawn bags 10 Solid fermentation growing bags
35 x 22 x 0.1 cm
Powdered CaC03 200 g Increase pH and prevent grains
Powdered CaS04 200 g sticking together
Silica zerogel powder, Gasil GM2 t 10% w Iw add to spores Complete and maintain desiccation
Black high density polyethylene 1-8 Storage
(0.001 inches thick)

Method
1 Grow seed broth 3 days at 150 r.p.m. and 24 'c. Check for contaminants by microscope and by plating
sample on agar
2 Soak buckwheat for 24 h
3 Drain and mix buckwheat with 5% CaC03 and 5% CaS04 in a drum mixer
4 Autoclave 400 g buckwheat per growing bag with the tops folded over at 120 cc for 40 min. Cool.
Contaminant check: plate some grains on agar
5 After cooling below 35 cC inoculate each bag with 70 ml seed broth, using sterile technique, and heat
seal. Shake to spread inoculum
6 Incubate bags 14 days at 25-30 DC (thermostat settings: heat cut-in at 24 DC, cooling in at 32 T). Aerate
room during week 2 to lower humidity and dry the grain to encourage conidiation. Pummel
periodically to prevent caking by mycelium bridging grains
7 To harvest conidia, pass the buckwheat through a fluid bed separator (see text), after discarding any
contaminated bags
8 Dry conidia in thin layers in ventilated cabinet dryer to < 6% moisture, checking the moisture content
periodically by a rapid method. Avoid moist spots caused by poor air circulation
9 Sieve through encased 100 J.im screen. Expected yield ca 1 kg spores (1-5 x109 sporeslg dry weight
grain)
10 Mix with 10% silica powder and store at 4-10 DC in black bags, not permeable to water vapour,
preferably vacuum packed
11 If, for the strain in use, there is an additional market for a technical granule or powder by-product (see
text), use the 30% of conidia left on the grain, by drying and bagging the residual grain
* This protocol combines the optimum features of many production systems and needs research adjustments for
specified strains and species of fungi (Bartlett and Jaronski, 1988; Bateman, 1996; Daoust and Roberts, 1983; Ferron,
1981; Goettel, 1984; N.E. Jenkins, personal communication, 1996; Jenkins and Goettel, 1997; Jenkins and Lomer, 1994; Jin
et al., 1993; Mendon"a, 1992).
t Crossfield Chemicals, Poole, UK.
168 Formulation of mycoinsecticides
various means. Heat has the advantage that
cooking increases availability of nutrients to
the fungus. Use of excess water for soaking,
then draining off the excess, partially removes
any potentially toxic crop-spray residues of
fungicides and insecticides. The sterile grain
is inoculate'ti with broth culture.
In nature, the fungus builds up biomass
inside the insect. This is mimicked (Table
4.14) by growth in the broth, then on the cer-
eal nutrients. The grain must be small with a
favourable surface area-to-volume ratio. Opti-
mal water content depends on substrate, ide-
ally just forming a surface film. These factors
facilitate nutrient absorption by the fungus
(which may be minimal), gas exchange and
heat transfer. Addition of CaC03 and CaS04
can increase pH and prevent grains sticking to
each other without affecting yield (Moor-
house, 1990). Rice is widely used, being ideal
in shape, size and open texture. Millet is good,
while buckwheat retains its structure and
does not agglomerate. These are among the
best of the nutrient substrates for harvest con-
venience (Bartlett and Jaronski, 1988; Moor-
house, 1990; Ibrahim et al., 1993). The grain
can be supplemented with nutrients or it can
be replaced by nutrient-soaked porous
mineral granules, such as pumice and exfol-
iated vermiculite (Ferron, 1981; Bartlett and
Jaronski, 1988) and clay, which can be
retained as a granular formulation or will dis-
integrate in water to form a spray (Guillon,
1997).
Re-usable, autoclavable, heat-sealable
spawn-growing bags, used in the mushroom
industry, allow efficient aeration during incu-
bation (Table 4.14). Opaque, white, Valmic
microporous polypropylene film has win-
dows of clear non-porous polypropylene
through which the grain can be seen. The
area ratio of the two polypropylenes is
adjusted to customer specification in order to
obtain any desired rate of grain drying, which
can be fine tuned by altering the growing
room humidity. Micropores of < 0.4mm pre-
vent contamination, while allowing good
transmission of air (ca 25cm3 /cm 2 /min at
1 atm) and water vapour (ca 500 g/ m 2 /24 h
at 23C and 50% RH). The microporous film
is protected by a coarsely perforated pOlypro-
pylene film. Growing mycelium produces
enough heat to cause hot spots and premature
drying if substrate bulks are too large; circula-
tion of air in the room, even a cooling system,
may be needed to dissipate heat. Slowly the
high moisture content optimal for mycelial
growth falls to the lower optimum for coni-
dial production. Since relative humidity in the
bags is near 100%, which is excellent for con-
idial survival (section 4.6.3), the first-formed
conidia are well preserved during the spor-
ulation period until harvest (section 4.7.3).
Opening the bags for harvesting and for-
mulation allows contamination by other
microorganisms, whose development is best
prevented by drying. The most natural
method of harvest is extraction of dry spores
by an air stream in a fluid bed device. This
mimics the dehiscence of dry conidia from
phialides growing from insect cadavers and
their dissemination - dry - to other insect
targets. Cyclones in the extraction system
separate conidia from debris to form a vir-
tually pure spore powder that does not need
screening (Bateman, 1994b, 1997; S. P. Mer-
melstein and R. P. Bateman, International
Institute for Biological Control, Ascot, perso-
nal communication).
Spores are further dried (Table 4.14)
because only very dry spores have a good
shelf-life (sections 4.6.3, 4.6.5). If necessary,
they are then screened (841 mm = 20 mesh,
McCabe and Soper, 1985; 100mm = ca 140
mesh, Lomer et al., 1997; 300mm = ca 50
mesh, Moore et al., 1996a) to prevent spore
aggregations and foreign particles later block-
ing spray nozzles. Material retained by the
screen may be lightly ground and re-screened.
Storage with silica powder completes drying
(section 4.6.3). The resulting product has
minimal bulk and weight for convenient stor-
age, transport and formulation. For example,
the M. flavoviride LUBILOSA conidia powder,

Green Muscle '189'TC technical concentrate,
contains practically no particles >30 Jlm in
diameter (R. P. Bateman, personal commun-
ication) and is suitable for formulation in oil
as a ULV spray to produce droplets in the 40-
120 Jlm size range, each containing a load of
conidia (100-10000) lethal to grasshoppers
(Bateman, 1996). Thus this follows industrial
flow line 1 (Fig. 4.2; section 4.3.1).
As an alternative to dry-harvesting, conidia
may be harvested by washing-off in water,
concentrated by filtering or centrifuging,
then air-dried. This has three disadvantages:
(1) the water may leach out important sub-
stances from the spores, particularly if there
is some germination (sections 4.6.6a, 4.6.7);
(2) the amount of water to be dried off is
great and the rate, method and temperature
of drying are critical (section 4.6.2), while trig-
gering of germination must be avoided (sec-
tion 4.3.2); and (3) the dried spores tend to
cake and require grinding. Thus harvest in
water is more suitable for the production of
technical powder for formulation and use in
aqueous sprays in flow line 2, than for formu-
lation in oil sprays in line 1 (Fig. 4.2). In Brazil,
conidia of Metarhizium flavoviride are harv-
ested in kerosene and formulated in oil
(Magalhaes and da Silva Frazao, 1996); survi-
val in storage may be poorer than as dry
powder (section 4.6.5).
The need to extract spores from the produc-
tion medium can sometimes be avoided. The
whole contents of the growing bags or the
residual grain (Table 4.14) may be dried and
ground as the technical base product for
dusts, wettable powders and water-dispersi-
ble granules (sections 4.3.2, 4.6.4; Table 3.5 in
section 3.3.1; Table 3.11 in section 3.3.4).
Grinding is costly; grinders must be carefully
chosen to avoid damaging the spores both
physically and by the production of heat. Dif-
ferent cereal grains and different varieties of
anyone cereal have different milling qualities;
grain can be selected to facilitate grinding and
formulation. On re-wetting in water or oil
spray, cereal particles swell and may block
4.7.1 Production on solid substrates 169
nozzles, so screens must be fine enough to
accommodate swelling. Swelling and sheer
bulk of the product make this harvest method
unsuitable for ULV use in flow line 1 (Fig.
4.2). Even harvest of conidia in an air stream
may pick up substrate particles that may later
swell. From this viewpoint, mineral granules
soaked in nutrient solution are better than
cereals and also enable better definition of
media. The grain or granules may be mar-
keted unground for use in soil or for corn
borer control (Table 4.14; sections 4.4.2, 4.4.3).
Expensive drying and harvesting can be
eliminated by marketing the growing bags as
end-use containers, even before sporulation
has peaked. The grain/ granules are undried
for use in soil (flow line 3, Fig. 4.2). This
increases shelf-life by taking advantage of
the excellent survival power of conidia at
100% RH (section 4.6.3). Before distribution,
autoclavable bags need addition of another
cover, permeable to 02 and CO2 but not to
water vapour, to prevent on-shelf drying. For
example, conidiation of M. anisopliae on millet
at 25C was rapid over 14 days, when the
bags would be usable for application to soil,
followed by slower conidiation for a further
14 days and beyond, which could be allowed
to occur after distribution during shelf storage
(Moorhouse, 1990).
There are many alternatives to the bag sys-
tem (Bartlett and Jaronski, 1988; Jenkins and
Goettel, 1997). Milner et al. (1993) used auto-
clavable contaminant-proof boxes that permit
gas exchange. The biomass may be grown by
liquid fermentation (section 4.7.2) and inocu-
lated onto granules or membranes on frames
for surface conidiation (Bailey and Rath, 1994;
Jenkins and Lomer, 1994). These methods
should allow efficient debris-free harvest of
conidia and control of contamination. In Bra-
zil, open trays were discarded due to serious
contamination (Mendon\a, 1992). Rotary fer-
menters tend to impair conidia by excessive
agitation of the granules. Prill (section 5.3.3),
bearing P. fumosoroseus mycelium, requires
activation by wetting and incubation for 7
170 Formulation of mycoinseetieides
days to produce conidia for harvest at site of produce, so a method has been patented to
use (Osborne and Landa, 1994). This time grow mycelium in deep liquid media and
interval is an inconvenience when timing preserve it by filtration after harvest at the
and anticipating treatments in the course of a peak growth phase (McCabe and Soper,
growing plant crop. 1985). Vacuum-filtered mycelium is detached
from the filter and formulated by spraying
with 10% aqueous maltose and glucose for
4.7.2 PRODUCTION BY LIQUID
protection during drying (section 4.6.2). The
FERMENTATION
drying routine is probably critical.
Hyphomycetes with heads of sticky conidia, Production of Metarhizium mycelium has
e.g. Verticillium, are less amenable to harvest been fully mechanized in a two-step fermen-
from solid media than those with dusty, tation that aggregates mycelium into compact
hydrophobic conidia. Mycelial biomass of carrier-free pellets or granules (Table 4.15).
both types of fungus is grown more rapidly These granules mimic the durability of fungal
by deep liquid fermentation, which may be biomass in insect cadavers. They can be
much more easy to handle, scale-up and con- applied in industrial flow line 3, with the
trol while using established fermentation formulation advantage of still holding some
technology and equipment. From the form- nutrients (Fig. 4.2). In fermentation step 1 in a
ulation viewpoint, water is inevitably present shake flask, the germ tubes of conidia strongly
as the fermentation carrier. Some strains of knot together to form little beads, 0.1 mm in
B. bassiana, M. flavoviride and H. thomsonii diameter. In step 2 in a fermenter, more
can produce submerged conidia, which may mycelium grows on each bead, enlarging it
or may not equal aerial conidia in virulence to a granule ea 1 mm in diameter. Gentle dry-
and longevity. Like the blastospores produced ing in a fluid-bed granulator maintains the
by all species, submerged conidia may be integrity of each granule (Table 4.15). The sys-
relatively hydrophilic and more difficult to tem is flexible. Dry granules can be used unal-
formulate in oil than the corresponding lipo- tered or formulated into a wettable powder
philic aerial conidia (Roberts and Sweeney, (Table 3.11 in section 3.3.4), which can be
1982; Bartlett and Jaronski, 1988; Jenkins and made low in bulk by omitting the carrier,
Prior, 1993; Jenkins and Goettel, 1997). To since the granules themselves are carrier-
produce aerial conidia, mycelium grown in free. In addition, the granules can be made to
broth can be applied to solid substrates conidiate before harvest so that they are
(section 4.7.1) or allowed to conidiate on the infective immediately on application without
surface of shallow layers of broth (Bartlett and waiting for granules to conidiate in situ. Alter-
Jaronski, 1988). natively, pure conidia can be harvested and
Blastospores, the spores normally produced stored with Gasil GM2 powder (section 4.6.5)
in deep agitated liquid, are more delicate and to formulate into a minimal bulk ULV spray
short-lived than conidia, but can be equally or (Table 3.10 in section 3.3.3; Table 4.9 in section
more virulent (Hall, 1981; Bartlett and Jar- 4.6.5). Scale-up of the fermentation is easy and
onski, 1988; Jenkins and Goettel, 1997). Blas- industrial production beyond pilot stage was
tospores can be formulated as an effective, frustrated only by the small market size. The
durable, wettable powder by a sophisticated system can be modified for other fungal
but commercially viable process (section 4.6.4 strains and species with spores and hyhae of
and Table 4.7 in that section). differing hydrophobicity, with the aid of floc-
Entomophthorales have very delicate con- culants, complexing substances, more surfac-
idia which are extremely difficult to store, tant and different pH values (Andersch et al.,
while the tough resting spores are difficult to 1990).
 4.7.3 Optimizing spore vigour 171
Table 4.15 Production of dry carrier-free granules of Metarhizium anisopliae by a fully mechanized process
(Andersch et aI., 1990)

Ingredient Percentage Function

M. anisopliae POOl (DSM 3884) Fungus strain slants
Malt extract-glucose-peptone agar 5.6 Production of seed conidia
Tween 20 1.0 Non-ionic surfactant solution
Glucose-yeast autoloysate broth 2.2 Solid-free growth medium
Baysilicone E, 30% 0.05 Antifoam
Glucose, aqueous 10% Nutrient wash

Method
1 Make a 106 conidia/ml suspension in the surfactant solution from 16-day agar plate cultures at 25C
2 For step 1 fermentation, seed the suspension at 1% vol/vol into 100 ml broth, pH 7.5, in 1-1 flasks and
incubate at 25C for 24 h on a rotary shaker at 100 r.p.m., with antifoam, to form beads of germinated
conidia
3 For step 2 fermentation, transfer the flask culture complex of step 1 (3% vol/vol) in 10 I broth in a 15 I
air-lift fermenter and grow for 60-80 h at 25C, 400 r.p.m., 51 air/min, forming compact granules.
4 Concentrate the granules by sieving (0.1 mm pore diameter) and suction filtration
5 Dry in 200 g portions in a fluid bed granulator (filling volume 16.51, airflow 1300 l/ min, 40 cq. Monitor.
Stop drying at 10% moisture
6 Store in dry conditions
7 Alternative production of conidia. After (4), wash with 100 ml glucose solution per 50 g granules,
refilter, incubate 60-70 h at 100% RH, 25C, in a humidity chamber, monitoring conidia formation by
depth of green colour, dryas (5). Each granule forms about 106 conidia

In Hyphomycete fermentations, granule or Some examples are given below. When

filamentous growth - as well as blastopore
yield and desiccation tolerance after formula-
tion - can be controlled by factors such as
modification of water activity (Humphreys et
al., 1989), nutrient ratio and pH, as well as
Tween 80 and PEG concentration (Kleespies
and Zimmermann, 1992; Jackson et al., 1997).
4.7.3 OPTIMIZING SPORE VIGOUR
It is important to grow vigorous spores.
Poor production conditions may not reduce
germination percentage after brief storage,
but may stress spores enough to limit shelf-
life and impair response to formulation.
Media ingredients, light (Hall and Papierok,
1982), temperature and humidity during
growth, duration of culture, minimizing
agitation during the sporulation period on
solid substrates, and conditions during drying
and harvest (section 4.6.2), all repay attention.
stored in Ringer's solution, B. bassiana blastos-
pores from nitrogen-limited media survived
longer than those from carbon-limited media
(Lane et al., 1991). Increasing the sucrose
content of agar-based growth media from 2
to 8% and decreasing the growth temperature
from 30 to 26C, reduced tolerance of
M. flavoviride conidia to high temperature
(McClatchie et al., 1994). In three fungal spe-
cies, media were modified to reduce content
of low M r polyols (glycerol and erythritol) or
trehalose. The resulting conidia survived
longer in storage at 75% RH, germinated
both more quickly and at relatively low
water activity, and were more virulent
(Hallsworth and Magan, 1994, 1995). M. flavo-
viride conidia, harvested after culture for 24
days on rice at 28-32 0c, tolerated long storage
better than conidia from 12-day cultures;
possibly more of the conidia grown in the
shorter time were immature with incomplete
172 Formulation of mycoinsecticides
food reserves. Conidia of M. anisopliae, P.
fumosoroseus and V lecanii - but not B. bassiana
- from young cultures germinated more
quickly than those from older cultures. Possi-
bly this was because first-formed conidia in
linear chains on phialides in the first three
species differ in structure rather than in vig-
our from those formed later (Hall et al., 1994).
This might not happen in B. bassiana because
its conidia are not formed sequentially in
chains (Fig. 4.1). Means of maintaining vigour
during and after storage are discussed in sec-
tion 4.6.7.
4.8 FUTURE USE, DEVELOPMENT AND
RESEARCH
The development of ULV-CDA formulations
of conidia to control locusts in dry climates
(Fig. 4.2) has refuted early belief that fungi can
be used successfully only in moist conditions.
A 2-year shelf-life of conidia formulated as a
very dry powder has been achieved in tempe-
rate ambient conditions. This dual progress is
the key to the future and should enable fungi
to compete in efficacy with chemical insecti-
cides on nearly equal terms, and should
increase projected market sizes towards
industrial viability. The first oil-flowable pro-
duct, Mycotrol GH-OFD, has been registered
in the USA for grasshopper and cricket con-
trol (Mycotech, 1995). However, strain-spec-
ificity is a constraint. Large strain differences
have been a feature in most research areas
mentioned in this chapter, including strain
reaction to formulation. New products
consistently able to survive competition in
the market are sorely needed (section 4.1).
New industrial ventures must plan facilities
flexible enough for use with different strains
for different markets, to assemble a volume of
products large enough to be industrially
viable. Budgets should cater for adequate
research on strain variability, including reac-
tion to formulation requirements. Research
should target three areas that need improve-
ment: application, storage and production.
4.8.1 APPLICATION
4.8.1a Application in dry climates CDA oil for-
mulation is the method of choice for applica-
tion in dry climates. Research priority should
be given to maximizing foliage cover, direct
hits of droplets on insects and cover on lower
leaf surfaces to shield spores against UV
radiation (Appendix Tables II.7 and II.8); or,
if no more progress in these subjects seems
likely, confirmation that the research has
reached its useful limit. Deposits on leaves
and insects can be visualized with tracer
dyes (Appendix Table 1.9). Rapid loading of
sprayers is critical to fully utilize the brief
daily period of favourable inversion condi-
tions, easiest to do with ready-to-use
formulations. Droplet size is affected by visc-
osity of the spray (Appendix Fig. II.4);
most aerial sprayers have pumped feeds
able to use viscous sprays. By designing pro-
ducts to be diluted with a light oil when
necessary, the same products could be
used in hand-held applicators with gravity
feed, which may need reduction in spray vis-
cosity. Prevention of settling during storage
and in spray tanks without agitation is prob-
lematic.
Oil establishes infection by spores better
than water. Research should find the best
droplet size (Appendix Table II.s; Appendix
Fig. II.2); viscosity (Appendix Fig. II.4); oil
type (Appendix Table 11.4); charge; and nutri-
ent content for suspensions to spread spores
to the vulnerable intersegmental membranes,
then nourish them there to encourage germ-
ination (possibly by adding low-bulk nutri-
ents). The charge on locust cuticle varies,
negative predominating on the rigid cuticle
and positive on the flexible cuticle (Charnley,
1992). The charge on flexible cuticle is the
more important because it is desirable to
increase accumulation of spores between
intersegmental membranes. Equally, pick-up
of spores from the leaf (Fig. 4.3 in section 4.3)
by the insect could be improved by modify-
ing these factors, as well as by ensuring

palatability of the oil, since many spores stick
to insect appendages during feeding.
4.8.1b Application in moist climates In very
humid conditions, insect control by fungi can
be devastating. This was demonstrated when
clear plastic cups were inverted over small
individual aphid-infested chrysanthemum
plants in a glasshouse (H. D. B., unpublished
data). All aphids were dead or fatally infected
with naturally occurring V. lecanii within 10
days, whatever the species and the number of
aphids placed on the plants. It was impossible
to maintain an infestation. Natural outdoor
epizootics inspired early workers, but results
of trials varied with changes in weather. Fre-
quent modern CDA fungal applications are
effective in moist climates, while single sprays
sometimes give lasting effective control due to
natural spread during the most humid peri-
ods of \Veather. The challenge to formulation
research is to ameliorate the curbs of lower
humidities in drier weather enough to give
reliably predictable insect control from single
or few fungal applications. Improvements are
needed in spore survival and replication on
leaves, and in reduction of spore mortality
due to low daytime humidity. Also, speed of
germination needs increasing, in order to use
diurnal humid periods more effectively and
to decrease plant damage while infection is
developing.
Spore survival on leaves can be improved
by humectants and oil to protect spores from
drying, and by sunscreens. Evaporation from
an aqueous spray can be reduced most, pos-
sibly for 4 days, by an overspray of an invert
emulsion, which is sophisticated and expens-
ive (Quimby et aI., 1989), but would the resul-
tant improvement of fungal survival and
growth on leaves be sufficient to justify the
cost of the emulsion and of double spraying?
The great differences in effect of sunscreens
with similar UV absorptance ranges, already
observed in laboratory and field, need more
investigation to take into account interactions
of the leaf with screens and carriers. The
4.8.1 Application 173
intimate position of screen particles in relation
to spores after application may be very im-
portant (Appendix Fig. II.6). The UV energy
reaching the target should be measured in
order to improve comparability of different
experiments. Actual effects on insect control
obtainable in the field need quantification, e.g.
Inglis et al. (1997b). Sunscreens may be eco-
nomic to use only under the fiercest and long-
est exposure to sun, although realistically
their increase in spore half-life may be enough
only in the shielded position on the underside
of a leaf, because intense sunlight begins kill-
ing spores within minutes of exposure.
Application late in the day should be recom-
mended to escape severe initial exposure to
sunlight. Stilbene sunscreens unexpectedly
synergized the rate at which insect virus
killed larvae by promoting infection of gut
cells (section 3.4.4e); a search for unexpected
effects on fungi may be rewarding (although
it should not be for synergists of gut infection
as fungi rarely infect by this route).
Germination of conidia may be accelerated
by various formulation tactics. A product may
be soaked in water before mixing the spray. A
surfactant may be added to facilitate spore
rehydration, or colloidal chitin (Samuels,
1986) to stimulate and synchronize germina-
tion, or some fatty acids to stimulate germina-
tion, but others inhibit it (Barnes and Moore,
1997). Methods analogous to seed priming
may be used, e.g. soaking in PEG (Knudsen
et aI., 1991). These formulation tactics need
trial - are they adverse if there is a diurnal
dry period and the conidia reach only the pre-
swelling stage of germination? Comparison is
needed with the alternative strategy of retard-
ing insect growth and moulting by co-applica-
tion of insect antifeedants (Sanyang and van
Emden, 1996), growth retardants or moulting
inhibitors. Ca2+ ions are deeply involved in
conidial germination of Beauveria bassiana,
which is speeded by pretreatment with a
Ca2+ ionophore (A23187) + CaCl2 (Lamina
1998). Further physiological study on conidia
may be inspirational.
174 Formulation of mycoinsecticides
Pest control has been enhanced by formu-
lating spores with nutrients so that the fungus
grows and sporulates on the plant. But
improvements are needed. Agent-specific
nutrients might avoid possible side effects of
supporting plant disease or disfiguring organ-
isms, or of causing extensive unnatural fungal
growth on insect cuticle while repressing
penetration and chymoelastase synthesis (St
Leger et al., 1989). Unlike the other fungi, V.
Iecanii mycelium normally grows over the
insect cuticle during the infection process
(Schreiter et al., 1995). Whether treatments
affect this growth to enhance or delay host
death needs investigation.
Fungicides used to control plant disease
threaten fungal agents, though some of the
most specific can be formulated in the same
tank mix. Laboratory tests with insects on
leaves have been misleading, and in vitro
tests of the fungus on treated agar even more
so. The only reliable information comes from
spray trials on infested plants in the field (sec-
tion 4.3.7).
Insects may cure Beauveria infection by rais-
ing their preferred peak body temperatures
after moving into high-temperature zones in
vegetation (grasshoppers, laboratory study,
Inglis et al., 1996b; field study, Inglis et al.,
1997b). Grasshoppers infected with M. flavo-
viride in a natural infestation in Senegal raised
their preferred peak body temperature by ca
4C during sunny periods, but this had little
therapeutic advantage as shown in popula-
tion counts in the particular weather condi-
tions prevailing over 7 days (Blandford et al.,
1998). Research on formulation of spores with
an irritant, or on formulation of strains with
different temperature ranges together (Inglis
et al., 1997a), as well as a search for virulent
strains with higher temperature ranges, may
solve this disconcerting problem.
The favourable nature of the soil environ-
ment for fungal survival suggests that use of
fungal control of soil pests should increase.
Although long-term control can be expected,
continued research is best directed at giving
the fungi the best start. For most pest situa-
tions, bait, dust, granule and drench formula-
tions need comparison. Research subjects
include baiting materials; nutrients and pro-
tectants in granules to combat fungistasis; the
improvement of the power of aqueous spore
suspension drenches to penetrate into soil
with surfactants (e.g. the new organosili-
cones); buffers and materials to control the
electrical charge on spores; and combination
with free-flow additives to prevent spores
clumping, minimize particle size and reduce
adsorption to soil.
4.8.2 STORAGE
Recent key work with M. flavoviride shows
that the future for long shelf-life lies particu-
larly with formulation to keep spores ultra-
dry. Before this was realized, work was done
with conidia of various moisture contents and
dubious quality, stored moist. This research
needs repeating under well defined condi-
tions, using synchronized batches of well
defined, high-quality conidia of various spe-
cies to decide the best moisture content(s),
best oils, value of the absence of O 2 , best
desiccants and the effect of absorbent clays,
predried or not. The process of maturation in
storage over a long period should be traced by
regular measurements of moisture content,
spore size, respiration, chemical composition,
germination (including speed of germination)
and potency in insects. A quality control test
should be developed from the easiest of these
methods, e.g. microscopic measurement of
spore size, or mean spore weight, if size can
be correlated with quality. The predictive
value of accelerated tests, i.e. at high tempera-
ture, needs examination. Whether silica gel
acts as an absorbent of metabolic wastes, as
well as a desiccant, needs study.
Inbibition damage, as with many seeds,
may have unwittingly been caused by plung-
ing fully dried conidia directly into water
(section 4.6.7). Gentle rehydration of the con-
idia by exposure to moist air could have

avoided this damage. Such confusion of
results should be avoided in future work by
exposure of spores to moist air before setting
up germination tests on agar.
An alternative method of harvest - with
aqueous surfactant, concentration, then dry-
ing - altered the relationship of survival of
M. anisopliae conidia to storage humidity
(Clerk and Madelin, 1965). This needs com-
parative research, including on the effects of
cryoprotectants.
No entomopathogenic fungi, formulated or
not, have consistently stored well in water
above 0 DC; the future for this system is to
hold spores briefly in the laboratory before
experiment. However, some work has been
flawed by the failure to recognize possible
harmful effects of O 2 depletion and CO2 accu-
mulation in sealed containers, probable bene-
ficial effects of the low spore concentrations
usually used in tests, and the possibility that
additives were really protecting M. anisopliae
conidia at 4 DC and V lecanii at 2 DC from the
deleterious effects of cold. Selected research
should be repeated, particularly to avoid dete-
rioration of spores before experiments. Harm-
ful effects of storing conidia in dense slurries,
not detected in tests with low concentrations,
must be remedied for commercial storage, a
potentially serious difficulty with storage of
emulsions.
Surfactants are essential for the formulation
of hydrophobic conidia in water, but doubt
remains as to whether some cause harm.
Because exposure is long-term, the most sens-
itive experiments to investigate these doubts
involve storage, in both wet and dry formula-
tions. Data from the food industry provide
useful information about potential physiolog-
ical effects of surfactants.
4.8.3 PRODUCTION AND QUALITY OF
SPORES
It is unclear whether solid or liquid produc-
tion systems will predominate in future. Key
advantages are the 2-year shelf-life of conidia
4.8.3 Production and quality of spores 175
realizable with solid systems against the easy
technical operation and scale-up of some
strains in liquid systems. Where no factor
dominates, the choice may be a question of
cost. More examples of cost analysis (e.g.
Guillon, 1997) would be valuable assets to
the public domain, especially to illustrate the
cost of striving for spore quality and optimal,
possibly sophisticated, formulation. The con-
sequences of compromise, particularly with
formulation, to lower the cost must be studied
in terms of performance.
Performance may be primarily a function of
conidium condition. One can postulate that
since the conidium evolved as the natural aer-
ial dispersive stage, the prime condition
occurs at natural dehiscence from the phial-
ide, triggered by favourable atmospheric con-
ditions. At this time, the conidium has
probably swollen to its largest size, suggest-
ing that size may be the best single measure of
quality. Metarhizium conidia are produced in
chains, youngest nearest to the phialide, pos-
sibly involving differences in structure (sec-
tion 4.7.3). Alternatively, it can be argued that,
in the stillness of a fermentation bag, conidia
remain attached to the chain past the 'prime'
condition postulated above, and that they
might undergo a beneficial maturation
beyond that stage. In either alternative, con-
idia must eventually undergo detrimental
ageing. Thus harvest from solid substrates
yields a mixture of conidia in prime condition,
of immature conidia - detached prematurely
from phialides - and of conidia past their
prime. The presence of immature conidia
may explain why, in many storage and for-
mulation tests, there is an early, small but
variable loss of viability (e.g. 5-10%) in the
first few days and then viability stabilizes.
Extending the length of a production run,
e.g. from 10 to 30 days at 30C, would reduce
the proportion of immatures, albeit at the
expense of the oldest conidia dissipating
their endogenous reserves by respiration in
their moist state (section 4.6.6a). These
considerations may influence the timing of
176 Formulation of mycoinsecticides
commercial harvest, at present a compromise
between maximizing accumulated spore yield
at the cost of prolonging production time.
Production costs in end-use containers are
minimal. The proportion of prime conidia
depends on how soon the product is used.
Conidia are, in effect, formulated in the
growth medium and shelf-life is good, bene-
fiting from excellent storage humidity near
100% RH (section 4.6.3). Moist spores age
relatively rapidly as a result of their compara-
tively high rate of metabolism, which
presumably lowers performance. Changing
performance needs monitoring and research
on incorporation of slow-release nutrients
may maintain the proportion of prime conidia
while storage continues.
The content of low-Mr polyols and treha-
lose in conidia can be increased by altering the
nutritional balance and water activity during
fungal growth. This increases shelf-life and
germination speed, and lowers the humidity
at which germination can occur, but also low-
ers yield (Hallsworth and Magan, 1994, 1995).
Means need to be found of overcoming or
avoiding the yield reduction, e.g. by lowering
media water activity only at the sporulation
stage, or by formulation of normal conidia
with added polyols and trehalose. The intri-
guing results of this study encourage further
research on conidial physiology, particularly
fundamental research to improve our under-
standing of spore quality and to balance the
increasing volume of empirical data.
The solid-substrate production system sug-
gested in Table 4.14 (section 4.7) facilitates
formulation and combines ideas, often vital
minutiae, from different sources. This poses
many research questions and, of course, the
system must be researched for each different
fungal strain involved.
In deep liquid fermentation, strains with
amenable growth habit produce blastospores,
mimicking the delicate spores evolved to
spread the fungus within the insect. When
applied to insects fresh, and in ideal condi-
tions, these can be more potent than conidia.
However, delicacy makes them more difficult
to dry and formulate; continued research in
this area should be rewarding, particularly a
search for useful additives to growth media,
as well as to slurries at harvest and drying.
These additives also function as formulation
ingredients.
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FORMULATION OF MICROORGANISMS 5
TO CONTROL PLANT DISEASES
Deborah R. Fravel, William J. Connick Jr and Jack A. Lewis

CONTENTS
5.1 Introduction 187
5.2 Commercial biocontrol products 188
5.2.1 Products containing fungi 188
5.2.2 Products containing bacteria or actinomycetes 193
5.3 Types of formulations 193
5.3.1 Seed treatments 193
5.3.2 Wettable powders and liquids 194
5.3.3 Granular formulations 195
5.3.4 Carriers 197
5.3.5 Tolerance of the environment 198
5.4 Research and the future 199
Acknowledgements 200
References 200

5.1 INTRODUCTION research on the development of these

Formulations affect many aspects of the
success of biocontrol organisms, including
the shelf-life of a product, ability of a biocon-
trol organism to proliferate and survive in
the environment, effectiveness for disease
control, ease of preparation and application,
and expense. Recent review articles have
discussed these topics as well as production
of biocontrol organisms (Lumsden and
Lewis, 1989; Connick et al., 1990; Fravel
and Lewis, 1992; Harris, 1994; Lumsden et
al., 1995). This chapter discusses formulation
of commercially available biocontrol organ-
isms for control of plant pathogens and
organisms.
Formulation can influence the quality of a
product in several ways (Lisansky, 1985). It
stabilizes the product for storage until needed
by providing adequate shelf-life. It makes a
biocontrol organism both convenient to use
and safe to handle. Choice of type of formula-
tion depends primarily on the delivery target,
as well as on the biology and ecology of the
biocontrol agent, pathogen and host, and the
cropping system. For example, a granular
material would be appropriate for distribut-
ing in-furrow or for incorporation into a
horticultural potting mix, while a wettable
powder would be suspended in water for

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
188 Formulation of microorganisms to control plant diseases
drenches or root dips. Beyond these consid-
erations there are several other desirable
characteristics of a formulation, including
compatibility with agricultural machinery,
ease of integration into a pest management
system, and cost to produce and purchase.
Since the use of microbes to control plant
pathogens is a newly emerging field, the best
means of formulating these organisms is just
now beginning to be explored. Biocontrol
organisms have been formulated in a variety
of ways (Chapter 2; Appendix III) to result in
wettable powders, dusts, gels, emulsions,
prill, pellets and granules, for seed treatments,
sprays, dips, drenches and incorporation into
soil and potting mix. Some formulations, such
as dusts, have long been used for chemical
pesticides and are useful for biocontrol organ-
isms also, while others, such as pesta (section
5.4), have been developed specifically for
microbial pest control agents.
5.2 COMMERCIAL BIOCONTROL
PRODUCTS
Some of the decisions that determine whether
a biocontrol product is commercialized are
business decisions not based on science.
Before scaling-up for commercial production,
a company must assess many factors includ-
ing demand for the product, potential market
size, and existing competing products
(Lisansky, 1985). Another factor in question
is the amount and type of data that may be
required for registration and sales permits.
Regulations differ from country to country
and sometimes between states in a country.
Evolution of regulations has progressed as
more biologicals are being developed (Ricard,
1981).
5.2.1 PRODUCTS COI;'JTAINING FUNGI
There are nearly 40 products on sale world-
wide for biocontrol of plant pathogens (Table
5.1). In addition, there are several products,
such as Promot, sold for growth promotion
rather than for biocontrol. Although not
labelled for biocontrol, these products prob-
ably provide some disease control. The fun-
gus Trichoderma is most frequently used for
control of plant pathogens. At least 12
products (Bio-Fungus, Binab-T, RootShield,
Supresivit, T-22G, T-22HB, Trichodex, Tricho-
pel, Trichoseal, Trichoject, Trichodowels, Tri-
choderma 2000) contain Trichoderma spp. to
control a variety of pathogens, including
Botrytis, Fusarium, Gaeumannomyces, Pythium,
Rhizoctonia, Sclerotinia, Sclerotium, Verticillium,
and wood-rot fungi. Formulation of Tricho-
derma varies considerably depending on the
intended use. For example, a combination of
T. viride and T. harzianwn is formulated as a
pellet for soil incorporation (Trichopel), as
dowels for insertion into wood (Trichodo-
wels), as a wettable powder in a syringe for
injection into grape vines (Trichoject), and as a
wettable powder which is resuspended and
applied with a paintbrush to wounds (Tricho-
seal). Bio-Fungus is available as a granule, as a
wettable powder, impregnated in sticks, and
as 'crumbles' for mixing into soil. A similar
antagonist, Gliocladium virens in SoilGard, is
formulated in alginate prill to control damp-
ing-off caused by Pythillln spp. and Rhizocto-
nia solani. SoilGard is mixed with soilless
potting mix before seeding. Another fungus,
Myrothecium verrucnria, is available as a wett-
able powder, emulsifiable liquid or granules
containing killed cells for control of plant
parasitic nematodes.
Non-pathogenic Fusarium oxysporum (Bio-
fox C and Fusaclean) controls pathogenic
F. oxysporum and Fusarium moniliforme on
basil, carnation, cyclamen and tomato. Biofox
C is formulated as a dust or alginate prill, and
Fusaclean is supplied as a microgranule.
Aspire, a wettable powder, contains the
yeast Candida oleophila for post-harvest appli-
cation to citrus and pome fruit to control
Botrytis and Penicillium spp. A pycnidial para-
site of powdery mildew fungi, Ampelomyces
quisqualis, formulated as a water dispersible
granule (AQ10), is sprayed onto leaves of
Table 5.1 Commercial biocontrol products for use against soilborne crop diseases

Product/biocontrolorganism Target pathogen/disease Crop Formulation Application method

1
AQ10 Biofungicide Powdery mildew Apples, cucurbits, grapes, Water-dispersible Spray
Ampelomyces quisqllalis ornamentals, strawberries, granule
tomatoes,
Aspire1 Botrytis spp., Citrus and pome fruit Wettable powder Post-harvest application
Candida oleophila Penicillium spp. to fruit as drench, drip or
spray
Binab T2 Pathogenic fungi causing Flowers, fruit, ornamentals, Wettable powder Post-harvest application
Trichoderma harzianum wilt, take-all, root rot, turf and vegetables and pellets to fruit as drench, drip or
T. polysporlll1l internal decay of wood spray
products and decay in tree
wounds
Biofox C 3 Fusarium oxysporum Basil, carnation, cyclamen, Dust or alginate Seed treatment or soil
Fusarium oxysporum Fusarium moniliforme tomato granule incorporation
(non-pathogenic)
Bio-Fungus (formerly Sclerotinia, Phytophthora, Flowers, strawberries, trees, Granules, wettable Applied after fumigation,
Anti-Fungus)4 Rhizoctonia solani, Pythium vegetables powders, sticks and incorporated in soil,
Trichoderma spp. spp., Fusarium, Verticillium crumbles sprayed or injected
Bio-Save 105 Botrytis cinerea, Penicillium Citrus and pome fruit Wettable powder Post-harvest application
Pseudomonas syringac spp., Mucor pyroformis, to fruit as drench, dip or
Geotrichum candidum spray
Bio-Save 11 5 Botrytis cinerea, Penicillium Citrus and pome fruit Wettable powder Post-harvest application
Pseudomonas syringae spp., Mucor pyroforruis, to fruit as drench, dip or
Geotrichum caudidul1l spray
BlightBan A506 6 Frost, Erwinia amylouorn Almond, apple, cherry, Wettable powder Post-harvest application
Pseudomonas fluorescens peach, pear, potato, to fruit as drench, dip or
strawberry, tomato spray
Blue Circle 7 Fusarillm, Pythiunl, lesion, Vegetables Peat carrier or Seed treatment (peat) or
Burkholderia cepacia spiral, lance, and sting liquid drip irrigation
nematodes
ConquerS = Victus 26 Pseudomonas to/assii Mushrooms Aqueous biomass Spray
Pseudomonas fluoresccns suspension
Contans9 Sclerotinia sclerotiorum and Canola, sunflower, peanut, Spray
Coniothyrium minitalls S. minor soybeans and vegetables
(lettuce, bean, tomato)
Deny (formerly PRECEPf Rhizoctonia, Pythium, Alfalfa, barley, beans, clover, Peat-based dried Applied to seeds with a
Burkho/deria cepacia (see Fusarium and lesion, spiral, cotton, peas, grain sorghum, biomass from solid sticker in planter box
Blue Circle) lance, and sting nematodes vegetable crops and wheat fermentation (an (aqueous suspension
aqueous formulation is for use in
suspension is drip irrigation or as a
awaiting EPA seedling drench) ......
approval) 00

'"
 ......
Table 5.1 (Contd.) \0
0

Product/biocontrol organism Target pathogen/disease Crop Formulation Application method

lO
DiTera Root-knot, citrus cyst, stubby Fruit, vegetable and Wettable powder,
Myrothecium verrucaria root, sting, lesion and ornamental crops, turf emulsifiable liquid
(killed cells) burrowing nematodes or granule
Epic l l Rhizoctonia solani, Fusarium Cotton, legumes Dry powder Added to a slurry, mix
Bacillus subtilis spp., Alternaria spp. and (5.5 x 1010 with a chemical fungicide
Aspergillus spp. spores/g) for commercial seed
treatment
Fusaclean12 Fusarium oxysporum Asparagus, basil, carnation, Spores, In drip to rock wool;
Fusarium oxysporum cyclamen, gerbera, tomato microgranule incorporate in potting
(non-pathogenic) mix; in row
Galltrol-A13 Crown gall disease, Fruit, nut and ornamental Petri dishes with Bacterial mass from one
Agrobacterium radiobacter Agrobacterium tumefaciens nursery stock pure culture grown plate transferred to one
on agar gallon non-chlorinated
(1.2 x lOll water, suspension applied
c.f.u./plate) to seeds, seedlings,
cuttings, roots, stems, and
as soil drench
Intercept 14 Rhizoctonia solani, Fusarium Maize, vegetables, cotton
Pseudomonas cepacia spp., Pythium sp.
Kodiak1!, Kodiak HB, Rhizoctonia solani, Fusarium Cotton, legumes Dry ~owder (5.5 Added to a slurry mix for
Kodiak AT spp., Alternaria spp. and X 10
1 spores/g); seed treatment; hopper
Bacillus subtilis Aspergillus spp. usually applied box treatment
with chemical
fungicides
Mycostop15 Fusarium spp., Alternaria Field, ornamental and Powder Drench, spray or through
Streptomyces griseoviridis brassicicola, Phomopsis spp., vegetable crops irrigation system
Botrytis spp., Pythium spp.
and Phytophthora spp.
Nogall, Diegall 16 Agrobacterium tumefaciens Trees Washed plates; Root dips
Agrobacterium radiobacter culture suspensions
Norbac 84C 17 Crown gall disease, Fruit, nut and ornamental Aqueous suspension Root, stem, cutting, dip or
Agrobacterium radiobacter Agrobacterium tumefaciens nursery stock containing bacterial spray
cells
(8 x 1010 c.f.u./IOO ml),
methyl cellulose, and
phosphate buffer
(refrigerate)
Phagus12 Pseudomonas tolassii Agaricus spp. Bacterial suspension
bacteriophage Pleurotus spp.
Polygandron 18 Pythium ultimum Sugarbeet Granule or powder Seed treatment or soil
Pythium oligandrum incorporation
pSSOL 12 Pseudomonas so/anacearum Vegetables
Pseudomonas so/anacearum
(non-~athogenic)
Rotstop 5, P. g. Suspension Heterobasidium annosum Trees Spores in inert Spray, chain saw oil
Ph/ebia g~antea powder
RootShield Pythium spp., Rhizoctonia Trees, shrubs, transplants, all Granules Mix with soil or potting
Trichoderma harzianum so/ani, Fusarium spp. ornamentals, cabbage, medium
strain T-22 tomato, cucumber
SoilGard (formerly Rhizoctonia so/ani and Ornamental and food plants Granules Granules are incorporated
GlioGard)2o Pythium spp. in greenhouses, nurseries, (1 X 106 c.f.u./g) in soil or soilless growing
Gliocladium virens homes and interiorscapes media prior to seeding
Supresivit21 Various fungi
Trichoderma harzianum
System 322 Seedling pathogens Barley, beans, cotton, peanut, Dust Seed treatment in planter
Bacillus subtilis and pea, rice, soybean box
chemical pesticides
T_22G 19 and T-22HB Pythium spp., Rhizoctonia Bean, cabbage, corn, cotton, Granules or dry Granules added in-
Trichoderma harzianum solani, Fusarium spp. and cucumber, peanut, sorghum, powder (both at furrow with granular
Sclerotinia homeocarpa soybean, sugar beet, tomato 1 x 10 7 c.f.u./g) applicator, by broadcast
and all ornamentals application to turf, mixed
with greenhouse soil, or
by mixing powder with
seeds in planter box, or in
commercial seed
treatment slurry
Trichodex 23 Primarily Botrytis cinerea, also Cucumber, grape, nectarine, Wettable powder Spray
Trichoderma harzianum Colletotrichum spp., Fu/via soybean, strawberry,
fulva, Monilia /axa, P/asmopara sunflower, tomato
viticola, Pseudoperonospora
cubensis, Rhizopus st%nifer,
Sclerotinia sclerotiorum
Trichopel 24, Trichoject, Armillaria, Botryosphaeria, See text, section 5.3 See text, section 5.3
Trichodowels, Trichoseal Chondrosterium, Fusarium,
Trichoderma harzianum and Nectria, Phytophthora,
T. viride Pythium, Rhizoctonia
Trichoderma 2000 25 Rhizoctonia so/ani, Sclerotium Nursery and field crops Incorporated into soil or
Trichoderma sp. rolfsii, Pythium sp. potting medium

.....
\0
.....
 ......
\0
N

Table 5.1 (Contd.)

Product/biocontrol organism Target pathogen/disease Crop Formulation Application method

26
Victus = ConquerS Pseudomonas tolassi i Mushrooms Aqueous Spray
Pseudomonas fluorescens suspension of
fermenter biomass
(3.5 x 109 cells/ml)

References:
1 Ecogen, Inc., Langhorne, Pennsylvania, USA: Jerusalem, Israel
2 Bio-Innovation AB, Bredholmen, Sweden, UK
3 SlAPA, GaIliera, Bologna, Italy
4 Grondortsmettingen DeCuester, St Katelijne-wavwe Belgium
5 EcoScience Corp., Worcester, USA
6 Plant Health Technologies, Boise, Idaho, USA
7 CTT Corp., Carlsbad, California, USA
8 Mauri Foods, North Ryde, Australia
9 Prophyta Biologischer Pflanzenschutz GmbH, Germany
10 Abbott Laboratories, Chicago, Illinois, USA
11 Gustafson, Inc., Dallas, Texas, USA
12 Natural Plant Protection, Nogueres, France
13 AgBioChem, Inc., Orinda, California, USA
14 Soil Technologies Corp., Fairfield, Iowa, USA
15 Kemira Agro Oy, Helsinki, Finland
16 Bio-Care Technology Pty Ltd, Somersby, New South Wales, Australia
17 New BioProducts, Inc., Corvallis, Oregon, USA
18 Vyskumny ustav rastlinnej [Plant Production Institute], Piestany, Slovak Republic
19 BioWorks, Inc. (formerly TGT, Inc.), Geneva, New York, USA
20 ThermoTriiogy (formerly W. R. Grace & Co.), Columbia, Maryland, USA
21 Borregaard and Reitzel, Denmark
22 Helena Chemical Co., Memphis, Tennessee, USA
23 Makhteshim Chemical Works, Ltd, Beer Sheva, Israel
24 Agrimm Technologies, Ltd, Christchurch, New Zealand
25 Mycontrol, Ltd, Israel
26 Sylvan Spawn Laboratory, Kittanning, Pennsylvania, USA
 5.2.2
grapes, apples, cucurbits, strawberries, tomat-
oes and ornamentals to control powdery mil-
dew. The mycoparasite Pythium oligandrum
(Polygandron) has been formulated as a gran-
ule or a powder for use as a seed treatment to
control pathogenic Pythium spp. on sugarbeet
(section 8.4.3c). One of the first microbes used
for control of a plant disease was Phlebia
gigantea (Peniophora gigantea) for control of
Heterobasidium annosum (Fomes annosus) on
trees (Rishbeth, 1979). Since the pathogen
can colonize freshly cut stumps then spread
through root grafts, the product Rotstop is
applied to freshly cut stumps.
5.2.2 PRODUCTS CONTAINING BACTERIA OR
ACTINOMYCETES
Bacteria (Table 5.1) have also been formulated
in a variety of ways to control plant pathogens
(see also section 8.4.2). Non-pathogenic Agro-
bacterium radiobacter (Galltrol-A, Nogall, Nor-
bac 84C, Diegall) is supplied on fresh or dried
Petri dish cultures to treat tree crops and
roses. Bacteria are suspended in non-chlori-
nated water and applied as dips or sprays to
seeds, seedlings, cuttings, stems or roots, or
they may be applied as a soil drench. Bio-Save
10 and Bio-Save 11 are wettable powders con-
taining different strains of Pseudomonas syrin-
gae. Although both are labelled for post-
harvest application to citrus and pome fruit
to control Botrytis, Penicillium, Mucor and Geo-
trichum, Bio-Save 10 is recommended for
citrus, and Bio-Save 11 for pome fruit. Pseudo-
monas fluorescens, formulated as an aqueous
suspension of fermenter biomass (Conquer,
Victus), is sprayed onto mushrooms to pre-
vent blotch caused by Pseudomonas tolassii.
Burkholderia cepacia (Pseudomonas cepacia), for-
mulated with a peat or clay carrier, is applied
with a sticker (Appendix Table 1.6) to seeds
(sections 8.3.3, 8.4.2c), and as a liquid suspen-
sion for seedling drench or application in drip
irrigation (Deny, PRECEP). Bacillus subtilis,
supplied as a dry powder (Epic, Kodiak), is
sometimes mixed with a chemical fungicide
Products containing bacteria or actinomycetes 193
for commercial seed treatment (section 8.4.2a).
Epic and Kodiak contain different strains of
the bacterium. Although Kodiak is sold by
itself, it is usually combined with chemical
fungicides. Kodiak HB is a hopper-box treat-
ment to overtreat commercially treated seed,
while Kodiak A . T is a hopper-box treatment
that also contains Apron and Terraclor. Penta-
chloronitrobenzene and metalaxyl are com-
bined with Kodiak for the seed treatment
System 3. BlightBan A506 (P. fluorescens), sup-
plied as a powder, is sprayed onto leaf sur-
faces and competes against indigenous strains
of P fluorescens to provide protection from
frost, and from Erwinia amylovora, cause of
fireblight on apples and pears.
The actinomycete Streptomyces griseoviridis,
Mycostop powder, can be used as a drench or
spray, or added through the irrigation system
to control Fusarium spp., Alternaria brassicicola,
Phomopsis spp., Botrytis spp., Pythium spp. and
Phytophthora spp. on field, vegetable and
ornamental crops.
5.3 TYPES OF FORMULAnONS
See also section 2.3 and Appendix III, and for
soil inoculants see section 7.5.
5.3.1 SEED TREATMENTS
In the simplest seed treatments, the biocontrol
formulation is applied to seed as a liquid or
powder (see also section 8.2.2). For example,
Burkholderia cepacia and Pseudomonas fluores-
cens were applied to pea seed with or without
captan for control of Pythium damping-off
and Aphanomyces root rot (Parke et al., 1991).
The bacteria provided control with or without
the captan. Ascospores of Talaromyces flavus
have been applied to potato seedpieces in a
pyrophyllite carrier (Fravel et al., 1985a).
Stickers (Appendix Table 1.6) are added to
most formulations for seed treatment in order
to stick propagules of biocontrol agents to the
seed (see also section 8.3.3). Pelgel has been
used to treat pea and soybean seeds with
194 Formulation of microorganisms to control plant diseases
Pseudomonas putida for control of Pythium ulti-
mum (Paulitz et al., 1992). Pelgel has also been
used to treat cucumber seeds with T. harzia-
num (Harman et al., 1991; Taylor et al., 1991).
This treatment was then overlaid with shale in
the sticker Polynox-N-I0, which was also
used to treat bean seed with conidia of T.
harzianum (Harman et al., 1991; Taylor et al.,
1991). Kloepper and Schroth (1981) mixed
various amounts of plant gums, xanthan
gum or methyl cellulose with talc to apply
plant growth-promoting rhizobacteria to
potato seedpieces. Populations of plant
growth-promoting rhizobacteria did not
decline in talc plus 20% xanthan gum for 2
months stored at 4 0c. This formulation pro-
vided significant increases in early plant
development in a field test on potatoes. The
bacteria did not survive in mixtures contain-
ing gum karaya or gum tragacanth. Carboxy-
methylcellulose has been used to apply
biocontrol agents to seeds and seedpieces to
control R. solani. Jager and Velvis (1985) used
1% carboxymethylcellulose with a clay carrier
to apply Verticillium biguttatum and other
antagonists to potato seedpieces. Plant
growth-promoting rhizobacteria have been
applied to seeds of sugarbeet using either
methyl cellulose or gum xanthan in combina-
tion with a neutralized peat or talc carrier
(Suslow and Schroth, 1982). Similarly, sur-
face-disinfested wheat seed was coated with
P. fIuorescens in 1% methyl cellulose for
control of take-all (Weller and Cook, 1983). A
polymer binder was used to pellet seeds of
Chinese aster and tomato with T. fIavus in
quartz flour (Nagtzaam and Bollen, 1994).
The T. fIavus was recovered from these seeds
after 17 years' storage under ambient condi-
tions.
Other technologies have been used to apply
seed treatments. Pythium oligandrum survived
the heat of a commercial seed-pelleting pro-
cess when used to treat various seeds (section
8.4.3c; Lutchmeah and Cooke,1985). Strains of
Trichoderma and Enterobacter cloacae were
delivered to tomato and cucumber seeds
through solid-matrix priming (Harman and
Taylor, 1988, 1990; section 8.2.5 and Table 8.2
in that section). In priming, seeds are brought
to a moisture level just below that required for
germination, moistened with water containing
Trichoderma or E. cloacae and then mixed with
either shale, bituminous coal or sphagnum
moss. The seeds plus carriers are then mixed
with water and incubated before planting.
Cucumber seeds were protected from
Pythium spp. by coating them with a liquid
formulation containing a binder (FeIgel or
Polyox N-I0), finely ground solid particulate
matter (Agro-Lig or muck soil) and T. harzia-
num. Plant stands comparable to those with
solid-matrix priming were obtained with this
formulation (Taylor et al., 1991). Fluid drilling
(section ~.2.5c) with an antagonist in a gel is
another way to increase emergence and
achieve a uniform plant stand. Germinated
pepper seeds were mixed with a hydrophobi-
cally modified hydroxyethyl cellulose (Poly-
surf-C gel, Appendix I) containing the
biocontrol agent Laetisaria arvalis (Conway,
1986). This treatment prevented damping-off
caused by R. solani (section 8.2.5c). Similarly,
T. harzianum and L. arvalis were applied in the
gel as a root dip to control Sclerotium rolfsii
(Conway, 1986).
A bio-priming seed treatment was devel-
oped for application of Pseudomonas fIuores-
cens to corn seed for control of Pythium
ultimum (Callan et al., 1990). The bacterium
was suspended in 1:5% medium-viscosity
methyl cellulose and coated onto previously
surface-disinfested, dry seed. Before planting,
coated seeds were incubated in moist vermi-
culite for 20-22 h, resulting in a seed moisture
content of 35-40%. During biopriming, the
population of P. fIuorescens increased from
lO-fold to over 10 OOO-fold, depending on
the initial inoculum level.
5.3.2 WETTABLE POWDERS AND LIQUIDS
Microorganisms such as P. putida, B. subtilis
and Trichoderma spp. - used to control post-

harvest diseases - are usually applied as
sprays, dips or drenches to fruit after harvest
(Tronsmo and Dennis, 1983; Colyer and
Mount, 1984; Pusey and Wilson, 1984; Wilson
and Pusey, 1985; sprays - sections 2.2.2b,
2.3.2a, b, 3.3.3, 3.3.4, 3.4, 4.3, 4.4.1). Spraying
pineapple fruit with attenuated strains of
Penicillium sp. in the field reduced post-har-
vest diseases (Lim and Rohrbach, 1980).
5.3.3 GRANULAR FORMULATIONS
Lignite silage has been used to make granules
containing Gliocladium roseum and T. harzia-
num for soil application to control of R. solani-
induced damping-off of peanut Oones et al.,
1984). Lignite was ground to produce gran-
ules 425-2000 f1.m in diameter, which were
amended with a thin silage, the product of
sorghum fermentation. Isolates of Gliocladium
virens and T. harzianum were grown on these
granules for 7 days. The granules were air-
dried and stored before adding in-furrow to
R. solan i-infested soil (see also sections 4.4.2,
7.5.3, 7.7).
Entrapment of biocontrol organisms in cal-
cium alginate granules, termed prill (Table
5.2; Table 6.7 in section 6.8), has been used
5.3.3 Granular formulations 195
widely (Connick, 1988). Alginates are biopo-
lymers that are stable when dry (Mugnier and
lung, 1985). Although most commercial algin-
ates are derived from kelp, other organisms
also produce alginates. Alginates produced
by Azotobacter vinelandii can substitute for
kelp alginate in the preparation of biocontrol
products to control plant pathogens (DeLucca
et al., 1990). Further developments may lead to
less expensive alginates. Sodium alginates dif-
fering greatly in purity and viscosity are avail-
able. We have found the more granular
sodium alginates, such as Kelgin, Kelgin HV
(Appendix Table 1.3) and sodium alginate IG-
350, easier to work with than the more pow-
dery alginic acids.
The general formulation for alginate is an
aqueous suspension of 1-5% sodium alginate,
10-20% bulking agent, and propagules of the
biocontrol agent (Fravel et al., 1985b; Lewis
and Papavizas, 1985). The suspension is
added dropwise into a gellant, usually
0.25M CaCh (calcium chloride) or 0.1 M
CaC12H22014 (calcium gluconate, Table 5.2).
Calcium chloride stays in solution, while the
calcium gluconate needs to be stirred fre-
quently to prevent precipitation. However,
many microbes retain greater viability in

Table 5.2 Preparation of alginate prill with antagonist fungal spores and a clay carrier (Fravel et aL, 1985b)

Ingredients Quantity Function

Antagonist suspension
Water 11 To create suspension for dripping
Sodium alginate 109 Gelant
Pyrophyllite 100g Carrier
Gelling solution
Water 11 To create a suspension to gel drops
Calcium chloride (dihydrate)" 36.8g To gel alginate suspension

Method
General remarks. Our laboratory usually makes the antagonist suspension in 4-1 batches, and the calcium
chloride solution in 2-1 batches. The amounts listed above are adjusted accordingly. We usually drip 50-
1501 per day using 3-6 carboys simultaneously. Thus we do some of the preparation the day before
forming prill, including refrigerating the carrier suspension. This suspension does not need to be
refrigerated and can be made just before use, providing it cools sufficiently that it does not damage the
antagonist. Some alginates can be made ahead of time and refrigerated, although many will not gel. Thus
we recommend autoclaving the alginate just before use.
196 Formulation of microorganisms to control plant diseases
The day before forlllillg prill. Autoclave equipment including lO-l-capacity carboys with spigots at the
base, rubber or autoclavable plastic tubing, Y-shaped tubing connectors, disposable plastic pipette tips (1
mm diameter orifice), spatulas, mixing bowls, mixing blades, screens for draining prill, blender
containers, and rectangular plastic tubs (48 x 27 x 13 cm high). Autoclave half of the water with the
carrier, then refrigerate overnight; we use 2 I water and 200 g pyrophyllite in each 4-1 beaker. If the
antagonist will be added as a spore or cell suspension, rather than biomass, autoclave water to use in this
suspension and adjust the amount of water added to the alginate suspension (below) accordingly. Cover
lab benches and the floor near the benches with kraft paper or newspaper.
The day prill are forllled. Mix the alginate with the remaining water. This is the most critical step in
production of prill, as lumps in the alginate will clog the tubing and tips, making dripping difficult. Place
the water in a blender and turn the blender on. With the blender running, remove the top and add the
alginate in a steady stream. Pour this suspension into a 4-1 beaker. Use remaining water to rinse the
blender container into the same beaker and autoclave the alginate suspension. After autoclaving, we often
place the alginate in a cold room or outside in winter to cool.
While the alginate is in the autoclave, remove the water-carrier suspension from the refrigerator. Attach
tubing and Y-shaped connectors to the bases of the carboys. The alginate suspension flows most evenly if
tubing connections at each level are the same length. The length of tubing between connections should be
2-4 cm. Make dichotomous connections until there are 16 ends for each carboy. Attach disposable pipette
tips to the ends of the tubing. Set carboys on the edge of the lab bench and place a rectangular tub on
the floor underneath each carboy. If necessary, pull out a lab bench drawer (protected with paper) to move
the dripping apparatus away from the bench so that drops will fall into the tub. It may be necessary to
tape tubing near the tips together so that all drops will fall within the tub. Mix the calcium chloride
solution and add 2 I of this solution to each tub.
Combine 2 I of the carrier suspension with 2 I of alginate suspension in a large bread mixer (Hobart
Corp., Troy, Ohio). Test the temperature of the mixture to make certain that it will not harm the
antagonist, and add the antagonist. The suspension will drip faster when warm, but tubing connections
may become loose if the suspension is too warm. Place the suspension in the carboy and open the spigot. It
is sometimes necessary to clamp off individual sections of the dripping apparatus to reconnect tubing or
replace clogged tips. Prill will form as the drops contact the calcium chloride solution. Periodically, shake
or stir the tub, otherwise 'mountains' of prill will form under each tip and these will rise above the height
of the calcium chloride solution. When the tub is full, replace it with a new tub of calcium chloride
solution. Drain the prill in window screen or net placed in a wire basket. Rinse the prill with water and let
them drain an additional 10-20 min. Spread the prill on kraft paper to dry. Ideally, the prill should be
dried under a hood, although we dry them on greenhouse benches with fans on when we make large
quantities. Prill will dry in 2-24 h, depending on how often the calcium chloride solution was changed
(frequent changes shorten drying time), fans and humidity.
* Anhydrous calcium chloride (27.7 gil), or calcium gluconate (43 gil) can be substituted.

calcium gluconate. When the drop of alginate
suspension enters the gellant, a shell of
calcium alginate gel is formed around the
drop. After a few minutes, the Ca 2 + diffuses
throughout the drop converting it to a gel
bead. These gel beads are rinsed and dried.
Most alginate formulations contain 104 to 106
viable propagules per gram of dry formula-
tion. The alginate formulation is amenable to
modification. The carrier may be inert (e.g.
pyrophyllite, vermiculite; Appendix Table
1.2), a nutrient source (e.g. wheat bran, maize
cobs), or any combination. Fungicides, fertili-
zers or other compounds may also be added.
For example, inclusion of dichloran, Rose
Bengal or cyfluthrin in alginate prill with
atoxigenic Aspergillus flavus did not affect the
number of spores produced after 2 years' stor-
age at 8C (Daigle and Cotty, 1995).
In order to develop a formulation, some-
times it is necessary to balance ease of pre-
paration with optimal conditions for the
biocontrol agent. For example, Daigle and
Cotty (1995) reported that wheat gluten
improved performance of atoxigenic A. flavus
but that concentrations above 5% were

difficult to process. The final formulation con-
tained 1% sodium alginate, 5% gluten and 5%
corn-cob grits. Similarly, Streptomyces sp. was
formulated in alginate using polyamide as a
carrier, since clay was difficult to keep in sus-
pension (Petrolini et al., 1988).
5.3.4 CARRIERS
Carriers (Appendix Tables I.1-I.3) are inert
ingredients in the sense that they do not
have pest control capabilities; however, they
can profoundly affect shelf-life and efficacy of
the product (see also sections 7.6.1, 8.3.2).
Polon (1973) lists 38 compounds that have
been used as carriers for chemical pesticides.
Although some of these are toxic to microbes,
most are likely to be useful for formulation of
biocontrol agents. During 12 weeks of storage
at 20C, Pseudomonas spp. survived better on
minerals with small particle size, such as
montmorillonite, zeolite and vermiculite,
than on minerals with larger particle size
such as pyrophyllite, talc and kaolinite (Dan-
durand et al., 1994). Addition of lactose
improved survival of the bacteria on the
minerals of large particle size. Backman and
Rodriguez-Kabana (1975) compared attapul-
gous clay and diatomaceous earth for their
water-holding properties, swelling of the
granules after wetting, disintegration when
wetted, water retention, and physical proper-
ties after autoclaving. Attapulgous clay gran-
ules swelled excessively in water and lost
their integrity if stirred while moist. In con-
trast, diatomaceous earth granules did not
swell significantly, withstood autoclaving,
and remained intact after stirring. The diato-
maceous earth could absorb up to 50% of its
volume of liquid, making it well suited to
absorb a molasses-based medium for delivery
of T. harzianum to soil. Bentonite clay en-
hanced the survival in soil of freshly cultured
or freeze-dried Rhizobium leguminosarum
(Heijnen et al., 1992).
Peat is commonly used to carry Rhizobium
spp. (sections 8.3.2a, 7.6.1) and is useful for
5.3.4 Carriers 197
seed coats and soil applications of biocontrol
agents. Huber et al. (1989) used finely ground
peat with a methyl cellulose sticker to apply
bacteria to wheat seed for control of take-all
root, crown and seed rot caused by Gaeuman-
nomyces graminis. Finely ground bark from
trees has also been used as a carrier (Stack et
al., 1988). Fusarium strains that induced resis-
tance to F. oxysporum f. sp. dianthi were mixed
with soil using a wheat bran-perlite mixture
or poplar bark compost (Garibaldi et al., 1987).
Alder bark has been used as a carrier to apply
Talaromyces flavus to potato seedpieces (Kei-
nathetal.,1990).
It has long been recognized that nutrition
affects virulence of plant pathogens (Huber
and Watson, 1974). Thus, it should not be
surprising that nutrients present during
production of biocontrol organisms and
added to the formulation can affect biocontrol
efficacy (section 7.6.9). Because many aspects
of antagonism depend on enzymes produced
by the biocontrol organisms, the amount and
type of nutrients in the formulation must
allow production of the desired enzymes
(Stack et al., 1988). The concentration and
source of carbon and nitrogen affected the
percentage of carrier granules from which
hyphae grew into soil, the number of hyphae
extending into the soil from each granule, and
the length of hyphaI strands (Stack et al.,
1988). Increased carbon:nitrogen ratios
(12:1 to 80:1) and increased molar concentra-
tions of both carbon and nitrogen sources
(0.02 to 0.18 M maltose and 0.006 to 0.024 M
arginine) enhanced proliferation of G. roseum,
Thelaviopsis basicola and Trichoderma spp. on
lignite granules (Stack et al., 1987). However,
ability to colonize the substrate-impregnated
granules was not related to performance in
soil. Similarly, when Talaromyces flavus was
formulated with eight different organic car-
riers, the best control of Verticillium dahliae on
eggplant was obtained in treatments with the
highest carbon:nitrogen ratios (159:1 for pyr-
ophyllite, 97:1 for corn cobs) (Fravel et al.,
1995). Carriers affect activity of the biocontrol
198 Formulation of microorganisms to control plant diseases
organism by providing a food base to aid
proliferation.
As more knowledge of mechanisms of bio-
control accumulates, it may be possible to
express desirable biocontrol traits by manip-
ulating nutrient composition of formulations.
For example, efficacy of G. virens GL-21
depends on the form of nitrogen in the for-
mulation. In general, inorganic nitrogen such
as nitrate inhibits conversion of active glio-
toxin to the inactive form, dimethylgliotoxin,
while organic nitrogen in the formulation
accelerates this conversion (R. D. Lumsden,
Plant Sciences Institute, USDA-ARS, Belts-
ville, Maryland, personal communication).
Some nutrients may even repress biocontrol
activity and should be avoided.
Alginate prill of G. virens with wheat bran
as a carrier gave more consistent control of
S. roifsii than when vermiculite plus wheat
bran was used as the carrier (Ristaino et al.,
1994). Alginate prill has been made using
either soya flour or oatmeal with F. solani f.
sp. cucurbitae for control of Texas gourd (Wei-
demann and Templeton, 1988; section 6.8 and
Table 6.7 in that section). After 6 weeks, the
alginate formulations of the mycoherbicide
provided 82-97% control, compared to 46%
control from the microconidia alone or 36%
control from a herbicide. There were no dif-
ferences between the soya flour and the oat-
meal. The addition of 2% oatmeal, corn meal
or soya flour to alginate prill increased soil
populations of the mycoherbicide F. solani f.
sp. cucurbitae compared to prill made without
food bases (Weidemann, 1988). The cost of
prill is relatively high (d. section 9.3.3).
Oils (Appendix Table 1.1) may provide a
food base as well as regulate water availabil-
ity in formulations that have been used for
application to soil (section 7.5.1d) and as
mycoinsecticides (section 4.3.2). Oils should
be explored for use in formulation of biocon-
trol organisms against plant diseases.
Although oils may be beneficial in some
instances, they may be deleterious in others.
Conidia of insect pathogenic fungi lost viabil-
ity in some oils but not others (section 4.6.5;
Tables 4.9 and 4.10 in section 4.6.5), while
conidia in other liquids also lost viability and
those in most dry formulations maintained
high viability (sections 4.6.6, 4.6.3 and 4.6.4;
Tables 4.11-4.13 in section 4.6.6; Table 4.8 in
section 4.6; Table 4.6 in section 4.6.4).
5.3.5 TOLERANCE OF THE ENVIRONMENT
Before formulation can begin, the biocontrol
organism must be cultured in such a way that
the desired propagules are produced. For bac-
teria and fungi, the long-term survival form is
usually the most durable and will often be the
preferred propagule for formulation. Most
microbes survive best when dried, and this
is the basis for laboratory techniques such as
storage of microbes on silica gel. One import-
ant function of a formulation is to regulate
water availability. Increased moisture often
causes microbes to break dormancy prema-
turely and encourages growth of contamin-
ants. Cells of Saccharomyces, Rhizobium,
Agrobacterium, Arthrobacter, and spores of
Penicillium spp. entrapped in polyacrylamide
gels remained viable for over 3 years when
stored at 28C and a water activity (a w ,
Appendix III) of <0.069 (Mugnier and lung,
1985; section 4.2). The microbes lost viability
as aw increased to 0.83. Influence of aw is
described and discussed in detail in sections
4.6.2, 4.8.2, 10.6. The addition of desiccants to
formulations of microbial pest control organ-
isms (sections 4.6.4, 4.6.5, 10.6; Appendix
Table 1.4) should be investigated more fully.
With some microbes, it may be necessary to
increase the water availability in a formula-
tion by addition of humectants (section 4.3.4;
Table 6.3 in section 6.5; Appendix Table 1.4).
Humectants in the fermentation medium can
also affect the subsequent shelf-life of a bio-
control agent. For example, growing T. harzia-
num in a medium containing polyethylene
glycol 8000 (PEG) increased the ability of con-
idia to withstand drying. In addition, conidia
produced on PEG-amended medium were

better able to protect cucumber seedlings
from attack by Pythium ultimum than those
grown in non-amended medium (Harman et
al., 1991). The effects of further media amend-
ments are described in section 4.7.2.
A novel method was developed to activate
biocontrol organisms. Dried, powdered bio-
mass of G. virens, Trichoderma hamatum or
T. harzianum was wetted with 0.05 N HCl.
This preparation was incubated for 2-3 days
at room temperature to stimulate the develop-
ment of young hyphae (Lewis et al., 1991;
1993). Treated biomass could be stored at
room temperature non-aseptically (under
cheesecloth) before incorporation into soil.
The activated biomass produced greater soil
populations of the biocontrol organisms,
which reduced survival and saprophytic
activity of R. solani more than non-activated
biomass (see also section 8.4.3f).
5.4 RESEARCH AND THE FUTURE
There are several experimental formulations
currently being investigated. In invert emul-
sions (section 2.3.2b; Appendix III), water dro-
plets (dispersed phase) are surrounded by oil
(continuous phase), reducing the evaporation
of water. Invert emulsions have been used
successfully for the formulation of mycoher-
bicides (Connick et al., 1991b; Womack et al.,
1996; section 6.6 and Table 6.5 in that section)
and should be explored for formulation of
biocontrol organisms against plant pathogens.
Invert emulsions can protect antagonist pro-
pagules on leaf surfaces after spray applica-
tion (Daigle et al., 1990). Pesta granular
formulations are based on extrusion of
wheat flour (gluten) dough with entrapped
biocontrol organisms and are promising for
application of mycoherbicides and entomo-
pathogenic nematodes (Connick et al. 1991a;
1993; section 6.8 and Tables 6.8, 6.9 in that
section; section 9.5.2;). Starch treated with
strong urea, alkali solutions or high heat
becomes gelatinized forming a cross-linked
structure in which biocontrol organisms can
5.4 Research and the future 199
be entrapped (Shasha and McGuire, 1992; sec-
tions 3.3.1, 3.3.5, 3.7.2a, b). Addition of isopro-
panol during mixing aids in granulation of
this formulation (B. S. Shasha, National Cen-
ter for Agriculture and Utilities Research,
USDA-ARS, Peoria, Illinois, personal com-
munication). This formulation has been used
to deliver G. virens and T. hamatum for control
of R. solani on peppers, eggplants and zinnia
in a soilless mix (Lewis et al., 1995).
The addition of feeding stimulants (Appen-
dix Table 1.7) such as Coax (Appendix I)
increases feeding by insects targeted with
Bacillus thuringiensis (section 3.4.3 and Table
3.14 in that section). For biocontrol organisms
that are insect disseminated, this technology
offers new opportunities for dissemination of
biocontrol organisms against plant pathogens.
Similarly, the addition of Congo Red, folic
acid or optical brighteners such as Leucophor
BS, Leucophor BBS, Phorwite AR, Phorwite
RKH and Tinopal LPW has been used to pro-
tect entomopathogens from ultraviolet light
(section 3.4.4e and Table 3.16 in that section;
section 4.3.3 and Table 4.4 in that section;
Appendix Tables 1.9-1.12) and these should
be explored for use with biocontrol organisms
against plant pathogens.
Compounds can be added to formulations
to give the biocontrol organism a selective
advantage in soil (section 7.6.9). For example,
an exotic nutrient source (Appendix Table 1.8)
in the formulation could favour growth of the
biocontrol agent. This could be a nutrient that
the biocontrol organism requires naturally,
or the trait could be specifically introduced
into the organism for the formulation. Thus,
a plasmid was introduced into Pseudomonas
putida rendering it capable of using salicylate
(Colbert et al., 1993). Bacterial cells, and later
salicylate, were applied through drip irriga-
tion lines. Salicylate significantly increased
populations of P. putida.
During the history of formulation of chemi-
cal pesticides and microbes used for nitrogen
fixation and insect biocontrot many methods
have been developed for formulation. In
200 Formulation of microorganisms to control plant diseases
addition, techniques developed in other areas,
such as microencapsulation of living cells for
use in medicine (Sun and O'Shea, 1985) may
have applications in agriculture, which have
not been investigated for use with biocontrol
microbes. Much of this work has not been
attempted due to the cost and amount of
labour involved. For example, if humectants
are needed, how many different compounds
should be tried? How many levels of each
compound should be tried? How often should
the formulated materials be checked, not just
for viability, but also for efficacy? In develop-
ing Colletotrichum gloeosporioides f. sp. aeschy-
nomene as a mycoherbicide, the Upjohn
Company encountered tvvo difficulties in for-
mulation (Bowers, 1982): many of the addi-
tives tested were toxic to spores, and
vigorous physical manipulations killed most
of the spores.
Research needs and future prospects are
correlated with those for other organisms in
Chapter 10.
ACKNOWLEDGEMENTS
Mention of a trademark or proprietary pro-
duct does not constitute a guarantee or war-
ranty of the product by the US Department of
Agriculture, and does not imply its approval
to the exclusion of other products that may
also be suitable. The authors thank the many
individuals who supplied information on
available products, the Biopesticides Division
of the US Environmental Protection Agency
for current information on registered
microbes, and Javier Gracia-Garza for transla-
tion from Italian.
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FORMULATION OF MICROBIAL
HERBICIDES
Michael P. Greaves, Peter J. Holloway and Bruce A. Auld
CONTENTS
6.1 Introduction
6.2 Propagule production
6.3 Factors affecting efficacy
6.4 Foliage application
6.5 Additive toxicity and synergy
6.6 Aqueous and oil-based sprays
6.7 Spray application
6.8 Solid formulations
6.9 The future and research requirements
Acknowledgements
References
6.1 INTRODUCTION
In his recent review, TeBeest (1996) points out
that 'the science of using plant pathogens to
control weeds is almost as old as the science of
plant pathology', and cites references show-
ing continuous effort in biological control of
weeds from about 1890. Regrettably, most of
the references report failures.
During the past four decades, much
research has focused on the discovery and
development of plant pathogenic microorgan-
isms as potential microbial herbicides. This
effort was rewarded quite early. In the 1960s,
the wilt fungus Acremonium diospyri was first
used to control persimmon trees (Diospyros
virginiana) in the Oklahoma rangelands
Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
6
203
206
208
210
210
216
223
223
227
229
229
(Griffith, 1970): a conidial suspension is
inoculated into wounds made by hand axes.
Similarly, a Cephalosporium sp. is wound-
inoculated to control Kolomona weed (Cassia
surratensis) in Hawaii (Trujillo and Obrero,
1976). Trujillo (1992) cites several other suc-
cessful biological control agents for woody
weeds. Although this form of microbial herbi-
cide does not involve formulation of the con-
trol agent in the accepted sense, Dorworth
(1995) has developed a novel inoculation
strategy to control red alder (Alnus rubra) by
inserting a pellet loaded with the fungus Nec-
tria ditissima into the stem of the tree using a
novel spring-loaded 'hammer'.
Clearly, the need for wound inoculation
limits the widespread use of a microbial
204 Formulation of microbial herbicides
herbicide. Accordingly, most research has
been directed at pathogens suitable for deliv-
ery from conventional spray equipment to the
foliage of target weeds, especially in agricul-
tural crops. Again, some success was soon
achieved: in the early 1980s, the first commer-
cial microbial herbicides were marketed.
DeVine, the first to be registered with the US
Environmental Protection Agency in 1981,
contains Phytophthora palmivora and controls
stranglervine (Morrenia odorata) in citrus
plantations in Florida. It is an aqueous
suspension of live mycelium and chlamydos-
pores, applied directly to the soil (Kenney,
1986). Collego, a wettable powder formula-
tion of spores of Colletotrichum gloeosporioides
f. sp. aeschynomene registered in 1982, controls
the legume weed northern jointvetch (Aeschy-
nomene virginica) in rice and soybeans.
Since the introduction of these two com-
mercial products, several other plant patho-
genic fungi have been developed as potential
microbial herbicides. However, none has yet
appeared on the market (Greaves, 1992, 1993).
Furthermore, DeVine and Collego were both
withdrawn in 1994, because their limited sales
were unable to sustain the costs of production
and marketing. Fortunately, DeVine has been
re-introduced, and possibly Collego will re-
appear in the near future. Even so, lack of
commercial success of microbial herbicides,
despite the intensive research stimulated by
the introduction of these two products, causes
concern. In 1989, at least 109 projects were
researching 105 fungal taxa with 69 target
weeds (Charudattan, 1991). Many other pro-
jects could not be included in this analysis
because of commercial confidentiality. Thus
the failure rate in the transition from research
project to commercial development is high.
Alternatively, lack of appropriate funding
curbs development (Charudattan, 1991). As
development funding is usually seen as
being more appropriate for the private sector,
its absence almost certainly indicates low
industrial confidence in the commercial viabil-
ity of potential products.
Failure of candidate organisms to achieve
product development status may be due to
many reasons. Inappropriate strain selection,
agent instability or selection of a non-
commercial weed target are but a few exam-
ples evident in the literature. However, far
more often it seems that unreliable perfor-
mance in the field is a major reason. This
also may have many causes. Production
difficulties can result in product variability;
storage may seriously reduce microbial
viability; poor method or timing of applica-
tion can reduce efficacy; and environmental
constraints may be present. Generally, storage
and, particularly, environmental constraints
are acknowledged to be the main causes of
unreliable performance (see analysis in Table
10.1, section 10.1). These two areas are most
likely to benefit from formulation. Indeed, the
1996 International Bioherbicide Symposium
in Stellenbosch, South Africa, agreed unani-
mously that formulation is a vital focus for
future research. Formulation is the combina-
tion of specific ingredients to provide a
practical crop protection product. Additives
(defined in Appendix III) are ingredients
added to maintain the long-term viability of
propagules and to facilitate their ease of hand-
ling, storage and application. Adjuvants are
proprietary products added to improve spray
delivery of a product or to enhance the patho-
logical effect of the propagules. Boyette et al.
(1996) have reviewed some aspects of the for-
mulation and application of mycoherbicides.
An effective biological control product
should be capable of uniform production
and have a shelf-life of 1-2 years, allowing
adequate time for it to pass through the
distribution and marketing network, as well
as for on-farm storage. Storage presents
particular problems arising from the wide
range of storage conditions. For example, tem-
peratures can range from 0 to 40C. A spore
is, in a sense, inherently formulated to resist
such conditions: fungi and bacteria have
evolved spores that will withstand severe
environmental fluctuations in order to ensure

dispersal and survival. Nonetheless, technical
formulation will, in most species, be essential
to ensure acceptable shelf-life.
By far the most frequently cited reason for
variable field performance of a microbial her-
bicide is the constraint imposed by climate
after application. All fungal spores require
free water, or at least exposure to high humid-
ity (sections 4.3.2, 4.3.4) to germinate, grow,
infect the host and express disease. Fre-
quently, moisture is not available for long
enough after application. Similarly, the spore
may be inhibited by low or high tempera-
tures, by UV radiation (sections 3.4.4, 4.3.3a)
or by lack of available nutrients. Most of these
constraints can be reduced or eliminated by
appropriate formulation (Table 10.1 in section
10.1). Unfortunately, formulation require-
ments have not been recognized, or have
been ignored, in much of the research on
microbial herbicides. Researchers have relied
on the use of aqueous suspensions of spores,
or other propagules, as inoculants for target
weeds. While this may give satisfactory
results in the controlled environments of
laboratory research, it will rarely - if ever -
produce a successful product for use in the
field. That goal will be achieved only by the
development of robust formulations designed
to meet stringent criteria. Such formulations
should enhance microbial health (sections
4.6.2, 4.6.7, 4.7.3) and performance relative to
the unformulated agent, and must be compat-
ible with standard agricultural practice. It
goes without saying that they must also be
economically practical and environmentally
acceptable. In practice, microbes will tend to
be formulated in much the same way as agro-
chemicals, because the industry is comforta-
ble with that system, but they do have some
special requirements beyond those of chemi-
cals. The unit processes and factors influen-
cing the infectivity of microbial herbicides
after application to foliage are summarized
in Fig. 6.1. The formulation of the microbe
must take into account all the performance
factors listed in the diagram.
6.1 Introduction 205
PROPAGULE DEPOSIT PERFORMANCE FACTORS
Weathering
Photodegradation
GERMINATION
Moisture content
Microclimate
Nutrients
APPRESSORIUM
FORMATION
Additives
Competition/antagonism
phylloplane organisms
PENETRATION OF
TARGET Number of propagules
Siting of propagules
INFECTION
Figure 6.1 Summary of unit processes and factors
influencing efficiency of spray applications of for-
mulations of microbial herbicides to foliage.
It is important to recognize that formulation
must be carefully integrated with the applica-
tion equipment and system to be used, either
in the laboratory or, more particularly, the
field. The current trend in pesticide applica-
tion is to use the lowest possible application
volume (Chapter 2; section 10.7). This con-
trasts markedly with the normal practice in
microbial herbicide research of spraying to
run-off. The continuous films of water on the
leaves, and the resultant inoculum distribu-
tion patterns, achieved by applying such
large volumes (sometimes>1000 l/ha), simply
cannot be achieved by conventional spraying
(section 6.4). This is another important factor
underlying the failure to repeat successful
laboratory results in the field. As yet, there
has been little attention paid to spray para-
meters (illustrated in AppendiX II), and it is
206 Formulation of microbial herbicides
BIOHERBICIDE FORMATION
Hydraulic
nozzle
DROPLET GENERATION 1II
o Number
PERFORMANCE o Size
FACTORS o Propagule content
o Additive content
IMPACTION
Reflection Run-off
I RETENTION ij . . .r-----t---~ I COVERAGE ~
I EVAPORATION ~
V
DEPOSIT FORMATION
Figure 6.2 Complex of interacting factors during
spray application of microbial herbicides.
beyond the scope of this chapter to review the
area. Suffice it to say that spray application
volume and spray distribution are critically
important for the efficacy of microbial herbi-
cides. Even the most sophisticated formula-
tion will not be effective if the spray
parameters are not optimized. The complex
of factors which interact, and which must be
accounted for, during spray application of
microbial herbicides to foliage are summar-
ized in Fig. 6.2.
Although relatively little research has
focused on formulation of microbial herbi-
cides, there have been several important
advances. These exploit both conventional
pesticide-type approaches and some novel
developments. However, living microbials
present complex problems with regard to
formulation and, often, conventional off-the-
shelf answers are unlikely to be appropriate
(Rhodes, 1990).
This chapter reviews the progress in formu-
lation of microbial herbicides, highlighting
innovative advances and assessing needs for
further strategic research.
Spinning
disc
6.2 PROPAGULE PRODUCTION
Production and formulation are closely inter-
related in the development of cost-effective
inoculants. Most microbial herbicides use
fungi which, in nature, depend on rain-splash
or wind for dispersal from the initial site of
infection. The need for lengthy reproduction
in the field and inefficient natural dispersal is
bypassed by inundative application. Indivi-
dual plants have physical and chemical bar-
riers, or a degree of tolerance to infection, that
require massive dosages, typically in the
order of 106 infective units per ml of spray
(Auld and Morin, 1995). Such dosages
dictate the minimum spray volumes and the
type of formulation. For example, they would
be unrealistic at ultra-low volume of, say,
10 1/ha, especially using biphasic formula-
tions such as an invert emulsion, in which the
inoculant may be partitioned into one phase.
Inoculant is usually mass produced by
submerged fermentation. However, as spores
are preferred for formulation, this method is
used only for those fungi that sporulate in the
submerged state, as with insect pathogenic
fungi (section 4.7.2 and Table 4.15 in that sec-
tion; Table 4.7 in section 4.6.4; Table 4.14 in
section 4.7.1). Mycelial inoculant can be effec-
tive, as with DeVine, but lack of shelf-life
precludes its widespread use. Alternatively,
mycelium can be harvested and treated to
induce sporulation, but this adds significantly
to the cost. Most commonly, inoculants are
spores separated from medium and mycelium
by filtration and centrifugation. Controlled
culture in the homogeneous medium in a
bioreactor is monitored and manipulated bio-
chemically to improve efficacy of the inocu-
lant. Propagules of the first two microbial
herbicides registered in North America have
been grown in submerged fermentation

(Bowers, 1986; Kenney, 1986; Cunningham
and Kuiack, 1989).
Dimorphism may occur in mass pro-
duction. Thus, in Collego, 80-85% of the
propagules are fission spores, 8-10% are con-
idia and 5% blastospores plus arthrospores
(Churchill, 1982). Also, Jackson and Schisler
(1995) have shown that microsclerotia, small
(200-400/lm), melanized, compact, hyphal
aggregates formed by Colletotrichum trunca-
tum in liquid culture, are effective inoculants.
Temperature, aeration and the balance
between nutritional elements control sporula-
tion of filamentous fungi in submerged cul-
ture. Extensive studies with C. truncatum
(Jackson et al., 1996a) have demonstrated
the potential complexities in optimizing
spore production. Carbon concentration and
carbon:nitrogen ratio strongly influenced
propagule production. Carbon concentration
regulated conidiation and microsclerotia
formation (Jackson and Bothast, 1990). Spore
concentrations were high at 4-16 g carbon per
litre and production was inhibited above 25 g
carbon per litre. This explained the failure of
early attempts to produce spores of C. trunca-
tum using a modified Richard's medium (ca
22 g carbon per litre). Similarly, Auld et al.
(1990) found that dilution of modified
Richard's medium to 10-20% of the normal
concentration increased spore production by
Colletotrichum orbiculare by more than six-fold.
Carbon:nitrogen ratio affects spore yield, as
well as fitness, in C. truncatum (Schisler et al.,
1991). Yields of fission spores were optimized
at 30:1, while 10:1 yielded spores which ger-
minated more rapidly, formed appressoria
more frequently and induced more disease
in the host. Also, conidia produced at 10:1
were longer and thinner than those at higher
ratios (Jackson and Bothast, 1990). Moreover,
at this ratio, conidia contained more prot~in
and less lipid than those from higher ratios,
implying a role for high protein in determin-
ing the infective efficacy of conidia (Jackson
and Schisler, 1992). Subsequently, it was
shown that a defined amino acid composition
6.2 Propagule production 207
of the nitrogen source improved production
of conidia (Jackson and Slininger, 1993).
Oxygen mass transfer is a major limitation
for aerobic processes in submerged culture,
because the solubility of O 2 in water is only
ca 6p.p.m. Slininger and Jackson (1992)
showed that spore germination, mycelial
growth and sporulation of C. truncatum had
increasing dissolved O 2 tension (DOT) re-
quirements for maximum production. How-
ever, although the DOT optimum for
sporulation (55%) was four times higher
than that for mycelial growth, the specific
O 2 demand of sporulating mycelium (4.9
(10 4 moll g/h) was an order of magnitude
below that of growing mycelium.
In vivo, spore production may be associated
with a mucilaginous matrix which may con-
tain several enzymes playing a part in appres-
soriaI adhesion and pathogenicity. The
enzyme composition of the matrix of C. orbi-
culare varies with changes in the composition
of the culture medium (McRae and Stevens,
1990). In submerged culture, any matrix pro-
duced would generally be lost in the dis-
carded liquor. Incorporation of concentrated
fermentation liquor into formulations, or sub-
stitution of an appropriate polysaccharide,
may be a useful means of increasing inoculum
efficacy (see also section 10.9).
The target shelf-life of 1-2 years has been
achieved by drying spores harvested from
submerged culture and mixed with an inert
filler such as kaolin (Mortensen, 1988). How-
ever, difficulties have been encountered in
stabilizing some fungal spores, including
those of C. truncatum (Jackson et al., 1996a)
and Phytophthora palmivora (Kenney, 1986).
This did not prevent commercialization of
the latter (as DeVine), which is custom-
produced for each order and supplied as
fresh spores and mycelium suspended in
liquid. It can be stored for only a few days in
the refrigerator before use. Collego is sold as a
wettable powder, consisting of conidia in an
undisclosed inert carrier. This may be diato-
maceous earth, which is probably used for
208 Formulation of microbial herbicides
filtration and helps to stabilize the conidia
during drying, as well as bulking up the
final product and aiding packaging. The pro-
duct package also contains bottles of an osmot-
icum, possibly sucrose solution. The spore
powder is suspended in this solution, to facil-
itate gradual rehydration of the spores prior
to germination (Bowers, 1986), and then
diluted with water prior to use (section 4.6.7).
For species unable to sporulate in sub-
merged culture, mycelium can be produced
and spread as a thin slurry in trays and
induced to sporulate by exposure to approp-
riate temperature and light regimes (Walker
and Riley, 1982). Alternatively, mycelium can
be formulated as alginate pellets (Walker and
Connick, 1983; section 6.8; Table 5.2 in section
5.3) or vermiculite (Walker, 1981; section
7.5.3b). The pelletized mycelium is spread in
a thin layer and air-dried. Alginate pellets
must contain a filler, such as kaolin, to pre-
vent them from drying to a thin disc and
becoming hydrophobic. In the field, the dry
pellets may be applied directly on the soil or
foliage rosettes, sporulation occurring when
they rehydrate with rain, dew or irrigation.
Alternatively, they may be stored for ca 6
months and sporulation induced by re-
wetting under controlled light and tempera-
ture regimes before application to the target.
Some microbial herbicides have been pro-
duced on materials such as wheat, straw (Hil-
debrand and McCain, 1978), oat grains and
corn meal (Boyette, 1982; Boyette et al., 1984).
However, large-scale fermentation systems
that use solid substrates are not readily avail-
able in the western world (Stowell, 1991).
Moreover, propagules in solid substrates
may be difficult to separate or extract for for-
mulation; alternatively, the substrate may
become part of the final formulated product,
as with some insect pathogenic fungi (section
4.7.1 and Table 4.14 in that section). Never-
theless, solid substrate fermentation may be
the only method to produce some fungi.
Indeed, it may be the preferred method in
some countries with a tradition of producing
certain foods by solid state fermentation
(Auld, 1993a).
6.3 FACTORS AFFECTING EFFICACY
To ensure that the most appropriate formula-
tion is developed for a particular microbial
herbicide, it is essential to understand the fac-
tors which can affect efficacy. Each factor may
need a specific formulation requirement, all
the requirements having to be balanced,
usually through compromise. Probably the
most important factor is distribution of the
inoculant in the target area (section 2.2.2).
This is the result of a series of processes
which independently and interactively influ-
ence deposit formation (Fig. 6.2 in section 6.1).
Just as with spraying chemical herbicides,
much of the inoculant will miss the target
plants (Combellack, 1981). Some may drift as
far as 20 m for ground sprays or up to 160 m
for aerial sprays (Marrs et al., 1993). However,
unlike some chemical herbicides, the active
ingredients of microbial herbicides are parti-
culate and do not vaporize. Thus they can
travel great distances, sometimes up to 90 km
(Robinson and Fox, 1978). Nonetheless, inocu-
lant efficiency should be maximized by select-
ing a formulation and application system
which minimizes losses from the target area
(Appendix Table II.3). This requires attention
to both retention and distribution on the tar-
get plant.
Distribution of inoculum on individual
plants has received little critical attention.
However, Potyka (1996) has shown that when
aqueous spore suspensions are sprayed
onto the leaves of Viola arvensis at large appli-
cation volumes (ca 1000 l/ha), much spray
accumulates at the leaf tips and edges, result-
ing in concentration of spores at these points.
Also, many spores are likely to be lost from
the target in run-off. On the other hand,
movement of inoculant from leaf blade to
axillary buds or to the stem base may be
desirable. It is a common experience that a
microbial herbicide kills all the leaves on its

target but that the weed re-grows from axil-
lary buds, especially those low on the stem,
producing a multi-stemmed plant which
eventually becomes more competitive than if
it had not been treated. Movement of sus-
pended spores from sites of deposition to axil-
lary buds is not easy to achieve in a controlled
way. While the liquid phase can be spread to a
certain degree by addition of, for example,
surfactants, the movement of spores is likely
to be restricted by frictional drag on the leaf
surface, particularly on hairy or otherwise
rough leaves, and if the spores are large.
Also, spores may be trapped in the bound-
aries of applied droplets and then deposited
as annular aggregates when the droplets eva-
porate (Potyka, 1996; sections 3.3.3a, 4.3.1; Fig.
4.3 in section 4.3). Surfactants tend to increase
the rate of evaporation by reducing surface
tension. Oil-based formulations are more vis-
cous than aqueous formulations (Appendix
Table II.4) and may be more effective at mov-
ing spores across hydrophobic plant surfaces,
which they readily wet (section 4.3.1). Also,
they are generally less volatile than water and
should thus avoid the clumping of spores that
occurs when aqueous deposits evaporate.
This remains to be demonstrated in practice.
Another approach to distributing inoculum
to the base of the plant is to apply a sticky
formulation from a nozzle delivering an
angled spray close to ground level. In prac-
tice, such 'drop-leg' sprayers are not popular
because they are difficult to adjust and main-
tain at low heights - they tend to act as culti-
vators rather than sprayers. Electrostatic
spraying may increase deposition on the
lower parts of plants. As yet, however, no
appropriate commercial form of such a
sprayer is available.
As a general rule, plant species with a
determinate growth habit are easier to control
with a foliar pathogen than those with inde-
terminate growth that enables survival, parti-
cularly through rhizomes or stolons. Weeds of
the latter type, such as the water hyacinth
Eichhornia crassipes, are formidable targets as
6.3 Factors affecting efficacy 209
growth of the weed may exceed that of the
infecting fungus (Charudattan et al., 1985). For
these, formulation with suitable plant growth
retardants may offer a way forward (section
6.5).
Any weed population is likely to contain
genetically diverse biotypes which may
include some with resistance to the microbial
herbicide. So far this has been encountered
only as a difference between sites widely
separated geographically. Should it occur
within one site, as it often does for chemical
herbicides, it might be countered by formulat-
ing mixtures of selected biotypes of the
biological control agent (Weidemann and
TeBeest, 1990) or, perhaps, by adding an
ultra-low concentration of a selective herbi-
cide to the formulation.
Apart from intrinsic plant factors, there are
many others which act separately or together
to influence the performance of a microbial
herbicide (Fig. 6.1 in section 6.1). These affect
one or more of the events between propagule
deposition on the target and establishment of
infection. Formulation attempts to overcome
any constraints they may impose.
In the aerial environment of plants, by far
the greatest factor influencing efficacy of most
microbial herbicides is the requirement for
extended periods of dew or free water (Say,
1990; Auld and Morin, 1995), or sequential
periods of dew (Walker and Boyette, 1986),
before severe disease can develop. Further-
more, a delay in the onset of dew after spray-
ing can result in low levels of infection (Auld
et al., 1988; Boyette, 1994). Timing sprays to
take advantage of rain, dew and irrigation is
one strategy to overcome this problem (Auld
et al., 1990; Makowski, 1993). Unfortunately,
precipitation often cannot be predicted accu-
rately enough to rely on this approach; dew or
rain may not occur when weed control is
needed.
Dew requirement and temperature typ-
ically interact (McRae and Auld, 1988) so
that, at high temperatures, shorter dew peri-
ods may suffice and vice versa. This suggests
210 FormuLation of microbiaL herbicides
that the hot, humid tropics, with predictable
rainfall, are the best areas for microbial herbi-
cide use (Auld, 1993a). They are, also, the
regions whose ecosystems are most sensitive
to perturbation and whose people are increas-
ingly opposed to chemical herbicides.
Formulation research to overcome or
reduce dew requirements in microbial herbi-
cides is now an urgent need. A need for
shorter dew periods can improve reliability
and efficacy, as well as reduce inoculant
dose requirement (Amsellem et aL., 1990).
This, in turn, may reduce the output required
from mass production and improve the eco-
nomics of product development.
6.4 FOLIAGE APPLICATION
Although it is possible to use granular form-
ulations to inoculate plant foliage, especially if
the granules have a sticky surface and the
plant has a rosette of leaves at or near the
soil surface, this approach has been limited
to soil inoculation. Microbial herbicides, typ-
ically, have been sprayed in the field as aqu-
eous suspensions using similar equipment to
that used for conventional herbicides (Table
2.2 in section 2.2.2), but often using consider-
ably greater application volumes. Laboratory
experiments have almost exclusively used
some form of atomizer (e.g. an air-brush or
aerosol spray). Conventionally, target plants
have been sprayed to run-off, an application
volume equivalent to 1000l/ha or more.
While this optimizes the chances of the microb-
ial herbicide reliably producing successful
infections, and so facilitates experimental pro-
gress, it seriously impedes assessment of the
real potential of the agent to perform satis-
factorily in field conditions. The reason for
the use of such high application volumes is
obscure. It may have something to do with the
requirement by fungal plant pathogens, the
commonest organisms used as microbial her-
bicides, for lengthy exposure" to very high
humidity or to free water to achieve maxi-
mum infection, perhaps by perpetuating the
techniques traditionally used in crop patho-
logy. Often, it may simply be an attempt to
ensure that even target cover is achieved.
However, high volume rates do not necessa-
rily ensure persistence of the applied water or
even distribution of inoculant. Much of the
water can run off the leaf, moving spores to
the edges of the leaf and, sometimes, remov-
ing them. Klein and Auld (1995) have shown
that 1000l/ha was, generally, not superior to
500 and 250l/ha with Colletotrichum orbicuLare
to control Xanthium spinosum. Presumably the
rate of evaporation from the plant surfaces
was influenced more by the ambient humidity
and temperature than by the volume of water
retained on the leaf. Potyka (1996) has shown
that even very large (2 pJ) droplets placed on
the leaf surface will evaporate completely in a
very short time. In still air at 21C and 65%
RH, evaporation was complete in 45 min.
Clearly, the application of fungal inoculant
onto target plants is beset with many difficul-
ties which formulation may help to overcome,
especially if the application technique is fully
taken into account when designing the pro-
duct. In the following sections, the different
additives used to develop formulations of
microbial herbicides will be considered.
6.5 ADDITIVE TOXICITY AND SYNERGY
Obviously, any additive should not exert
adverse effects on the microbe it is supposed
to support. Known adverse effects of indivi-
dual additives are assembled in Appendix I.
Surprisingly, most papers in the literature do
not seem to have considered adverse effects,
although they may have been assessed and
not reported. This seems unlikely, however,
as much valuable information would, thus, be
repressed. More likely, particular additives
have been selected because of use by others
with apparent success. This is a potentially
dangerous route to follow, because additives
can exert a wide range of effects on different
fungal species, or even on different strains of
the same species. Additives to be used with a

Table 6.1 Effects of formulation ingredients on production and germination of conidia of Colletotrichum
gloeosporioides f. sp. malvae (Grant et a!., 1990a)
Ingredient (0.1 % wlv)
Agral 90 (nonylphenoxy polyethoxy ethanol)
Assist (paraffinic mineral oil)
Bio-Veg (vegetable oil)
Enhance (tallow amine ethoxylate + nonylphenoxy
polyethoxy ethanol)
Starch*
Tween 20 sorbitan monolaurate
ethoxylate
Water (control)
* Tested concentration = 0.1 gil.
particular fungus must be checked for intrin-
sic toxicity to that strain, both singly and in
combination with any other additive which is
to be included in the final product (Daigle and
Cotty, 1991; sections 3.4.1, 4.3.6). Many meth-
ods have been used to measure toxicity, as
with fungal entomopathogens (sections 4.3.6,
4.3.7). These range from simple growth tests
on agar media to more complex assessments
of spore germination, appressorium forma-
tion (Tables 6.1 and 6.2), infection and disease
expression on the host plant.
In general, methods involving some mea-
sure of the organism's interaction with its
host are to be favoured. Conditions in agar
culture are so far from those met during prac-
tical use of the organism as to make the results
obtained of dubious value (sections 10.6, 10.7).
Among the factors to be considered is concen-
tration. It seems naive to test only the concen-
tration to be applied in a spray, or that plus
one other, usually five or ten times higher,
when the water in the spray all evaporates
within minutes after arrival on the leaf sur-
face. Thus, at least in aqueous formulations,
dissolved additives concentrate rapidly, satu-
rate the solvent and dry out. Then the microbe
is exposed, albeit briefly, to very high concen-
trations, and finally to the additive dried to
equilibrium with air humidity, which may be
more damaging than long exposure to lower
6.5 Additive toxicity and synergy 211
Spore production Spore germination
(% of control) (% of control)
0.0 52.6
100.3 5.4
101.9 238.9
4.3 3.7
101.9 463.4
100.5 123.6
100.0 100.0
concentrations. It is now well established that
osmotic conditions, temperature and rate of
drying all have to be critically controlled if
fungal spores are to be preserved without
damage in culture collections and in stored
microbial products (sections 4.6.2, 4.6.4, 4.6.5,
4.7.3). The requirements for ensuring that
spores survive on the leaf surface and pro-
duce infections are no less stringent. This
stresses the absolute need for much more
attention to be paid to proper assessment of
additive toxicity.
Most attention to date has been given to
pesticides. This reflects the concern that
active ingredients and accompanying addi-
tives applied during normal crop husbandry,
as well as any used to increase the efficacy of
the microbial herbicide, may be damaging.
Prasad (1993, 1994) examined the effects of
several pesticides on radial growth on agar
of Chondrosterium purpureum, a potential
microbial herbicide for controlling woody
weed species. All the pesticides tested were
toxic at 0.1% w/v (in the agar) in this assess-
ment. However, while reduction of extension
growth may not be of great significance to a
microbial herbicide, spore germination and
formation of appressoria are important. Cer-
tainly, Prasad's results cannot justify his con-
clusion that many additives impair the
viability of microbial herbicides; in fact, very
212 Formulation of microbial herbicides
Table 6.2 Effects of some herbicides on the growth and behaviour of Colletotrichum gloeosporioides f.sp.
malvae on agar plates (Grant et aI., 1990b)

Herbicide (kg/ha) Mycelium t Germination (%) Appressorium t (%)

Control 0 82.2 67.7

2,4-0 amine (0.9) 0 85.9 54.4
2,4-0 ester (1.0) 0 91.8 48.0
2,4-0B (1.1) + 2.8 4.2
Benazolin (0.7) 0 86.8 18.3
Bentazone (1.1) 0 79.0 81.0
Bromoxynil (=B) (0.4) 0 74.1 53.5
B+MCPA* (0.3+0.3) 0 80.2 15.1
B+MCPA* (0.3+0.3) 0 42.1 34.2
Clopyralid (0.3) 0 66.1 94.6
Cyanazine (=C) (1.4) 0 83.7 93.5
C+MCPA-K (0.3+0.5) 0 82.8 81.4
Oicamba* (0.4) 0 83.8 77.1
Oicamba* (0.4) 0 61.8 98.4
Oiclofop (0.8) ++ 0 0
Oifenzoquat (0.9) +++ 0.4 0
Fenoxaprop-ethyl (0.2) ++ 0 0
Flamprop-methyl (0.4) ++ 0 0
Imazethapyr (1.1) 0 82.1 88.1
Mecoprop+2, 4-0 (1.4+1.4) 0 72.8 79.9
Metribuzin (0.2) + 88.2 40.9
MCPA-Na (0.5) 0 81.1 62.0
Picloram (1.1) 0 60.8 93.6
Propanil (1.0) +++ 0 0
Sethoxydim (0.8) ++ 0.2 0

* Different formulations.
t 0 = no effect, +, ++, +++ = moderate, severe or total inhibition of growth.
t Appressorium formation expressed as percentage of total spore germination.

few completely prevented growth. Fortui-
tously, his conclusion is the correct one.
Grant et al. (1990a) studied in detail the effects
of 45 pesticides on survival of Colletotrichum
gloeosporioides f. sp. malvae, a microbial
herbicide for the control of Malva pusilla
(round-leaved mallow). They applied spore
suspensions containing the pesticide under
test to the surface of agar plates at a rate
equivalent to 780 l/ha and at pesticide con-
centrations equating to x0.1-2 of the recom-
mended field dosage rate. They claimed that
'Once on the plates, the water from the test
suspension was readily absorbed by the agar,
and thus, the spores and pestiCide remaining
was equivalent to what would be left after
spraying in the field for the given area'.
While this may be true for wettable powder
suspensions, it cannot be so for soluble pesti-
cides. The latter will diffuse into the agar
along with the water and be effectively
diluted, as opposed to being concentrated, as
occurs on the leaf surface during drying of
spray deposits. Despite this, the results
obtained do show clearly the range of effects
that can be encountered (Table 6.2). All the
chemical herbicides for grass weed control,
at recommended rates, totally inhibited spore
germination and appressorium formation.
A similar result was found with herbicides
registered for control of both grasses and
broadleaf weeds (Table 6.2). In contrast,
herbicides registered for control of only broad-
leaf weeds, at recommended field dosage,

caused no more than a 20% reduction in
spore germination, while appressorium for-
mation was significantly reduced by only
two of these herbicides. Three further herbi-
cides (cyanazine, cyanazine+MCPA-K, and
imazethapyr) increased appressorium forma-
tion without affecting spore germination,
offering an opportunity to enhance the effi-
cacy of the microbial herbicide. Predictably,
most of the fungicides were strongly inhibi-
tory to the fungus. A more realistic assessment
of herbicide-fungus interactions was made
by applying fungus to its host weed before,
mixed with and after a number of herbicides
(Grant et al., 1990b). This approach gave a
similar range of results to those obtained on
agar (Table 6.2). Split applications of four
herbicides with the fungus improved weed
control compared with the fungus alone; six
herbicides could be mixed with the fungus at
recommended rates without affecting its effi-
cacy (giving opportunity to extend the spec-
trum of weeds controlled by the formulation);
and two significantly impaired fungal activity,
assessed as disease development on the plant.
Enhancement of microbial herbicide action by
a crop protection chemical was exploited by
Wymore et a!. (1987) and Wymore and Watson
(1989) who used a synergistic mixture of the
plant growth regulator thidiazuron with
Colletotrichum coccodes to increase seedling
mortality in velvetleaf (Abutilon theophrasti).
Thidiazuron stimulates axillary bud develop-
ment, causes cupping and curling of leaves
and reduces shoot height, weight and leaf
area. The resulting slow-growing, short,
bushy plants provide a microclimate with
less air movement than that through the
canopy of an untreated plant. Also, the weed
will remain within the microclimate of the
crop canopy. These two factors are likely to
provide a better, more humid, environment
for the fungus, with better prospects of
establishing a lethal epidemic. Furthermore,
the reduction of stem elongation rates also
reduces the chances of the plant outgrowing,
and surviving, the developing disease. The
6.5 Additive toxicity and synergy 213
same principle was exploited by Scheepens
(1987) to aid control of barnyard grass (Echino-
chloa crus-galli) by Cochliobolus Iunatus. Here,
the synergistic effect between fungus and her-
bicide was achieved with a low dose of the
chemical (2.5 mg/m 2 ). Gohbara and Yamagu-
chi (1993) showed that the herbicide pyrazo-
sulfuron-ethyl synergizes the control of
barnyard grass by Dreschlera monoceras at the
much lower dose of 0.044 mg/m 2
Synergism between plant pathogenic fungi
and crop protection chemicals has long been
recognized in relation to crop diseases. Iatro-
genic diseases, that is diseases resulting
from man's management of the crop, espe-
cially in relation to chemical inputs, have
been reviewed by Griffiths (1981). Much of
the information about chemically induced
increases in crop disease is directly relevant
to formulation of microbial herbicides and,
when taken in conjunction with the sort of
results presented above, is a powerful argu-
ment for focusing significant effort on this
potentially highly rewarding area (Christy et
a!., 1993; Hoagland, 1996). It also strengthens
the case for paying more attention to testing
interactions between any proposed additives
and the fungus needing formulation. Efforts
to avoid toxic effects on the fungus may pro-
duce a bonus in finding an additive that
significantly enhances one or more fungal
characteristics essential to the infection pro-
cess and establishment of disease.
Surfactants (listed in Appendix Table 1.5),
probably the most commonly used additives
at present, are usually regarded as essential
to ensure relatively uniform distribution of
inoculant on the leaf, especially if its surface
is hydrophobic. They may also serve as emul-
sifiers, spreaders and stabilizers. They can
penetrate lipophilic barriers, such as the cuti-
cle, which may aid retention of inoculant and
increase the probability of infection. In view
of this penetrative ability it is not surprising
that many are toxic to some fungi. In general,
non-ionic surfactants are less toxic than the
ionic forms, and are preferred for the formu-
214 Formulation of microbial herbicides
lation of microbial herbicides. Tween 20, sor-
bitan monolaurate ethoxylate (Mitchell, 1986,
1988; Cardina et al., 1988), Tween 80, sorbitan
monooleate ethoxylate (Walker, 1981;
Andersen and Walker, 1985; Boyette and
Walker, 1985) and non-oxynol surfactants
(Walker, 1981; Boyette, 1986, 1988; Charudat-
tan et aI., 1986) are, probably, the most fre-
quently used non-ionic surfactants. Despite
such widespread use, these surfactants can
be toxic to some fungi (Boyette et al., 1991;
section 4.3.6). Thus, Table 6.3 shows that
0.1% (v/v) Tween 20 significantly reduced
formation of both conidia and appressoria by
Colletotrichum dematium, a pathogen of fathen
(Chenopodium album). At 1.0% (v Iv), spore
germination was completely inhibited. In con-
trast, Tween 80 at 0.1 % significantly increased
spore germination in this species. Potyka
(1996) obtained similar results with Mycocen-
trosporum acerina, a leaf spot pathogen of field
pansy (Viola arvensis). McElwee et al. (1990)
used the non-ionic, silicone-based surfactant
Silwet L77 (known to enhance uptake of the
herbicide asulam by bracken, Pteridium aquili-
num) to formulate conidia of Ascochyta pteridis,
a potential microbial herbicide for bracken.
Silwet L77 slightly increased damage by the
pathogen. Other experience suggests that this
surfactant may, however, be toxic to a wide
range of fungi, including C. dematium, M. acer-
ina, Dreschlera sp., Alternaria spp. and Phoma
exigua (M. P. G., J. Lawrie and I. Potyka, Long
Ashton Research Station, Bristol, unpublished
data).
Humectants (listed in Appendix Table 1.4),
with their affinity for water, can stabilize the
water content of materials despite humidity
fluctuations. They include a wide range of dif-
ferent molecules, such as glycerol, sorbitol and
acetamide-,6-mercaptoethylamine (AMEA)
(section 4.3.4). Despite their stabilizing poten-
tial, they have not yet been widely used in
microbial herbicide formulation, perhaps
because they can be costly; they can also be
toxic. Potyka (1996) found that, although
~ 10% (v Iv) glycerol had no effect on conidial
germination in M. acerina, it significantly sti-
mulated it in C. dematium (Table 6.3). Appres-
sorium formation in this species was not
affected by glycerol at any concentration, but
was completely inhibited at 10% (v Iv) in M.
acerina. Both AMEA and lactamide-,6-mercap-
toethylaminel AMEA blend (LAMEA) had no

Table 6.3 Effects of some surfactants and humectants on

conidial germination and appressorium formation by
Colletotrichum dematium (Potyka, 1996)

Additive Concentration (% w/vt

0.01 0.1

Surfactants
Tween 20 + (0) -(-)
Tween 40 + (0) + (0)
Tween 60 + (0) + (0)
Tween 80 + (0) + (0)
Tween 85 + (0) + (0)
Humectants
AMEA 0(0) -(0)
LAMEA 0(0) -(-)
Glycerol + (0) + (0)

Figures in brackets are appressorium formation; -, inhibition:

0, no effect: +, stimulation, all compared with control.

effect on conidial germination at very low
(0.01 % w Iv) concentration (Table 6.3) but
completely inhibited it at higher concentra-
tions in both fungi. The low concentration
reduced appressorium formation by both spe-
cies. Tested only against M. acerina, sorbitol
significantly stimulated germination at con-
centrations up to 10% (w Iv) but had little or
no effect on appressorium formation.
Stickers have not been used widely (section
3.4.2; Table 3.13 in section 3.4.2; Appendix
Table 1.6). They increase spore adhesion to
the target leaf and prevent wash-off by rain,
while many also have humectant properties.
Solutions of sodium alginate are particularly
useful. McElwee (1987) formulated mixed
conidia of Ascochyta pteridis and Phoma aqui-
lina in 0.5 and 1.0% (w Iv) aqueous Manucol
OM (Kelcol AlL International Ltd), fortified
with 1% malt broth as a germination stimu-
lant, for the control of bracken (Pteridium aqui-
/inum). This gave a small but significant
increase in damage to the bracken, and a
higher incidence of pathogen re-isolation
6.5 Additive toxicity and synergy 215
from inoculated tissue, suggesting improved
spore retention andlor host penetration.
As most stickers are complex polysacchar-
ides of biological origin, it might be expected
that the majority would have little toxicity.
Unfortunately, this is not so. Potyka (1996)
found varying effects on spore germination
and appressorium formation in C. dematium
and M. acerina. Thus, both medium-and
high-viscosity alginic acids at 0.1% (w/v)
inhibited germination of C. dematium conidia,
while the low-viscosity form had no effect
(Table 6.4). The alginates Manucol OMF and
Manutex RH stimulated germination at 0.01 %
w Iv, whereas Manugel GHB inhibited it.
Similarly, microbial xanthan gum inhibited
germination at a concentration as low as
0.01% (w Iv), whereas plant gums (acacia,
ghatti, guar and locust bean) tended to
enhance it, the effect usually increasing
with increasing concentration. Appressorium
formation was significantly inhibited by only
one of the gums (locust bean). With M. acerina
conidia, all viscosity types of alginic acids

Table 6.4 Effects of some stickers on conidial germination and

appressorium formation by Colletotrichum dematium (Potyka, 1996)

Sticker Concentration (% wlv)'

0.01 0.1

Gums
Acacia + (0) + (0)
Ghatti + (0) + (0)
Guar + (0) + (0)
Karaya + (0) 0(0)
Locust bean + (-) + (0)
Xanthan -(0) 0(0)
Manucol DMF + (0) + (0)
Manugel GHB -(0) 0(+)
Manutex KPR 0(0) + (0)
Manutex RH + (0) 0(0)
.Alginic acids
Low viscosity 0(0) 0(0)
Medium viscosity 0(0) -(0)
High viscosity 0(0) -(0)

Figures in brackets are appressorium formation; -, inhibition; 0, no effect;

+, stimulation, all compared with controL
216 Formulation of microbial herbicides
inhibited germination at only 0.01% (w Iv)
but, surprisingly, at 0.1% (w/v) only the med-
ium-viscosity type was inhibitory. Alginates
caused similar effects to those noted on
C. dematium, as did xanthan gum. Some stick-
ers tested (acacia and ghatti gums) seemed
consistentl}j to increase appressorium forma-
tion in M. acerina, but the effect was small and
not statistically significant.
Shabana et al. (1997) evaluated eight hydro-
philic polymers for their capacity for hydra-
tion, duration of hydration, promotion of
spore germination and support of germling
viability in two species of Alternaria. At some
concentrations at least, all eight (gellan and
xanthan gums, Metamucil, Kelgin-HV, -MV
and -LV, polyacrylamide and N-gel) reduced
the percentage of germlings formed from
viable spores. Effects on appressorium forma-
tion were not assessed, but assay of the viru-
lence of mycelial inoculum in the presence of
the gels revealed no toxic effects.
Recently, attention has focused on oils and
oil emulsions (listed in Appendix Table 1.1) as
carriers for microbial herbicides. Generally,
mineral oils may be toxic to fungi, although
the inoculation of hardwood tree stumps with
slurries of Chondrosterium purpureum spores in
motor oil and chainsaw oil has given promis-
ing results (Wall and Simpson, 1989). Potyka
(1996) examined ten vegetable oils - four that
dried to form a tough, elastic skin (grape seed,
linseed, sunflower and walnut oils); three
non-drying (corn, sesame and soybean oils),
which slowly become rancid; three intermedi-
ate forms (groundnut, olive and rapeseed or
canola oils). Colletotrichum dematium conidia
failed to germinate in any of the oils on glass
slides or in tissue culture plates, and only a
maximum of 2% of those of M. acerina suc-
ceeded. Spore germination of both species
was much greater in an emulsion of these
oils in solutions of Tween 40, that of C. dema-
tium increasing with surfactant concentration
to a maximum of 63%, while that of M. acerina
decreased with increasing concentration. In
both species, appressorium formation was
severely reduced to only 4% in the emulsions.
This result may not indicate toxicity of the
oils, but may merely reflect the formation of
an oil coat around the spore, preventing
hydration or oxygen uptake, or both. This
contrasts with experience with the entomo-
pathogenic fungus, Metarhizium, which is
effective when its conidia are applied to
insects in oil (sections 4.3.1, 4.3.2). Here, the
conidia are stored as a dry powder 5%
moisture) at 1O-20C and mixed with the oil
before use.
From the foregoing, it is obvious that tox-
icity assessment of additives proposed for
incorporation in a formulation of a microbial
herbicide is essential. This should include not
only the individual additives, but also all
combinations of them, as there may well be
unpredictable toxic interactions. Certainly, it
seems a real possibility that a surfactant
might increase the side effects of other co-
formulants. That each fungal species may
well require a unique formulation to ensure
maximum efficacy, and that different strains
of the same species may not respond in the
same way to the same additives, cannot be
stressed too much. While it may seem a
daunting task to have to examine all the
additives singly and in all combinations,
tests for spore germination, appressorium for-
mation and host infection, in either detached
leaf or whole plant assays, are not difficult
and the results should certainly be worth-
while.
6.6 AQUEOUS AND OIL-BASED SPRAYS
Microbial herbicides have, typically, been
applied in laboratory and field as aqueous
suspensions of spores (wettable powders)
with sprayers similar to those used for chem-
ical herbicides. Undoubtedly, such sprays can
be highly successful, as shown by the com-
mercial product Collego. However, Collego is
applied in rice paddy or irrigated soybean,
both crops with very humid microenviron-
ments which favour disease establishment

and expression. Most crops will be dry for
long periods, especially if there is air move-
ment, so spore germination, germling growth
and appressorium development will not be
favoured. This is probably the main reason
why so many promising microbial herbicides
have been unreliable in the field and why
formulation is, belatedly, receiving attention.
As a typical example of the unreliability of
simple aqueous suspensions, Auld et al.
(1990) found that spiny cockleburr (Xanthium
spinosum) was controlled effectively by Colle-
totrichum orbiculare in a dryland grazing site
with no dew but with >84% RH for 9 h after
spraying and heavy rainfall 3-6 weeks later.
In contrast, in a drier area the fungus per-
formed erratically, killing only up to half the
treated plants. The authors concluded that 'In
drier areas the formulation of the spore sus-
pension into a product with a low rate of
evaporation could playa major role in achiev-
ing a consistent effect with the fungus in the
field'.
Formulation is recognized as essential in
patented microbial herbicides. Thus, in a
patent application for control of field bind-
weed by Phoma proboscis, Heiny (1992) claims
that 'the pathogen can be formulated as a
spray using a wettable powder and various
spreader / sticker or emulsion additives
obvious to those skilled in the art'. It is frus-
trating that the application then only details
formulation as an emulsion in corn oil or with
two surfactants, all of which enhanced effi-
cacy. Perhaps other stickers and emulsion
additives are not so obvious?
Simple suspensions in a wide range of sur-
factant solutions are the commonest bioherbi-
cide formulations cited in the literature. As
pointed out in section 6.5, the reasons for
using a particular surfactant are rarely given,
but it would certainly be useful to know them.
Potyka (1996) has reported a selection process,
with a limited range of alternative com-
poundS. After assessing spore germination,
appressorium formation, drying time and dis-
tribution of the inoculant on the target, she
6.6 Aqueous and oil-based sprays 217
concluded that, although Tweens 20, 40, 60,
80 and 85 - at one or more concentrations - all
enhanced germination of M. acerina conidia,
and did not impair appressorium formation;
only Tween 80 was non-toxic at a wide range
of concentrations and actually increased
germination. It was therefore the surfactant
of choice for this propagule, and one of two
first choices for many insect pathogens,
although some toxicity has been recorded
(sections 3.4.1, 4.3.6). Furthermore, there was
no evidence of undesirable interactions with
co-formulants, e.g. vegetable oils, used to pre-
pare emulsions. Tween 20 has been formu-
lated successfully with a number of different
fungi, including Fusarium lateritium and Gib-
bago trianthemae (Walker, 1981; Boyette and
Walker, 1985; Mitchell, 1986, 1988), while
Tween 80 is compatible with Colletotrichum
coccodes (Andersen and Walker, 1985). Heiny
(1992) used a complex blend of alkyl-
polyethoxyethanol, n-butanol, iso-propanol
(IPA) and dimethylpolysiloxane (Activate 9-
0, Uniroyal Chemical) and a mixture of alky-
larylpolyoxyethylene glycols, free fatty acids
and IPA (Riverside/Terra Corp., Iowa) as sur-
factants for Phoma proboscis. Both of these
adjuvants increased necrosis of seedlings
relative to treatments with spores in water;
no significant necrosis was caused by the
adjuvant alone.
Walker and Boyette (1985) and Charudattan
et al. (1986) used 0.02 or 0.04% (v Iv)
nonoxynol [C(]-(p-nonylphenyl)-w-hydroxy-
poly (oxyethylene) (POE)] with conidia of
Alternaria cassiae. This surfactant was recom-
mended by Walker and Riley (1982), who
found that the fungus did not germinate
consistently in either Tween 20 or 80. While
effective under glass, 0.02% was not sufficient
to wet leaves of the sicklepod (Cassia obtusifo-
lia) target in the field, and control was poor.
Increase to 0.04% gave acceptable control. A
similar surfactant was used successfully by
Walker (1980) with Alternaria macrospora on
spurred anoda (Anoda cristata); by Boyette
(1986) with Alternaria crassa on jimsonweed
218 Formulation of microbial herbicides
(Datura stramonium) and with Colletotrichum
truncatum on Florida beggarweed (Desmonium
tortuosum); and by Boyette et al. (1993b) with
C. truncatum on hemp sesbania (Sesbania exal-
tata).
Huber-Meinicke et al. (1989) suspended
spores of Ramularia rubella, a pathogen of
docks (Rumex spp.), in 0.05% (v Iv) Etalfix
(25% Citowett [iso-octyl-phenyl ether of poly-
ethylene glycol] diluted in 20% (v Iv) methyl
alcohol) (Maag AG, Switzerland). Despite the
presence of two compounds known to be
toxic to many fungi, 90% of the applied spores
germinated during each inoculation. No com-
parison with spores in water was given, so it
is not clear why formulation with this parti-
cular surfactant was selected.
A bacterial herbicide (Xanthomonas campes-
tris) is being developed in the USA for control
of annual bluegrass (Poa annua ssp. annua) in
turf (Connick et al., 1993; Savage, 1993). When
used as an aqueous suspension it requires
wounding of the grass by mowing to ensure
infection. However, formulation in a non-
ionic organosilicone surfactant reduced the
surface tension of the water sufficiently to
deliver the bacteria directly into the weed
(without wounding) via open stomata and
hydathodes. Mowing or harrowing another
weed, Bathurst burr (X. spinosum), signific-
antly increased plant death when sub-
sequently sprayed with C. orbiculare conidia
in aqueous suspension (Klein and Auld,
1996). Bacteria which enter plants via natural
openings such as stomata or wounds may
require wounding in conjunction with appli-
cation or, more economically, entry will need
facilitation by an appropriate surfactant
(Savage, 1993).
Apart from distributing a spray as evenly as
possible and minimizing run-off, surfactants
also aid dispersion of spores in water. This
can also be achieved by preparing the spores,
which are usually hydrophobic, as a wettable
powder (sections 2.3.1c, 3.3.4, 4.6.4). For
example, conidia of Colletotrichum gloeospor-
ioides were mixed with water and kaolin,
dried and then re-suspended in water as
needed for application (Mortensen, 1988). A
similar dispersible spray product was pre-
pared by mixing macroconidia of Fusarium
lateritium with hydrated silica powder, pep-
tone and starch (Quimby, 1985). BioMal, a
microbial herbicide containing C. gloeospor-
ioides f. sp. malvae, has been produced as an
experimental wettable powder with a silica
gel carrier (Boyette et al., 1991; d. sections
4.6.2, 4.6.4). It routinely gave over 90% control
of the weed in the field. This mixing of hydro-
phobic spores and hydrophilic carrier mate-
rial, followed by drying, usually seems to be
sufficient to permit wetting of the spores
when the mixture is suspended in water (d.
section 3.3.4). A more recent extension of this
approach (Connick, 1994) is to formulate a C.
truncatum spore/carrier mixture as tablets or
in water-soluble bags. These provide pre-
measured doses for making up the spray sus-
pensions. Although pressures necessary to
form the tablets harmed the fungus, good
control of hemp sesbania (Sesbania exaltata)
was obtained with the bags. Pressure damage
could be avoided by agglomeration in a dish
granulator (section 7.7.3a).
Although it is clear that formulation in sur-
factant solution, or as wettable powder, can
improve reliability and efficacy, it is equally
clear that many fungi require more robust,
and more complex, products. In particular, it
has been necessary to try to prevent desicca-
tion of the inoculant during the critical period
between spraying and penetration. Com-
monly, plant pathogenic fungi require a
period of exposure to free water or high
humidity, often in excess of 12 h, before they
germinate and infect the host (TeBeest et al.,
1978; Boyette, 1982; McRae and Auld, 1988;
Morin et al., 1990; Heiny and Templeton,
1991; section 6.3). In temperate climates, at
least, this is not always available when micro-
bial herbicides are likely to be applied. While
some surfactants may have some humectant
properties, it is usually necessary to add
a humectant (Morin et al., 1990; Appendix

Table 1.4). In practice, humectants, such as
glycerol and sorbitol, have found little favour,
possibly because their effects are generally not
pronounced (Potyka, 1996). More success has
been achieved by adding nutrients to promote
fungal activity (d. section 4.3.2). Thus addi-
tion of sucrose, which is hygroscopic, signific-
antly increased severity of disease caused by
A. macrospora on spurred anoda (Anoda cris-
tata) (Walker, 1981; Walker and Boyette, 1985).
Similarly, sucrose increased spore germina-
tion and disease severity on Florida beggar-
weed (Desmodium tortuosum) caused by C.
truncatum (Cardina, 1986). Infection of John-
son grass (Sorghum halepense) by Bipolaris sor-
ghicola was significantly increased by adding
1% (w/v) Soy-Dex to the spray mixture
(Winder and Van Dyke, 1987). Sorbitol
increased the numbers of viable spores re-
isolated from inoculated velvetleaf (Abutilon
theophrasti) by x20 (Wymore and Watson,
1986). This nutrient also resulted in three 9-h
dew periods on consecutive nights being as
effective as a single 18-h dew treatment, sug-
gesting that the sorbitol was exerting some
humectant action. Indeed, most sugars, and
other nutrients, are likely to have similar
action, especially as they become concen-
trated during drying.
It is often stated, usually by agrochemical
company personnel, that the specificity of
microbial herbicides - a major factor in their
environmental safety - is a significant barrier
to their commercial development. Crops
rarely have monocultures of weeds, so several
different microbial herbicides and/or chem-
icals are needed for complete weed control.
This results in the microbial agent being
judged to be uneconomic. While there are
situations where this assertion may not be
true, such as infestation of plantation crops
with itch grass (Rottboellia cochinchinensis), a
recent report that host range could be
expanded by selected additives (Boyette and
Abbas, 1994) offers potential to counter the
specificity problem. Formulating spores of
Alternaria crassa in aqueous filtrates of jimson-
6.6 Aqueous and oil-based sprays 219
weed (Datura stramonium), the pathogen's
normal host, or hemp sesbania (Sesbania exal-
tata), or in fruit pectin, induced infection of a
range of otherwise non-susceptible crop and
weed species. As well as jimsonweed, the
fungus killed hemp sesbania, showy crota-
laria (Crotalaria spectabilis) and eastern black
nightshade (Solanum ptycanthum). Although it
is well established that formulation of micro-
bial herbicides and adjustment of their culture
medium can increase virulence (Schisler et al.,
1991, 1996), Boyette and Abbas (1994) were
the first to report increase of host range
by formulation. They concluded, cautiously,
that 'if used judiciously with proper timing
and application constraints, it is possible that
this system could be used in a practical
method to improve the weed control spec-
trum, and thus the biocontrol potential, of
Alternaria crassa'. Certainly, if the method
proves to be reliable and robust it will be a
major advance in microbial herbicide form-
ulation.
Perhaps the most important development in
formulation of microbial herbicides is the
recent introduction of invert (water-in-oil)
emulsions. These have given significant pro-
tection to the fungus against desiccation.
The first invert emulsion was devised as a
spore-free overspray to follow inoculation of
sicklepod (Cassia obtusijolia) with conidia of
Alternaria cassiae (Quimby et aI., 1988a). It con-
tained paraffin wax, paraffin oil and soybean
oil emulsified in an equal volume of water
using lecithin as emulsifier. By providing a
water source and retarding water evaporation
long enough to allow spore germination and
disease establishment, the formulation signifi-
cantly reduced the dew period requirement of
the fungus. However, the emulsion was very
viscous, needing specially designed spray
equipment (Quimby et al., 1988b) and spray-
ing was a two-stage process. Quimby (1990)
subsequently patented the invert emulsion
concept under the banner of control of unde-
sirable vegetation 'using a fungal pathogen
applied to weeds in such a manner that
220 Formulation of microbial herbicides
misting or an extended dew period is not
necessary to germinate and grow the fungus.
In particular, an oil-in-water invert emul-
sion ....,. Daigle et al. (1990) attempted to
improve the emulsion characteristics by incor-
porating the fungal spores, so that only one
spray operation was required. However, this
gave only low to moderate weed control with-
out dew. The authors concluded that, while
invert emulsions were promising carriers,
much more research was required to improve
emulsion stability, retain water longer,
optimize droplet integrity on the leaf surface,
incorporate co-formulants to increase patho-
genicity, and facilitate mixing and spraying.
Efficacy of invert emulsions has since been
confirmed in several publications (Table 6.5).
The need for very high inoculum levels, com-
monly associated with spore suspensions
applied in water, was eliminated by the emul-
sion. Whereas dosages of tens to hundreds of
spores per spray droplet might be needed
with aqueous sprays, invert emulsions con-
taining only one spore in a 2 j.1J droplet were
sufficient to infect sicklepod (Cassia obtusifolia)
with Alternaria cassiae and jimsonweed (Datura
stramonium) with Alternaria crassa (Amsellem
et al., 1990). The same formulation greatly
increased the host range of these two fungi,
and a saprophytic Cephalosporium species
became pathogenic (Amsellem et al., 1991).
Similarly, host ranges of C. truncatum and
C. gloeosporioides f. sp. aeschynomene (Collego)
were extended when spores of either patho-
gen were formulated in an invert emulsion.
Hemp sesbania (Sesbania exaltata), immune to
Collego applied in water, was highly suscept-
ible when the invert was used. A similar
response occurred with C. truncatum which,
in the invert, infected the normally immune
northern jointvetch (Aeschynomene virginica)
(Boyette et al., 1991). A further improved
invert was developed by Connick et al.
(1991b), with lower viscosity and improved
water retention properties (Table 6.5). The
paraffin wax and oil were retained but the
soybean oil and lecithin were replaced with
an unsaturated monoglyceride emulsifier

Table 6.5 Preparation of typical invert emulsion formulations of microbial herbicides

Ingredients Concentration ('Yo w/v)' Function

1 2 2 3

Spore suspension As required Pathogen

Paraffin wax 5 5.2 10 10 Thickener
Paraffin oil 28 28.1 40 87 Carrier
Edible soybean oil 55 54.7 40 Carrier
Soybean lecithin 12 11.9 10 Emulsifier
Monoglyceride 3 Emulsifier
(Myverol 18-99)
Sodium alginate 0.5 Carrier
Method
1 Melt wax at 50-60 C and stir in paraffin oil and monoglyceride
2 Homogenize soybean oil and lecithin in high speed blender at full speed for 2 min
3 Mix these two oil phases and cool
4 Add spores directly to the mixed oil phases with stirring (2, 3), or:
5 Add spores to 0.5% w Iv sodium alginate solution (1)
6 Mix equal volumes of oil phase mixture and water (2, 3) or spore suspension in alginate (1) and emulsify
using a paddle stirrer (2, 3) or a vortex mixer (1).
* From: 1, Amsellem et al., 1990; 2, Daigle et al., 1990; 3, Connick et al., 1991b.
 6.6 Aqueous and oil-based sprays 221
Table 6.6 Preparation of simple oil emulsion formulations* of microbial herbicides (Boyette, 1994; Potyka,
1996)

Ingredients Concentration (% v/v)* Function

Spore suspension As required Pathogen
Tween 40 0.1 Surfactant
Rapeseed oil (Potyka) 10 Anti-desiccant
Unrefined corn oil (Boyette) 50 Anti-desiccant
Method
1 Harvest spores from suitable agar medium in water (Boyette, 1994) or 0.1% (v/v) Tween 40 (Potyka,
1996) and adjust to desired concentration
2 Mix spore suspension with oil using a high-speed mixer

* Oil emulsions prepared in this way will separate after a few hours. They should be used immediately after
preparation.

(Myverol18-99). Alternaria cassiae proliferated period requirement from 12 to 2 h. A striking

after spraying in this emulsion. The water con-
tent of deposits 24 h after applying to a glass
plate was 22.3%, well above the 10% needed to
ensure spore germination. The emulsifier
must be selected with care: a highly unsatu-
rated monoglyceride seems to be the best type.
Some, e.g. the saturated monoglyceride
Myverol 18-06, have relatively poor water-
holding and viscosity and can inhibit spore
germination (Connick et al., 1991b). This, and
variants, were later used on a wide range of
fungi and target weeds with apparent success
(Boyette et al., 1993b; Egley et al., 1993; Yang et
al., 1993; Womack and Burge, 1993; Womack et
al., 1996).
A simpler approach, using straightforward
oil-in-water emulsions (Table 6.6), has had
some partial successes. Auld (1993b) showed
that formulation in vegetable oil emulsions
significantly improved the infection of Bath-
urst burr (Xanthium spinosum) by C. orbiculare
without dew, though all but canola (rapeseed)
oil did not match the level obtained with a 24-
h dew period. Boyette (1994) improved activ-
ity of C. truncatum on hemp sesbania (Sesbania
exaltata) by applying the conidia in unrefined
corn oil. A 1:1 (v Iv) emulsion of fungus spore
suspension and corn oil extended, by at least
70 h, the time during which the fungus
remains infective between spraying and the
onset of dew, as well as reducing the dew
feature of these results was that the spray
volume required for weed control was
reduced from 500 l/ha of an aqueous spore
suspension to only 51lha of emulsion. Potyka
(1996) also found that formulating Mycocen-
trospora acerina in rapeseed oil-water emul-
sion (1:9 v Iv) reduced its dew period
requirement for infection of field pansy
(Viola arvensis) from >36 h to 12 h. Even this
much-reduced period of exposure to free
water allowed enough infection to kill all trea-
ted plants in glasshouse experiments where
there was no natural dew and humidity was
<75%. The emulsion was probably having a
greater effect than merely reducing the dew
period requirement. A. Chassot and M. P. G.
(Long Ashton Research Station, unpublished
data) obtained similar results with Staganos-
pora sp. on field bindweed (Convolvulus arven-
sis). Weed infection was increased x30
compared to that with an aqueous spore sus-
pension, when the fungus was formulated in
an emulsion of rapeseed oil in water (1:9 v Iv)
and given an 8-h dew period. Even so, infec-
tion was doubled if the dew period was
extended to 24 h, a clear indication that the
emulsion did not completely protect the fun-
gus from desiccation. Auld (1993b) concluded
that the precise role of the oil in these emul-
sions awaits elucidation. Electron microscope
examination of transverse sections of Viola
222 Formulation of microbial herbicides
arvensis leaves sprayed with a suspension of
Mycocentrospora acerina conidia emulsified in
rapeseed oil (1:9 v Iv), with Tween 40 as emuls-
ifier (M. P. G., R. J. Pring and J. Lawrie, Long
Ashton Research Station, Bristol, unpublished
data), showed that the oil phase had unex-
pectedly penetrated into the leaf tissue and
appeared to contain many small droplets of
water, both in the deposits on the leaf surface
and the intercellular oil (Fig. 6.3). This appar-
ent inversion of the emulsion occurred only
when infection of the plant had been estab-
lished. It did not occur when emulsion, with
or without spores, was sprayed onto inert
surfaces such as glass or Mylar film, and was
not detected when wheat leaves, which are
resistant to infection by the fungus, were trea-
ted. Possibly, secretions from the conidia
before or during infection of the host cause
cell solution leakage and incorporation into
the oil phase in sufficient quantity to increase
spore germination and infection. It must be
stressed that these are initial observations
and verification is needed, although they do
offer an attractive explanation for the effect of
simple emulsions, which apparently lose their
water phase as quickly after application as
aqueous sprays (Potyka, 1996).

25.,.

Figure 6.3 Transmission electron micrograph of a transverse section through a leaf of Viola arvensis after
spray application of, and infection by, a rapeseed oil emulsion formulation of Mycocentrosporum acerina.
The oil has penetrated the stomate and formed an invert emulsion inside the plant tissue, as well as on the
surface of the cuticle (M. P. Greaves, R. J. Pring and J. Lawrie, Long Ashton Research Station, Bristol,
unpublished). c, cuticle; e, epidermis; f, fungus; 0, oil; sg, stomate guard cell; ss, sub-stomatal space;
w, water; bar = 25 (J.Lm).

6.7 SPRAY APPUCAnON
Egley et al. (1993), working with Colletotrichum
truncatum on hemp sesbania (Sesbania exal-
tata), attempted to optimize weed control by
determining the influence of spray droplet
size on germination of the pathogen on
detached leaves. Conidial germination
decreased from 46 to only 5% as droplet dia-
meter decreased from 2700 to 900 Ji-m. Droplet
size did not affect appressorium formation.
Assessed on a per conidium basis, 900-Ji-m
droplets were more infective than 2100-Ji-m
droplets. Conidia applied to plants in green-
house trials using an air-assist spray system,
producing droplet spectra with volume mean
diameters (VMDs) of 421 and 104 Ji-m, gave 90
and 94% control, respectively. The smaller
droplet spectrum covered the plant better
than one composed of larger droplets. Clearly,
sufficient conidia germinated in droplets of a
range of sizes to give adequate infection and
disease expression.
Chassot and Greaves (unpublished), in
similar experiments, found that the germina-
tion of spores of Staganospara sp. in rapeseed
oil emulsion was unaffected by VMDs in the
range 175-275 Ji-m, whether incubated at low
or high humidity. However, droplet size
did have a marked effect on several other
important parameters. Thus, at 280 l/ha,
increasing the VMD of the droplets from 175
to 275 Ji-m approximately halved the number
of deposits per unit area, but doubled the
volume of spray retained on the leaf, and the
leaf area covered. Conversely, and somewhat
surprisingly, the larger drops caused less
infection, as measured by leaf necrosis, than
the smaller drops. This unexpected result
was compounded by 10% of the smallest
drops containing no spores, and 50% contain-
ing two or less. In contrast, all the biggest
drops contained at least six spores, and 50%
contained >20. Possibly this high droplet
loading with spores resulted in some auto-
inhibition of spore germination. It is not
the purpose of this chapter to present a
6.8 Solid formulations 223
fully detailed analysis of the available litera-
ture on the spray application of microbial
pesticides, which is assessed in Chapter 2
and illustrated in Appendix II. However,
these examples clearly demonstrate the
complexity of spray parameters and, more
importantly, the crucial need to fully recog-
nize and account for them in experiments
on efficacy. In practice, the most important
objective is to maximize the number of
applied droplets that produce an infection.
The failure of many pathogens, thought to be
highly promising when sprayed to run-off in
the laboratory, is probably the outcome of
inadequate attention to integration of the
pathogen's dose, formulation and spray para-
meters.
6.8 SOUD FORMULAnONS
Solid inocula are in use to control certain
woody weeds (section 6.1). These include
agar-based preparations of Chondrosterium
purpureum to control black cherry (Prunus ser-
atina) in pine forests in the Netherlands
(Scheepens, 1980, 1990) and hardwood trees
in Canadian forests (Wall, 1990, 1994).
None of these solid formulations, however,
is economic or convenient to use in conven-
tional agriculture or horticulture. Some plant
residues, such as wheat bran (Morris, 1989) or
sorghum straw (Ciotola et al., 1995), can be
used successfully to both grow and distribute
the fungus. However, these carriers are bulky
and, more importantly, of inconsistent qual-
ity. They may also be contaminated with pest-
icides unless grown, albeit expensively, as
organic crops. The first reproducibly uniform
and reliable solid carrier was an alginate-
based granular formulation of fungal spores
(section 5.3.3 and Table 5.2 in that section).
Microbial herbicide granules are generally
0.3-2.0 mm in diameter and designed for
application to the soil surface or to be incor-
porated into the soil. Alternatively, the solid
inoculant can be prepared as a dust, with
particles of 3-50 Ji-m diameter, but these are
224 Formulation of microbial herbicides
prone to drift and thus are not favoured (sec-
tion 2.3.1a).
Walker (1981) described a method for large-
scale production of a mineral-based granular
formulation of Alternaria macrospora for con-
trol of spurred anoda (Anoda cristata). The
granules, nutrient-soaked vermiculite aggre-
gates on which the fungus had grown to pro-
duce both mycelium and conidia, were
applied to soil pre-or post-emergence of the
weed. A modification was successful for pro-
duction and application of Dreschlera sp.,
Fusarium spp., Colletotrichum spp. and Phyllos-
ticta sp. This solid inoculant not only infected
the target weed but also, due to its residual
activity, enhanced the pathogen's effect by
concentrating it in a band on top of the seed
furrow. Table 7.6 in section 7.5.3 describes
production of compacted vermiculite gran-
ules containing nitrogen-fixing Rhizobium for
soil application. Undoubtedly other inert car-
riers, e.g. kaolin, silica gel or diatomaceous
earth, could also be effective.
Other sustained-release (or sustained-pro-
duction) formulations have been reported.
Walker and Connick (1983) and Fravel et al.
(1985) described a process using sodium
algin-ate to prepare pellet formulations of con-
idia of five different fungi (Table 6.7; for
detailed example see Table 5.2 in section
5.3.3). When pellets were dried and then re-
wetted, abundant conidia formed on the sur-
face of the pellets 24-48 h after they were spread
on trays and incubated with 10 min exposure
every 12 h to 275-W sun lamps, so giving a
simple means of producing large numbers of
spores. The new conidia germinated (90-100%)
and infected their hosts readily even after sto-
rage of dry pellets for 6-8 months at 4 or 25C.
This method was intended primarily as a
means of soil inoculation, using pellets that
had been dried immediately after incorpora-
tion of fungal conidia in the alginate. After
application they take up soil water and the fun-
gus germinates, grows to the pellet surface and
either forms spores, which survive until a sui-
table host is present when they germinate and
infect the host, or grows directly to a nearby
host and infects it. Although the pellets tend to
collapse to a disc when air-dried undisturbed,
this was easily prevented by tumble-drying the
pellets or by incorporating 10% (w Iv) kaolin.
Drying reduced pellet diameter from a wet 3-
4 mm to only 1 mm. Obviously a pellet of this
size can carry other co-formulants in addition
to the spores and clay. Use of alginates to for-
mulate microbial herbicides has been reviewed
by Connick (1988, 1990).
Subsequently, Weidemann and Templeton
(1988) formulated macroconidia of Fusarium

Table 6.7 Preparation of alginate granule formulations of microbial herbicides (Walker and Connick,
1983)

Ingredients Concentration (%) Function

Mycelial homogenate 20 (v Iv) Agent
Sodium alginate solution 1.33 (w Iv) Anti-desiccant
Kaolin 10 (w/v) Bulking agent
CaCl 2 (0.25 M) Skinning agent
Method
1 Harvest mycelium by filtration
2 Re-suspend mycelium in equal volume of fresh medium
3 Homogenize for 30 s in high-speed blender
4 Dilute 1:4 with sodium alginate solution in distilled water (final pH 6.6-7.3)
5 Mix in 10% kaolin by stirring
6 Add suspension dropwise to CaCh to skin over and form granules
7 Collect granules on a sieve and air-dry (20-25 0c) for 24 h

solani f. sp. cucurbitae in alginate to control
Texas gourd (Cucurbita texana). Granules con-
taining spores, mycelium and soya flour (2%
w Iv), applied to soil in the field pre-or post-
emergence at 220 kg/ha, resulted in 70% or
higher mortality of the weed seedlings
through damping off.
In a detailed study of alginate pellets con-
taining Alternaria cassiae, a potential microbial
herbicide to control sicklepod (Cassia obtusifo-
lia), Daigle and Cotty (1992) confirmed the
utility of the system. They concluded that the
spore yield of the product is the primary
determinant of its efficacy as a soil inoculant.
There were several important variables, e.g.
composition of the fermentation medium, for-
mulation and fillers, as well as the fermenta-
tion time. Addition of nutrients to the
mycelial homogenate after fermentation
increased its sporulation before pelleting, but
the amount and ratio of nutrients had a
greater influence on spore yield after the
mycelium had been pelleted, dried and re-
wetted. These results suggested that respira-
tion of entrapped mycelium is reduced in
pellets made with high-nutrient liquid fer-
mentation media compared with that on
solid media.
Daigle and Cotty (1992) pointed out that the
kaolin filler in alginate pellets, though cheap
and compatible with the fungus, adds no
nutrient value. Wheat bran and rice flour at
5% (w Iv), though slightly nutritious, were
too viscous or lumpy after autoclaving and
unsuitable for pelleting; also they gave low
spore yields after the pellets were wetted. In
contrast, corn cob grits, a waste product of the
corn industry, yielded four times as many
spores after 2 or 3 days as the unsupplemen-
ted kaolin-based pellets. The alginate pellet-
ing process, like many other approaches to
formulation, is complex and several factors
interact to influence performance. This very
complexity, however, opens the door to
exploitation and the development of varia-
tions on a theme that can significantly
improve the efficacy of the fungus.
6.8 Solid formulations 225
The benefits of nutrient addition to a gran-
ular formulation are perhaps seen most clearly
in the recent development of the so-called
'pesta' granules, which encapsulate fungal
propagules in a wheat gluten matrix (Connick
et al., 1991a). Essentially the process is a mod-
ification of that for making pasta (Table 6.8). It
is simple to use, water-based and amenable to
a range of co-formulants. Connick et al. (1991a)
tested pesta with Alternaria cassiae, Alternaria
crassa, Colletotrichum truncatum and Fusarium
lateritium. All the fungi grew well and sporul-
ated on the granules after addition to soil.
Colletotrichum truncatum produced acervuli
with conidia embedded in a protective muci-
laginous matrix.
In greenhouse tests (Table 6.9), with pesta
granules spread by hand on the soil surface,
0.6-1.4mm granules with Alternaria spp. or
Colletotrichum sp. killed more weeds (68-
100%) than smaller granules. Granule size
did not affect the efficacy of F. lateritium
which killed only ca 30% of its target. In this
initial study, kaolin was used as the inert fil-
ler, as with the alginate granules. Connick et
al. (1991a) acknowledge that this is not the
best choice. Inexpensive fillers can be used to
replace up to ca 30% (w /w) of the wheat flour
before the dough-forming ability of the mix-
ture becomes inadequate. Fillers can reduce
product cost, add nutrient and alter hardness,
density and water retention/absorption. Co-
formulants compatible with pesta include ver-
miculite, pyrophyllite, bentonite, peat, corn
cob grits, wheat bran, glycerol and sucrose.
A particular advantage of pesta is that it can
be extruded or moulded easily to a variety of
shapes and sizes appropriate for different
applications (section 7.7.3c). For example
long, spaghetti-like strings can be applied to
water to tangle with submerged target aquatic
plants. The granules store well (see below)
and the microbial inoculant can be mixed
with chemical herbicides, giving slow release,
which may aid the action of the microbe.
Because gluten proteins are denatured by
autoclaving, the pesta carrier is not heat
226 Formulation of microbial herbicides
Table 6.8 Preparation of granular pesta microbial herbicides using high gluten wheat flour (Connick et al.,
1991a)
Ingredients Amount Function
Mycelial homogenate 52ml Pathogen
Durum wheat flour (semolina) 80 g Anti-desiccant/carrier
Kaolin 20 g Bulking agent
Method
1 A 7 to 10-day-old liquid culture of selected fungus is homogenized in a high-speed blender
2 Semolina and kaolin are mixed together by shaking and spread in a shallow layer in a dish
3 Fungal homogenate is mixed with flour to produce a cohesive dough
4 Dough is rolled in a pasta maker to a thickness of 2.5 mm
5 Dough sheet is folded and re-rolled repeatedly until it has an even consistency: it is then rolled to 1.8
mm and, finally, to 1.1 mm thick
6 Air-dry (20-23 DC, 55-70% RH) the sheet on polyester net with air circulation above and below until it
will break crisply (24-48 h)
7 Grind sheet by hand; material passing a 14-mesh sieve but retained on 18-mesh (mesh sizes may be
different for different fungi) is used as pesta inoculant

sterilized. This does not appear to be a pro-
blem. The high level of inoculant present,
relative to the potentially competitive organ-
isms in the gluten and soil, allows the
entrapped mycelium to proliferate in most of
the granules.
Connick et al. (1993) confirmed their earlier
results with pesta granules containing Fusar-
ium oxysporum or C. truncatum. With both,
granules rapidly became covered with new
fungal growth when moistened, resulting in
sustained production, release of new spores
and control of target weeds. Subsequent
investigation of the potential of F. oxysporum
as a microbial herbicide for sicklepod (Cassia
obtusifolia), coffee senna (Cassia occidentalis)
and hemp sesbania (Sesbania exaltata) showed
that pre-emergence applications of this patho-
gen killed plants by damping-off (Boyette et
al., 1993a). Post-emergence applications were
considerably less effective. This particular
organism offers a significant advantage over
most potential microbial herbicides in that it
infects and controls three different weed spe-
cies, overcoming the deterrent to commercia-
lization engendered by absolute specificity.

Table 6.9 Efficacy of pesta granules containing microbial herbicide propagules (Connick et aI., 1991a)
Pathogen Granule size (mesh) Target Mortality (%)

Alternaria cassiae 14-18 Sicklepod 68

18-30 70
> 30 40
Colletotrichum truncatum 14-18 Hemp sesbania 100
18-30 87
> 30 40
Fusarium lateritium 14-18 Velvetleaf 30
18-30 28
> 30 5

Further recent studies (Connick et aI., 1997)
have concentrated on the shelf-life of pesta
products. There is a critical relative humidity,
between 33 and 53%, below which pesta-
formulated C. truncatum maintains high via-
bility and above which shelf-life is reduced.
Co-formulants such as sucrose counteracted
the adverse effects of high-humidity storage,
at least partially. Although it is early in the
development of this type of product, results to
date are very promising. Shelf-lives of more
than 18 months have so far been achieved
with both Fusarium oxysporum and C. trunca-
tum (W. J. Connick and D. J. Daigle, Commod-
ity Utilisation Unit, USDA-ARS, New
Orleans, personal communication).
Current research (Boyette, 1994) is develop-
ing slow-release, alginate-encapsulated for-
mulations of rhizobacteria to control leafy
spurge (Euphorbia esuIa) and foxtail species
(Setaria spp.). This work is also developing
encapsulation of precursors of phytotoxic
indoles which are produced by, and enhance,
the rhizobacteria in the rhizosphere of soy-
bean seedlings. Another variant on the gran-
ule theme involves the invert emulsion
technology used for foliar sprays. Quimby et
aI. (1994) patented the encapsulation of bio-
control agents in granules coated with oil and
an absorbent. These granules consist of algin-
ate, starch or wheat gluten with a coating of
an oil, that forms an invert emulsion with
water, and an absorbent for the oil (hydrated
silica, fumed silica, clay, bran, diatomaceous
earth, zeolite, absorbent starch or mixtures
thereof). The coated granules are dried to 1-
10% moisture content, during which it is
claimed that, despite the presence of the
absorbent, the oil slows drying of the patho-
gen, so maintaining viability for extended
storage periods at 3-10C. The granules are
flowable and can easily be suspended in
water for spraying. A further, unexpected,
finding was that after storage and re-wetting
during spraying, the granules dry out slowly,
allowing for re-activation of the pathogen
with minimal dew or mist. The granules
6.9 The future and research requirements 227
retain more than 10% moisture for as long as
8-12h.
The encapsulation of entomopathogens in
starch has been established experimentally
for some years (Dunkle and Shasha, 1988;
McGuire and Shasha, 1990, 1992; sections
3.3.1, 3.3.4). Recently, the technology has
been applied to weed pathogens. Bothast et
aI. (1993), Jackson et al. (1996b) and Schisler
et aI. (1996) formulated C. truncatum micro-
sclerotia in pre-gelatinized corn starch or
pre-gelatinized corn flour, or a mixture of
both. Microsclerotia germination rates were
still high (>68%) after 9 months' storage at
4C and dried microsclerotia were capable of
myceliogenic and sporogenic germination on
water agar. Significantly more disease was
induced in seedlings of hemp sesbania (Sesba-
nia exaitata) by formulated than by unform-
ulated microsclerotia. If entomopathogen
experience is a guide, these encapsulated for-
mulations will find application to treat both
soil and foliage.
Hopefully, granular formulations will
kindle greater interest in application of
microbial herbicides to soil - a strategy
which has much to offer in overcoming
many of the problems presented by applica-
tion of fungi to the often highly hostile envir-
onment of the leaf surface.
6.9 THE FUTURE AND RESEARCH
REQUIREMENTS
It is clear from the literature available that the
development of microbial herbicide formula-
tions has been a somewhat piecemeal process
in all but one or two instances. The notable
exception is the sustained effort of the laborat-
ory of W. J. Connick and colleagues in the
USA, which has produced several significant
developments. It remains to be seen whether
these developments will carry through to the
market. Work elsewhere is characterized by
short-term efforts, each resulting in a formu-
lation that functions adequately in the experi-
mental programme in question, but has not
228 Formulation of microbial herbicides
been rigorously assessed in a range of
different arenas. Consequently, it is hardly
surprising that so many initially promising
pathogens disappear without apparent trace.
It is widely asserted that successful transi-
tion of pathogens from laboratory to field will
occur only if they are properly formulated to
make them reliable in a wide range of climates
and environments. Thus it is all the more
surprising that there has not been a major
emphasis on formulation by more research-
ers. Furthermore, technologies developed in
the food (Connick et al., 1991a) and cosmetics
(Womack et al., 1996) industries appear
promising in the development of microbial
herbicide products. This may call for the
establishment of new research partnerships
which, in turn, might stimulate innovative
thinking and action.
Similarly, productive partnership could be
established with those working on biocontrol
of insects. Detailed research on effects of stor-
age moisture content, starting at spore harvest
and continuing to post-storage activation, has
made a breakthrough in the dry storage of
some entomopathogenic fungi (Chapter 4).
Connick et al. (1997) have made a similar
advance with granular formulations of weed
pathogens. How much easier would the
advances have been, and how much sooner,
if the two studies had been collaborative?
Experience has proved that it is relatively
easy to find pathogenic fungi that show good
potential as microbial herbicides. Perhaps it is
now time for efforts to be diverted from what
appears to some as a simple 'stamp collecting'
exercise and put into a focused programme
designed to make the accumulated pathogens
work in a practical way in the field. It would
be naive, however, to assume that formulation
alone will solve the problem of achieving
reliability of action. No product will work in
a fully satisfactory way unless it is applied
correctly, with due regard to all the interact-
ing spray parameters that, together with for-
mulation, contribute to appropriate spray
distribution and retention on the target.
These are essential features in determining
whether a pathogen will function as intended,
and demand that a multidisciplinary
approach be taken to weed pathogen devel-
opment. It would also be naive to ignore other
research topics of a more fundamental biolog-
ical nature, which contribute significantly to
development and refinement of biological
control agents. For example, molecular biol-
ogy will undoubtedly, in the fullness of time,
make valuable inputs to the construction of
robust, highly effective, broad-spectrum
microbial herbicides. However, it is a truism
that no matter how well developed or well
engineered the pathogen, it will not work reli-
ably and effectively in practical agriculture
and horticulture unless it is properly formu-
lated and properly applied.
The following list, though not exhaustive,
indicates important applied research topics
which must be addressed:
develop practical, water-retaining formula-
tions;
improve shelf-life with detailed attention to
moisture content;
optimize spray application parameters for a
range of propagule sizes;
characterize spray application-formulation
interactions;
develop multi-component products effective
against important weed complexes;
find means of targeting susceptible sites, e.g.
meristems and stem bases;
develop pathogens and/or formulations
effective against underground organs.
It is axiomatic that the weed targets selected
for microbial herbicide research topics should
be economically important. That is, a real
market opportunity must exist. Without that
market opportunity, even if the research pro-
duct is robustly and reliably effective in field
conditions, there will be no practical or com-
mercial development and the research will, in
large part, be wasted. Research and the future
of microbial herbicides are discussed in rela-
tion to the other microbials in Chapter 10.

ACKNOWLEDGEMENTS
Research at IACR-Long Ashton Research
Station on microbial herbicides, including
formulation studies, is funded by the Ministry
of Agriculture, Fisheries and Food. IACR
receives grant-aided support from the Bio-
technology and Biological Sciences Research
Council of the United Kingdom.
REFERENCES
Amsellem, Z., Sharon, A and Gressel, J. (1991)
Abolition of selectivity of two mycoherbicidal
organisms and enhanced virulence of avirulent
fungi by an invert emulsion. Phytopathol. 81,
985-8.
Amsellem, Z., Sharon, A, Gressel, J. and Quimby,
P. C. (1990) Complete abolition of high inoculum
threshold of two mycoherbicides (Alternaria
cassiae and A. crassa) when applied in invert
emulsion. Phytopathol. 80, 925-9.
Andersen, R. N. and Walker, H. L. (1985) Colleto-
trichum coccodes: a pathogen of eastern black
nightshade (Solanum ptycanthum). Weed Sci. 33,
902-5.
Auld, B. A (l993a). Potential for bioherbicides in
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FORMULATION OF BENEFICIAL 7
ORGANISMS APPLIED TO SOIL
Alan S. Paau

CONTENTS
7.1 Introduction 236
7.2 Placement considerations for soil inoculants 237
7.3 Methods to apply inoculants to soil 237
7.4 Special considerations for soil inoculant development 238
7.5 Basic formulations to apply organisms to soil 238
7.5.1 Fluid suspensions for spray application 238
7.5.2 Powder formulations for various applications 241
7.5.3 Granules for soil and spray applications 243
7.5.4 Formulations using seeds as vehicles 247
7.6 Ingredients commonly found in formulations and
their use 247
7.6.1 Carriers, diluents, or bulking additives 247
7.6.2 Membrane stabilizers 247
7.6.3 Growth and contaminant suppressants 248
7.6.4 Buffering systems 248
7.6.5 Binders 249
7.6.6 Dispersants 249
7.6.7 Lubricants 249
7.6.8 Activators 249
7.6.9 Food sources 250
7.6.10 Coating compounds 250
7.7 Some common processing methods 250
7.7.1 Mixing 250
7.7.2 Drying 250
7.7.3 Size enlargement 251
7.7.4 Sterilization 252
7.8 Research requirements and future directions 253
References 253

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
236 Formulation of beneficial organisms applied to soil
7.1 INTRODUCTION
Unintentionally, man has long been an active
participant in the introduction of organisms
from one soil to another. In addition to natural
forces - including wind, water flow and ani-
mal movement - the participation by farmers
and horticulturists in accidental movement of
soil, harbouring its biota, from one field to
another, has long been recognized by patho-
logists as a major mechanism by which plant
diseases are spread. Use of soil-contaminated
field equipment and the shipment of diseased
plant materials carrying the infective organ-
isms account for most of these accidental
introductions.
The first intentional introduction or inocu-
lation of beneficial organisms to soil probably
occurred centuries before the start of micro-
biological research. Without knowing of the
Rhizobium-legume symbiosis and the under-
pinning symbiotic nitrogen fixation process,
farmers have long recognized that mixing
soil from a field previously producing a
good legume crop to a new field would
ensure the future leguminous crops in the
new field would have nodule outgrowths on
their roots and provide bountiful harvests. By
the late 1800s, the use of 'naturally inoculated'
soil to inoculate legume seeds and soils
became common practice (Smith, 1992). In
1896, a US patent was issued for the inocula-
tion of soil with Rhizobium for the cultivation
of legumes (Nobbe and Hiltner, 1896). As the
process of biological nitrogen fixation became
well known in the 1930s, spraying of the
asymbiotic nitrogen fixers Azotobacter to soil
became popular with farmers in Russia. Dec-
ades later, Frankia species were used in woody
shrubs; cyanobacteria in rice fields; and Azos-
pirillum in maize for nitrogen fixation, as well
as 'suppressive soil' - or microbial strains
from it - to combat take-all disease of wheat.
These historical examples illustrate the
major rationales for application of beneficial
organisms, especially microbes, to soil today.
Such rationales include
combating the causes of disease and infection,
currently termed biological control, with
microbes such as Pseudomonas, Bacillus,
Gliocladium, Trichoderma, Paecilomyces, Strep-
tomyces, Beauveria, and entomophilic nema-
tode species; and
promoting general plant growth and nutrition
by various mechanisms involving biolog-
ical nitrogen fixation (with Rhizobium,
Azotobacter, Frankia, cyanobacteria and
Azospirillum), phosphate utilization (with
mycorrhizae and Penicillium bilaii), and
improved moisture procurement with
mycorrhizae.
The popularity (or conversely, the lack
thereof for some species) of using any of
these beneficial organisms is highly depend-
ent on our ability both to mass produce the
organism economically, and to formulate a
stable final product which is easy to apply
and transport. Mass production of endomy-
corrhizae or vesicular-arbuscular mycorr-
hizae (Marx and Kenny, 1982) and, until
recently, of many entomophilic nematodes
(Chapter 9), has been problematic and chal-
lenging. In contrast, production of most
organisms for soil application, including Rhi-
zobium (Burton, 1967), Azotobacter, Frankia,
Azospirillum, Gliocladium, Trichoderma (Lewis
and Papavizas, 1985; Ricard, 1987), Paecilo-
myces, Pseudomonas (Connick et al., 1989),
Bacillus, Streptomyces (Yang and Ling, 1989),
Beauveria, cyanobacteria, Penicillium and
ectomycorrhizae (Marx et al., 1982; LeTacon
et al., 1984) has mostly been successful, rou-
tine and economical, and does not constitute a
significant technological barrier. A more
significant barrier to the popular use of these
organisms for soil application lies with formu-
lating a commercially viable final product.
This chapter presents selected formulations
and methods of their preparation developed
through the years for commercial and experi-
mental application of organisms to soil.
Emphasis is given to organisms applied to
soil to improve plant nutrition; applications
 7.3 Methods to apply inoculants to soil 237

to soil for the biocontrol of plant diseases are
covered in Chapter 5. The mention of specific
product names is strictly for illustration pur-
poses and is not an endorsement of the pro-
ducts. Technical terms are defined in
Appendix III.
7.2 PLACEMENT CONSIDERATIONS FOR
SOIL INOCULANTS
All inoculants formulated for delivery to soil
invariably aim either to place the active
organisms in direct contact with the roots of
the host plant, or to influence the biota of its
immediate rhizoplane or rhizosphere. The
specific court of action, be it inside the root,
on the rhizoplane or in the rhizosphere, is
determined by the mechanisms by which the
organisms will work to benefit the host plants
and is summarized in Table 7.1. Special con-
sideration, therefore, must be paid to courts
and mechanisms in order to place the organ-
isms in the appropriate proximity. Poor
understanding of benefiting mechanisms con-
tributed much to past failures of soil inocu-
lants. Inoculants inadvertently placed far
away from the court of action are often
wasted and use the product ineffectively.
The difference in product efficacy due to the
use of different application methods of place-
ment is well documented (Mohammadi and
Lahdenpera,1994).
7.3 METHODS TO APPLY INOCULANTS TO
SOIL
Many methods deliver inoculant products to
soil near roots or rhizosphere, with variable
efficiency. To protect the seed and to target
the main root system, inoculants can be fed
directly into the seed furrow at planting. Car-
riers for delivery to the furrow include the
seed itself, granular particles and fluid sus-
pension. Fluids can be water-based or poly-
mer gel-based (Mihuta-Grimm and Row,
1986). If the seed itself is not used as the
delivery vehicle, the inoculant is normally
fed directly into the seed furrow at planting,
following the placement of the seed. Granular
inoculants are delivered from an insecticide
(or Gandy) hopper, which through a gear
and sprocket (or chain) unit directly linked
to the drive shaft of a moving vehicle, meters
out a predetermined quantity of granules to

Table 7.1 Examples of mechanisms by which organisms applied to soil benefit the host species

Known or likely mechanism Organisms Preferred placement Critical action timing

Symbiotic nitrogen fixation Rhizobium Rhizoplane Root emergence, fast
Frankia Rhizoplane Root emergence, fast
Cyanobacterium General vicinity All the time
Asymbiotic nitrogen fixation Azospirillum Rhizosphere During host's lifespan
Azotobacter General vicinity All the time
Parasitism, pathogen infection, Gliocladium Rhizosphere Pathogens' presence
antagonism Trichoderma Rhizosphere Pathogens' presence
Paecilomyces Rhizosphere Pathogens' presence
Nematodes Rhizosphere Pathogens' presence
Suppressant growth of Bacillus Rhizosphere Pathogens' presence
competing organisms Pseudomonas Rhizosphere Pathogens' presence
Streptomyces Rhizosphere Pathogens' presence
Gliocladium Rhizosphere Pathogens' presence
Substrate solubilization and Penicillium Rhizoplane During host's lifespan
absorption Mycorrhizae Rhizoplane During host's lifespan
238 Formulation of beneficial organisms applied to soil
drop by gravity. Fluid suspension can be
delivered by a simple, passive drip irrigation
device or by a sprayer with a mechanical or
electrical pump. A great choice of nozzles to
control flow rate and band width is available.
Formulations suitable for using the seed itself
as a delivery vehicle (Paau, 1988) are the sub-
ject of Chapter 8 so discussion here will be
limited.
To deliver inoculants to the rhizosphere,
broadcasting granular particles with a sprea-
der or overhead spraying with a boom are the
most common methods. These applications
are normally followed by soil tilling to quickly
incorporate inoculants into the soil, in order to
protect the organisms from desiccation and
from harmful exposure to sunlight (sections
3.4.4a, 4.3.3a).
7.4 SPECIAL CONSIDERATIONS FOR SOIL
INOCULANT DEVELOPMENT
One key obstacle in the development of soil
inoculants is the extremely heterogeneous
nature of soil and its often unpredictably
harsh environment for introduced organisms
(van Elsas and van Overbeek, 1993). There are
few unoccupied niches in the soil for the
introduced species. A keen competitive war
may immediately develop among the intro-
duced and indigenous species for their
ecological space.
To achieve a desirably long shelf-life and
the required ease of transport and storage,
most organisms in commercial products for
soil application are propagated in a rich med-
ium, and later packaged as concentrates with
the organisms driven to a dormant or a
semi-dormant physiological state. Dormant
organisms can withstand relatively high
temperatures and wide temperature fluctua-
tions during transport and storage. These
organisms, however, are highly stressed,
alien to the natural soil environment, and
often physiologically not ready to compete in
soil with the indigenous species that have had
time, often generations, to adapt to the
specific ecological niche. Many formulations
specifically address these issues by including
a massive amount of carriers, selective
food sources, suppressants for indigenous
species, buffers and other ingredients which
can transiently alter the microphysical envir-
onment of the soil to provide a temporary
safe haven for the introduced species. This
haven allows the species time to reactivate,
adjust physiologically to the new environ-
ment, and propagate. A successful formula-
tion allows the introduced species to establish
itself on or inside host roots or, at least
temporarily, to shift biota dynamics in the
soil to favour host development in a timely
manner.
7.5 BASIC FORMULATIONS TO APPLY
ORGANISMS TO SOIL
This section complements Chapter 2, with the
emphasis on soil.
7.5.1 FLUID SUSPENSIONS FOR SPRAY
APPLICAnON
All formulated products in this section, com-
pared in Table 7.2, result in a suspension of
the living organisms for application to soil
either by in-furrow or overhead spray and
drip applications.
7.5.1a Agar cultures The earliest commercial
Rhizobium product was just a jar with an agar
substrate at the bottom and rhizobia growing
on the agar surface. Farmers would add a
little water into the jar, re-cap it, and shake
vigorously to obtain a concentrated suspen-
sion of the active bacteria, which was then
diluted with water and sprayed into the soil.
This unformulated product, although often
effective, was difficult to quantify for control
of the number of bacteria applied. It was also
very difficult to store and transport, and
required time-consuming handling before
use. It was very sensitive to temperature
fluctuations during storage and transport,
 7.5.1 Fluid suspensions for spray application 239
Table 7.2 Typical product characteristics of fluid suspension formulations for spray application

Formulation Physiological state of Storage and Production Product ease

organisms transport/cost costs of use
Agar cultures Active at various Difficult/high Intermediate Difficult
growth phases
Frozen and Dormant, mostly Difficult/high Intermediate Easy
refrigerated uniform growth
culture stage, easy to reactivate
concentrates
Dormant Dormant, need long lag Easy/low Low Easy
aqueous time to reactivate
concentrates
Dormant oil Dormant, most are easy Easy/low Intermediate Intermediate, need
concentrates to reactivate to high detergent to use with
conventional
hydraulic sprayer

and the agar occasionally dried, killing the
cultures. Also, the growth stage of the organ-
isms in the inoculant was not controlled and
the organisms may have been applied in var-
ious growth phases. Due to these limitations,
this simple inoculant product is no longer in
common commercial use today. It is, how-
ever, very popular for research with organ-
isms not yet equipped with a better
formulation for delivery to soil.
To produce an agar culture inoculant, a
Mason or canning jar can be filled to one-
quarter height with a molten 0.8-1% agar
and steam sterilized under pressure. After
cooling to solidify the agar at the bottom of
the jar, a small liquid inoculum is introduced
aseptically to the surface of the agar. The
inoculum is allowed to grow a culture of
the organisms on the agar surface. To increase
the growing surface area, the jar may be tilted
on its side during cooling. For laboratory
work, sterile Petri dishes may also be used.
Neither of these containers is recommended
for inoculants planned for long-term storage
or long-distance transport. Slanted agar
tends to slide down the side during transport
and cover the culture. Petri dishes generally
are not tight-fitting and lose moisture quickly,
drying the culture. Plastic dishes are
also easily broken or cracked, increasing con-
tamination.
7.5.1b Frozen or refrigerated culture concen-
trates To avoid harvesting agar cultures in
the field and to provide the organisms in a
more uniform growth phase, frozen concen-
trates of bacterial cultures are sometimes
used. This formulation also eliminates the
need to transport and store agar which may
dry and shorten the shelf-life of the inoculant.
Packaging is simpler. A heat-sealable plastic
bag or capped bottle sterilized (or not) by
steam, ethylene bromide or gamma irradia-
tion, is satisfactory as the high water content
of the formulation can sustain much moisture
loss without compromising the vitality of the
organisms. Storage and transport tempera-
tures are either below or just above freezing
to lower metabolism of the organisms,
achieving a physiologically uniform popula-
tion with extended shelf-life. The low tem-
peratures also discourage contaminant
growth. These culture concentrates are
diluted with water in the field to spray into
soil. The need for low storage and transport
temperature is costly and incompatible with
most agricultural field practices. The formula-
tion is now rarely used for soil inoculants, but
240 Formulation of beneficial organisms applied to soil
is still popular for commercial silage inocu-
lants and research.
Manufacture of a frozen formulation sim-
ply requires harvest from the culture medium
and storage at a predetermined low tem-
perature with various additives, especially
stabilizers of membrane integrity upon
freezing (section 7.6.2; Table 4.12 in section
4.6.6). Despite membrane stabilizers such as
skimmed milk, sugars, salts, glycerol, etc. and
manipulation of the rate of cooling, the recov-
ery rate of viable organisms after freezing is
highly organism-dependent and is often quite
low. This rate must therefore be figured into
the total cost/benefit analysis as manufac-
turers plan to deliver a specific number of
viable organisms per application.
7.5.Ic Dormant aqueous suspensions From
early on, plant pathologists learned that
many soil bacteria such as Xanthomonas and
Pseudomonas species can be harvested from
active cultures, rinsed clear of nutrient resi-
dues, and stored as a concentrated suspension
in sterile distilled water at ambient tempera-
tures. Bacteria in these concentrated suspen-
sions remain viable in a physiologically
dormant state for a long time and can be
recovered years later for experimentation.
This characteristic long-term viability has
been exploited to satisfy the long shelf-life
requirement of a commercial product.
In 1982, NifTAL (Paia, Hawaii, USA)
reported the first Rhizobium inoculant of this
type stored in sterile distilled water (Somase-
garan and Halliday, 1982) and paved the
way for several current commercial products.
Production is relatively simple. Rhizobia are
harvested from a liquid culture, washed free
of the spent medium, and stored at a prede-
termined concentration in sterile distilled
water at ambient temperature. The formula-
tion is ready for use as a spray by dilution
with water. Today, Rhizobium products such
as LIFT (LiphaTech, Milwaukee, Wisconsin,
USA), Liqui-Prep (Research Seeds, St Joseph,
Missouri, USA), and products containing
other organisms, are variations of this simple
formulation. The dormant organisms may be
in the form of bacteria, bacterial spores, fungal
spores or short fungal hyphal fragments,
often formulated with ingredients such as
growth suppressants, contaminant suppres-
sants (=preservatives, Appendix Table 1.8)
and surfactants (Appendix Table 1.5).
It is the author's experience, however, that
this formulation in particular is not suitable
for organisms whose fast actions are required
immediately upon application to soil. Experi-
ments with Rhizobium and soybean demon-
strated that the bacteria in this formulation
are in an extreme dormant stage. The bacteria
after storage are much smaller in size and, as
judged by the position of nodules formed by
the bacteria in soybean roots, require a pro-
longed reactivation time (A. S. P., unpub-
lished data). For most Rhizobium-legume
inoculants applied at planting or later, this
long lag time is not desirable. By the time
the inoculant strains are fully reactivated in
the soil and are physiologically ready to infect
the host legume plants, the plants are usually
fully nodulated by the indigenous strains,
often resulting in sucessful exclusion - at
least partial - of the inoculant strains.
7.5.Id Dormant oil suspension Organisms can
be suspended in oil at high concentration in
various degrees of dehydration and remain
viable (Johnston, 1962). This formulation deli-
vers organisms in a physiologically dormant
state and does not encourage the growth of
contaminants during storage or transport.
Rhizobium has been successfully dried by
continuous aeration as a suspension in oil to
provide inoculants with shelf-lives of several
years. The process requires mixing a culture
concentrate (cell paste) with oil and then pas-
sing dry air slowly through the suspension to
remove excessive moisture. This suspension
in oil can then be applied directly to seeds or
be sprayed with or without dilution with
water and a wetter. Alternatively, the bacteria
can be lyophilized prior to mixing with oil
 7.5.2
and other ingredients (Kremer and Peterson,
1982,1983; section 4.6.2). Lyophilization, how-
ever, is impractical on a large scale.
Due to high production costs, time-consum-
ing drying processes and slow action because
of the dormant state of the rhizobia, the
formulation is mostly used to deliver rhizobia
in combination products that also contain
chemicals such as fungicides, insecticides
and molybdenum compounds. In such pro-
ducts, the metabolically dormant state of the
rhizobia protects them from the high concen-
trations of hostile chemicals in the final
products until dilution at application. This
combination inoculant is useful in situations
where the benefits of the chemicals outweigh
the benefits of early action by the inoculant
Rhizobium strains. Such situations include
soils with high disease pressures, low molyb-
denum availability, and low indigenous rhi-
zobia presence.
Fungal spores are ideal candidates for oil
suspension formulations. Aerial spores of
fungi from species such as Gliocladium, Tricho-
derma, and Paecilomyces can easily be collected
from the top surface of solid-state (non-
submerged) cultures by vacuum suction
(Chet and Sivas, 1988; section 4.7.1 for insect
pathogens). These spores are relatively dry,
low in free water and directly suspendable
in oil without incurring significant costs for
moisture removal (sections 4.3.1, 4.6.5, 6.6).
Fungal spores and shortened hyphal frag-
ments can also be recovered from submerged
liquid cultures and then formulated into oil
suspensions (section 4.7.2). Such moist bio-
mass requires additional costs for drying,
which may affect the commercial viability of
the formulations. Regardless of whether the
dry aerial spores or the moist spores and
hyphal fragments are used as the starting
materials, the formulation normally requires
a wetter (a detergent or surfactant; sections
3.4.1, 4.3.6; Appendix Table 1.5) to allow the
oil suspension to be emulsified easily with
water and diluted for applications using con-
ventional hydraulic farm equipment. Some
Powder formulations for various applications 241
wetters stimulate spores, others are toxic
depending on species (sections 6.5, 4.7.3;
Appendix Table 1.5). For special applications
where a high-pressure, ultra-low-volume
applicator is available (Table 2.2 in section
2.2.2; Figs II.l and II.3 in Appendix 11), the
oil formulation can be sprayed and so will
not require a wetter, emulsification and dilu-
tion with water (sections 2.3.2a, 2.3.2b). A
commercial example for a dormant oil sus-
pension is the bioinsecticide Mycotrol GH,
for grasshopper control (Mycotech, Butte,
Montana, USA). It comes in formulations
both with or without a wetter (emulsifier) to
accommodate both types of application.
In general, dormant oil suspensions are dis-
tinguished from dormant aqueous suspen-
sions in that organisms suspended in oil
with low moisture are less prone to premature
regrowth until reactivation with moisture,
and in that the products are less likely to be
overtaken by contaminant growth during
storage and transport.
7.5.2 POWDER FORMULATIONS FOR
VARIOUS APPLICAnONS
Organisms can be formulated into concen-
trated dry or wet powders for easy storage,
transport and application. Depending on their
component ingredients, these powders can be
applied to soil by many methods. They can be
(i) applied directly to soil with no further
manipulations; (ii) suspended in water or
other carriers for spray applications; or (iii)
dusted onto seeds to deliver the organisms
using the seeds as vehicles. Method (i), how-
ever, is not advisable under windy conditions.
The two general types of powders are easily
distinguished by their moisture content.
7.5.2a Wettable dry powders These are nor-
mally spray-dried or lyophilized biomass
with practically no free moisture. For bacteria,
the biomass consists mostly of the bacterial
cells and spores. For fungi, the biomass con-
sists mostly of spores and some hyphal frag-
242 Formulation of beneficial organisms applied to soil
ments. Both lyophilization and spray-drying
are standard processes and will not be
described further here (section 4.6.2). Zamola
and Kaifez (1976) described the well-
developed process for spray-drying bacterial
spores. This is readily adaptable to formulate
fungal spores (section 4.6.2) and other organ-
isms amenable to drying at ambient or higher
temperatures. Lyophilization is seldom used
for large-scale production because of cost,
and its use is limited to research and develop-
ment.
In general commercial production, the bio-
mass is spray-dried or, more rarely, lyophil-
ized in the presence of a wetter and maybe
some bulking carrier materials (e.g. section
3.2.1 and Table 3.2 in that section; Table 3.11
in section 3.3.4; Appendix Tables 1.2 and 1.3).
The wetter, such as an ionic surfactant
(Appendix Table 1.5), allows the product to
mix easily with water or other liquids for
spraying. More importantly, the wetter allows
the powder to imbibe moisture easily once the
inoculant reaches the soil to facilitate the re-
activation of the organisms (section 4.6.7). The
bulking carrier, if used, is usually a light-
weight ionic material in fine particle sizes
(> 300 mesh). Examples include fine clay
powder (e.g. Pyrax or talc), vermiculite, peat
and activated charcoal. The carrier prevents
the biomass from caking or forming hard
aggregates, which frequently occur if the bio-
mass is dried alone directly. The cake will
render the biomass extremely difficult to
apply either as a spray suspension or as a
seed coating. Additional ingredients - includ-
ing dye and membrane stabilizers such as
skimmed milk, sugars and salts (Appendix
Table 1.8) - are often included before or
during the drying process. Examples of
commercial products include Bacillus thurin-
giensis for insect control (Chapter 3); Myco-
stop (Mohammadi, 1994), a Streptomyces
product for sprays on plant foliage or soil to
control Alternaria, Fusarium and other fungal
plant pathogens; and BioMal, a spray-dried
wettable powder bioherbicide for control of
round leaf mallow, Malva neglecta (S. Gleddie,
Philom Bios, Saskatoon, Canada, personal
communication).
Another method of making wettable dry
powder is physically to grind larger dry gran-
ules or aggregates carrying the organisms into
a fine powder. These granules or large aggre-
gates can be prepared either by drying bio-
mass and all necessary ingredients directly on
granules, or by culturing organisms in a moist
granular carrier and then allowing the fer-
ment to dry. However, the grinding process
is not suitable for organisms sensitive to phy-
sical shearing, and is often suitable only for
organisms which produce spores with thick,
tough walls.
Some dry-powder products, although also
wettable, are not designed for spraying. They
are dusted or coated onto seed, and use the
seeds as vehicles to carry the organisms into
soil. The wettable property, however, facilit-
ates rehydration of the organisms for activa-
tion. Such Bacillus products are currently sold
commercially in the USA for biological con-
trol of plant diseases (Table 5.1 in section 5.2).
They include Quantum 4000 and Kodiak (pro-
ducts of Gustafson, Plano, Texas, USA; section
8.4.2a). Liphatech (Milwaukee, Wisconsin,
USA) produces a Nitragin Gold alfalfa Rhizo-
bium dry powder for seed coating. The pro-
duct contains 2-4% final moisture in clay and
is sticky enough to coat seed without needing
a binder.
7.5.2b Moist powder cultures Organisms
which do not form filamentous or hyphal
growth and hence do not form a mat of bio-
mass can easily be formulated into a moist
powder suitable for spraying, seed coating or
direct discharge into soil. These organisms
include non-filamentous bacteria, yeasts, pro-
tozoans and other microscopic organisms.
Examples of suitable carriers include peat,
vermiculite, sawdust or other materials that
hold moisture well but do not form hard
aggregates. Moisture content in the powder
products may range up to 70% without the
 7.5.3
powder giving a wet feel with free-standing
liquid. Carrier particles are of very fine mesh
sizes (section 8.3.2).
The organisms can be fermented directly in
the fine powder (Graham-Weiss et al., 1987;
McCabe et al., 1994) or are added to the pow-
der after fermentation in a separate container.
The powder is kept moist in a sterile container
until application. Organisms in the formula-
tion remain metabolically active for fast action
once applied to soil. With the former method,
direct fermentation in the end-use container
also minimizes any risk of contamination
until the container is opened for use. Once
the in-container fermentation is complete,
invasion by contaminating microbes into
Granules for soil and spray applications 243
these 'fully occupied' environs is difficult.
Open containers of such products often
remain microbiologically pure for some time.
This method is now quite popular in southern
Europe and South America for producing
Rhizobium and Azospirillum inoculants. Table
7.3 is a brief guide to the production of this
simple but versatile inoculant.
7.5.3 GRANULES FOR SOIL AND SPRAY
APPLICAnONS
Granules are generally easier to handle and
apply, and are less dusty than powders.
They are, however, more bulky and have
higher material, storage and transport costs.

Table 7.3 Production of a moist vermiculite powder inoculant of Rhizobium japonicum for soybean
(adapted from Graham-Weiss et al., 1987)

Ingredients Percentage (w/w) Function

Inoculated yeast-mannitol broth 62 Active organism, moisture, nutrient
Vermiculite powder 33 Carrier, natural buffer
Water 5 Moisture, steam for sterilization

Method
1 Grind grade 3 vermiculite (W. R. Grace & Co.) in a Wiley mill and collect 45-80 mesh particles using
US standard sieves
2 Dispense 33 g of vermiculite in a polystyrene (2 mil thickness) plastic bag, add 5 ml water
3* Heat seal the plastic bag and avoid trapping air in it as much as possible. It should be of sufficient size
such that, upon autoclaving, the little trapped air and the steam created by the moisture inside may
expand without bursting the sealed bag
4 Stick a 5 x 2 cm piece of autoclave tape onto a flat corner of the bag. One end of the tape is folded back
to stick onto itself to create a 1 cm long 'pull tab'
5* Autoclave the sealed bag, then allow to cool. Vermiculite is sterilized by steam created by moisture
inside the bag
6 Using aseptic techniques, inoculate a sterile yeast-mannitol broth with a 1% Rhizobium japonicum
inoculum and mix thoroughly. Final bacterial density is around 104-5 per ml.
7 In a microbiological hood, pull the pull tab of the autoclaved bag to expose from under the tape a
small sterile surface area of the bag. Do not pull the tape completely away from the bag
8 Inject through this newly exposed, sterile surface area 62 ml of the broth prepared in (6) using a
needle on a sterile syringe or a peristaltic pump
9 After injection, re-seal the exposed area with the tape. Mix the content inside the bag to evenly
distribute the injected broth
10 Allow content to ferment in the bag at room temperature for 2 weeks or longer. Fermentation
continues during shipment or storage. Inoculant is ready for use or long-term storage at ambient
temperature for at least 2 years. Final product has 108-9 rhizobia/ g and can be used to inoculate
soybean by coating onto seed without a sticker
* Autoclaving can be replaced by a single exposure to gamma-irradiation at 2.5 M Rad or higher. For this, a heavy duty
polyethylene bag is preferred since a polystyrene bag will tum brittle upon exposure to gamma-irradiation.
244 Formulation of beneficial organisms applied to soil
Free-flowing granules, not sticky or tacky to
form bigger aggregates, are often referred to
as flowable granules. Most dry granules are
flowable granules, but moist granule products
often are less free-flowing. Both dry and moist
granules are suitable for broadcast and in-
furrow applications. Some granules can be
fabricated in such a way that they will disint-
egrate the instant they reach sufficient moist-
ure. Such granules, called water-dispersible
granules, are additionally suitable for spray
applications (section 2.3.1b).
7.5.3a Granular vehicles Granules in these
products are either not fabricated, or are fab-
ricated into granules before the addition of the
organisms. The commonest method of formu-
lation is to mix the organisms and ingredients
directly with the coarse granules. Such gran-
ules, depending on their intended uses, are
then either packaged directly or dried by
various methods before packaging. Other
than mixing and drying, they do not require
additional processing and are ready for soil
application. Examples include granular peat,
coarse vermiculite, perlite, clay, calcium
sulphate granules, corn cob and wheat bran
aggregates. They have varying moisture-
holding capacity, remain free-flowing and do
not form bigger aggregates under normal sto-
rage conditions.
Among the group, granular peat holds
30% or more moisture without significantly
compromising the flowability (section 8.3.2a).
The US commercial product Soil Implant
(LiphaTech, Milwaukee, Wisconsin, USA),
has a final moisture of > 30% and flows easily
through an insecticide hopper without aggre-
gating. It is made by sieving off granular peat
particles of a selected size from peat mined
from a bog and then mixing with a fermented
broth of Rhizobium to the desired final moist-
ure content. The wetted peat is then matured
for a couple of days to dissipate the heat of
hydration (Table 7.4).
Other materials are often used with less
moisture t9 maintain their free-flowing char-
acteristics (Table 8.3 in section 8.3.2). Also,
organisms in the drier granules can tolerate
higher storage temperatures due to their phy-
siologically dormant status. Table 7.5 illus-
trates the high temperature tolerance of some
experimental formulations prepared by dry-
ing fermented Rhizobium concentrates directly
on dry perlite granules (4-5 mm in diameter).
The porous nature of the granular carrier
provides a controlled drying rate, allows the
organism to acclimatize and produces an

Table 7.4 Production of a granular peat inoculant of Rhizobium japonicum for soybean

Ingredients Percentage (w/w) Function

Fermented broth 33 Active organism, moisture
Granular peat 67 Carrier, nutrient source

Method
1 Inoculate a yeast-mannitol broth (Graham-Weiss et al., 1987) with a 1% Rhizobium japonicum inoculum
and allow to ferment with continuous agitation and aeration at 26-30C for 10 days. Final rhizobia/ml
should be at least 109 Neutralize fermented broth with NaOH (amount required depends on the strain
used and the age of the fermented culture)
2 Sieve peat, pre-stabilized by storage at ambient room conditions, and collect granules 0.5-1 mm in size
3 Mix thoroughly peat granules with the fermented broth in a 2:1 (w /w) ratio
4 Level in trays the moist granules to no more than 10 em deep to allow efficient dissipation of heat
generated by peat hydration and residual fermentation. Allow to equilibrate for 2 days. Final product
should have at least 108 rhizobia I g
5 Granular peat inoculant is most effectively packaged in paper bags with a plastic liner for shipment and
storage. Inoculant is most effectively used for direct application into the seed furrow at 70 kg/ha
 7.5.3 Granules for soil and spray applications 245
Table 7.5 High-temperature tolerance of Rhizobium formulated on granular perlite for soil application

Strain/carrier" Initial cfu./g Treatment Survival! (cfu./g)

1325, perlite 108 22e, 45 min 108

1325, perlite 108 75'e, 45 min 108
2838, perlite 109 22 DC, 45 min 109
2838, perlite 109 75 cC, 45 min 109
A culture of Rhizobium japoniClim from a yeast-mannitol agar plate was harvested and mixed with an equal weight of
dry perlite particles 40-50 mm in diameter and allowed to slowly air-dry aseptically overnight in layers of ca 5 cm thick
(Paau, 1989) by covering the container with a sterile milk filter (ACME Mills Company, Detroit, Michigan, USA).
t Averages of six samples rounded to the nearest order of magnitude. All samples were stable in storage at room
temperature for at least 1 month without detectable decrease in c.f.u.

inoculant with a good initial survival recovery
(Paau, 1989). Although the formulations
provide good shelf-lives and tolerate high
temperatures, the perlite granules are very
abrasive and may damage conventional farm
machinery by repeated use. Gypsum (calcium
sulphate) granules, 3-4 mm in diameter, are
now being developed to dry rhizobia, bacillus
plant growth-promoting rhizobacteria, and
pseudomonad root-rot biocontrol agents
(J. H. Stephens, Microbio Rhizogen, Saska-
toon, Canada, personal communication). An
example of a clay granular product of vesicu-
lar-arbuscular endomycorrhizae is Nutri-
Link (Agridyne Technologies [formerly NPI],
Salt Lake City, Utah, USA). The product is
made by mixing spores and fragments of the
organisms collected from root cultures with
granular clay carriers and has a shelf-life of
at least several years. The product, however,
is no longer on the market because culturing
vesicular-arbuscular endomycorrhizae is
prohibitively expensive.
7.5.3b Fabricated granules These granules are
formed by processing components, often
powder or polymeric fluid carrying the organ-
isms and other ingredients, into hard aggre-
gates of increased sizes. Many methods are
used to fabricate these granules and some
typical processes are described in section 7.7
below.
Fabricated granules have some advantages
over granular vehicles in that they are often
made more uniform in size and composition;
offer a greater range of shape, size, composi-
tion and bulk density; and can be made from
many more types of raw materials including
various combinations of peat, vermiculite,
sawdust, clay, charcoal, wheat bran and poly-
mers such as alginate, carrageenan and even
polyacrylamide. The latter, however, is used
only for research because its toxicity makes
large-scale application to soil environmentally
unacceptable. Fabricated granules are gener-
ally more expensive than using natural gran-
ular vehicles.
A US product, GlioGard (formerly W. R.
Grace & Co., currently ThermoTrilogy,
Columbia, Maryland, USA), is a hard, dried,
alginate granule formulation containing
spores and hyphal fragments of the fungus
Gliocladiu11l virens. It is added to soil or other
potting mix for the suppression of root and
stem damping-off pathogens such as Pythiu11l
and Rhizoctonia. It is made by mixing a fer-
mented broth concentrate of the organisms
(spores, hyphal fragments and some spent
medium) and other ingredients with a solu-
tion of alginate, and processing into beads as
in section 7.7.3d. Upon drying in a fluidized
bed, the soft beads shrink and harden so
that they can be added to the soil to deliver
the fungal spores and hyphaI fragments
entrapped by the alginate. The product is
quite expensive to produce because of the
exhaustive drying required. The hard, solid
granule is also difficult to disintegrate.
An improved formulation of the same
organism was later introduced as SoilGard
246 Formulation of beneficial organisms applied to soil
(Table 5.1 in section 5.2). The product basically
is identical to GlioGard except that the
initial mixture contains a bulk of wheat
bran in addition to the organism and other
ingredients. The wheat bran not only serves
as a food reserve for the fungus once it hits
the soil, it also provides a light-weight bulk
and reduces the amount of moisture in the
soft beads that eventually requires drying off.
The inclusion of wheat bran bulk cuts down
substantially on the cost of drying and
improves the physical properties of the final
granule product, while the wheat bran food
source should also improve field survival of
the introduced organism. Table 5.2 in section
5.3; Table 6.7 in section 6.8; and sections 5.3.3
and 6.8 describe the preparation of alginate
granules.
A fabricated water-dispersible granular
product is exemplified by the entomophilic
nematode product BioSafe (ThermoTrilogy).
It is a clay granule containing a dispersant, a
surfactant and desiccated nematodes held
together by a binder (Georgis and Dunlop,
1993). Droplets of the nematodes are
encrusted with an outer coat of clay and dis-
ersant materials with a binder. Residual
moisture is then slowly removed to produce
a dry granule entrapping the desiccated
nematodes in the middle. When added to
water, the surfactant in the granule assists in
the wetting and imbibition of water, which in
turn allows the dispersant to physically disin-
tegrate the hard crust, releasing the nema-
todes in the centre for spray applications.
More details are given in section 9.3.2.
Various types of fabricated granule can be
obtained by simple rotary agglomeration of
materials such as sawdust and peat powder
by use of moisture or of a binder. Alterna-
tively, materials such as vermiculite powder
and clay powder can be extruded, pelletized
or tabletized into larger aggregates, as descri-
bed in section 7.7. Table 7.6 in section 7.5.3
describes the preparation of a simple extrud-
ed vermiculite granular inoculant.

Table 7.6 Extrusion of a granular* vermiculite inoculant of Rhizobium japonicum for soybean

Ingredients Percentage (w/w) Function

Vermiculite powder 35 Bulk carrier
Fermented broth 63 Active organism, moisture
Potato starch 2 Binder

Method
1I Prepare a vermiculite powder as described in Table 7.3
21 Prepare a fermented culture of rhizobia as described in Table 7.4
3 Mix 35 g of vermiculite powder, 2 g of potato starch, and 63 ml of a fermented broth of rhizobia in the
mixing bowl of a Kitchen Aid Pro Line heavy duty mixer/noodle-maker until the texture becomes
smooth and sticky (about 20 min at high setting)
4 Wearing gloves, hand-press the sticky bulk into nuggets (2 cm diameter x7 cm long cylinders) that can
pass through the feeder of the noodle-maker attachment unit
5 Slowly feed one by one the nuggets into the noodle-maker fitted with a template of 2 mm diameter
holes. Collect the extruded noodles in trays in layers of no more that 5 cm deep
6 Allow noodles to dry in a microbiological hood overnight with fan on
7 Rotate noodles in a drum (0.5-0.6 m diameter) on its side at about 45 r.p.m. until the long noodles break
into small oblong granules of 2 mm diameter x3 mm long. Loose dust and over-sized particles are
removed by sieving
Dispersible vermiculite granules suitable for spraying can be made by using grade 7 vermiculite (325 mesh, w. R.
Grace & Co.) in Step 1, and 2 g of microcrystalline cellulose (Avicel) instead of potato starch in Step 3. When added to
water, these granules disintegrate and the fine carrier particles do not interfere with spraying if the tank has an agitator.
t Steps 1 and 2 can be eliminated by using 98 g of a moist vermiculite powder inoculant as prepared in Table 7.3.

7.5.4 FORMULATIONS USING SEEDS AS
VEHICLES
All formulations useful in coating seeds (Paau,
1988) are suitable for delivering organisms to
soil using the seeds as carriers (Chapter 8).
7.6 INGREDIENTS COMMONLY FOUND IN
FORMULAnONS AND THEIR USE
Seldom does a product comprise one single
component. Many ingredients are added to for-
mulations for various purposes. The main pur-
poses include keeping organisms viable (either
by dormancy or active growth), manipulating
bulk for handling and delivery, promoting
the activity of the introduced organisms, and
arresting growth of potential contaminants.
Below are some examples of these components.
Appendix I gives a comprehensive list of ingre-
dients tried with microorganisms.
7.6.1 CARRIERS, DlLUENTS OR BULKING
ADDITIVES
The most common carriers are often seemingly
inert liquids, gels, or solids. These include
inorganic carriers such as water, vermiculite,
clay, calcium sulphate (various hydration
levels), mineral soil and sand; or organic car-
riers such as vegetable oil, compost, sawdust,
peat, wheat bran, corn cob, whey and synthetic
or purified natural polymers (alginate, K-car-
rageenan, polyacrylamide) (Appendix Tables
1.1-1.3). Carriers, however, are anything but
inert and greatly influence the performance
of the products (see also section 5.3.4).
Savoy and Breitenbeck (1988) reported that
use of an inorganic carrier, vermiculite, did
not stimulate the presence of predatory
protozoans in soil as much as the organic
peat carrier, resulting in better survival of
introduced Rhizobium and effectiveness of
the formulation. Vermiculite, because of its
ion-exchange capacity, also provides a good
buffered zone for introduced organisms in a
foreign soil environment. The same property,
however, renders the material unsuitable as a
7.6.2 Membrane stabilizers 247
carrier for organisms which release highly
ionic metabolites for the host's benefit. These
metabolites may bind to the carrier and not be
available to the hosts. Certain organic carriers
that have high lignin content also inhibit some
organisms, although they can also be a good
food source for the organisms upon decom-
position in the soil (section 7.6.9).
The nature of a carrier also limits one's
choice of processing methods. Steam steriliza-
tion of certain peats generated free radicals
and other organic by-products to the detri-
ment of Rhizobiul1l carried in the material
(Strijdom and van Rensburg, 1981; section
8.3.2a). Sand is non-porous and has low
organism-loading capacity without a binder.
In the formulation research and development
process, a good understanding of the carrier's
characteristics, both physical and chemical,
and of how it will react with the soil, organ-
isms, metabolites produced by the organisms
and the various processing protocols, is
important to a product's success.
7.6.2 MEMBRANE STABILIZERS
Many products contain desiccated or frozen
organisms which are reactivated after applica-
tion to soil. Both dehydration and freezing
may irreparably damage the membrane of
the organism and kill it. During dehydration,
water molecules which lodge themselves
among the hydrophilic tails of fatty acids in
the membrane bilayer are evaporated away.
Without these water molecules, fatty acid tails
are pulled together by van der Waal's force,
causing the fatty acids to gel and enter into the
liquid crystalline phase. This crystalline phase
shift causes the membrane to lose its fluidity,
some important transmembrane proteins, and
eventually its integrity. Trehalose is synthe-
sized by anhydrobiotic organisms, e.g. brine
shrimp cysts (Artemia salilla), slime mould
macrocysts (Dictyostelium), many fungal
spores and some nematode larvae which
naturally survive long periods of extreme
drought. Trehalose can move into the
248 Formulation of beneficial organisms applied to soil
Table 7.7 Effects of sucrose pretreatment on the survival of Rhizobium dried directly on soybean seeds in a
long term storage* test

Rhizobium/seedt
Storage period' No sucrose Sucrose'l

1 day 2.4 X 105 1.1 X 105

3 months 4.8 X 104 6.7 X 104
8 months 1.0 X 104 2.3 X 104
10 months 6.2 X 103 1.5 X 104
* Stored in a seed room at 5 'C in a paper seed bag with a plastic lining.
t Bacteria quantified by suspending three samples of 10 seeds, each randomly selected from the storage bags, in a
liquid medium, shaking, serially diluting and plating on agar.
t Since coating seeds at a rate of 13 g vermiculite inoculant per kg seeds.
Rhizobium fermented directly in vermiculite powder supplemented with a yeast-mannitol medium (Table 7.3).
~ Same as in () except also supplemented with a concentrated sucrose solution.

membrane to replace the water molecules
upon dehydration and allow the membrane
to maintain its integrity (Crowe et al., 1984).
With many organisms, addition of trehalose
prior to drying often increases their viability.
Many simple carbohydrates of similar mole-
cular size - such as maltose, sorbitol and
sucrose - have all found some success in
selected organisms depending on the organ-
ism's ability to utilize them (sections 4.6.2,
4.8.3; Appendix Table 1.8). Table 7.7 illustrates
the effects of sucrose on the survival of Rhizo-
bium upon drying directly on a seed surface
and during long-term storage.
Freezing has similar effects on membrane
integrity and kills cells. The water molecules
are not evaporated but are themselves crystal-
lized due to the low temperature. Many
agents such as glycerol, dimethyl sulphoxide,
magnesium sulphate and skimmed milk are
used to stabilize the membrane for freezing
(section 4.6.6d; Appendix Table 1.8). The exact
modes of action of these agents are not clear.
suppress the germination of fungal spores in
liquid formulations. When ready for use, the
suppressant is simply diluted out with the
addition of water and does not require com-
plete removal to allow spore germination.
Antibiotics such as cycloheximide (75-
100 p.g/ml) generally can be used to suppress
fungal contamination in cultures of bacteria
and other non-fungal organisms. Chloramphe-
nicol (100 p.g/ml) suppresses bacterial con-
taminants in fungal cultures. Crystal Violet
(1 p.g/ml) is sufficient to suppress growth of
most Gram-positive bacteria. Sodium azide
at (300 p.g/ml) can suppress most Gram-
negative bacteria and aerobic Gram-positive,
spore-forming bacilli. Rose Bengal (50 p.g/ml)
at neutral pH can generally suppress growth of
most soil bacteria. It can be used in soil inocu-
lants to enhance growth of fungi and some
bacteria, which are naturally resistant to Rose
Bengal.
7.6.4 BUFFERING SYSTEMS

Many products are loaded with buffer re-

7.6.3 GROWTH AND CONTAMINANT
SUPPRESSANTS
Often it is desirable to arrest the inoculant
organisms in a product at a certain dormant
state and/or to suppress the growth of
contaminants (sections 3.3.3b, 4.6.6b). Sorbic
acid and its various salts are often used to
agents such that, once introduced into soil, a
friendly microenvironment or safe haven is
created temporarily for the introduced organ-
isms. Inexpensive buffers such as phosphates
and carbonates/bicarbonates are often used.
As described earlier for vermiculite, many
carriers themselves also have a strong

ion-exchange capacity and may exert a pH-
stabilizing effect in the immediate proximity
of the introduced organisms. Consideration of
a strong buffering system is especially import-
ant in products carrying physiologically
active organisms that produce metabolites
which may shift the pH balance of the formu-
lation on prolonged storage. Maintaining the
pH at the optimum range for the inoculant
organisms also helps to discourage contami-
nant growth in the product.
7.6.5 BINDERS
Binders are normally used only in processing
granular soil inoculants or other large aggre-
gates. They are mostly water-soluble materials
such as starch, xanthan gum, acacia gum, glu-
cose, dextrin, gelatin, tragacanth, molasses,
polyvinylpyrrolidone, guar gum, methyl cellu-
lose, sodium carboxymethylcellulose, hydro-
xypropylmethylcellulose, cellulose acetate
and synthetic glues (Appendix Table 1.4).
After application, soil moisture dissolves the
binder and disperses the contents of granules
or aggregates. Binders are normally added to a
mix immediately prior to size-increasing pro-
cesses. Many hydrophilic colloids can also be
used. To make granules more resistant to dis-
integration in water, the water-insoluble bin-
der ethyl cellulose may be added.
Many carriers themselves serve as the
binding or entrapping agents. During their
gelation process, polymers like alginate and
K-carrageenan can gel together other ingredi-
ents into the aggregates (sections 5.3.3, 7.7.3d).
A good example is the alginate granule with
wheat bran in SoilGard. After gelling a drop
of alginate suspension of organisms and
wheat bran, both components are entrapped
or bound together by the alginate.
7.6.6 DISPERSANTS
To facilitate the disintegration of the large
aggregates or granules after application,
many products include a dispersant (Appen-
7.6.8 Activators 249
dix Table 1.4) tightly compacted among all the
other ingredients. Materials such as starches
(corn, potato, rice, wheat and partially prege-
latinized starch) and microcrystalline cellu-
lose (Avicel v, Emcocel, Ex-Cel) can swell
upon exposure to soil moisture. The swelling
loosens the aggregates or granules and im-
proves disbursement and release of the organ-
isms. Many effervescence systems, which
release carbon dioxide when added to water,
are also excellent disintegrating agents
(section 3.6.4). Other dispersant candidates
are amylose, alginic acid, amberlite, alumi-
nium silicate, sodium starch glycolate (Primo-
jet Explotab), croscarmellose (AC-DI-SOL),
crospovidone (Polyplasdone, Kollidon) and
soya polysaccharides (Emcosoy) (Table 3.22
in section 3.6.4).
7.6.7 LUBRICANTS
In some processes to increase the size of
aggregates, components are handled in a
tightly compressed environment. Some for-
mulators include a lubricant or glidant
(Appendix Table 1.4) to lower the impact of
friction which may damage the organisms. In
processes such as extrusion and tableting, it is
not uncommon to find lubricants such as
magnesium stearate in compositions being
mixed or pushed through the compression
and shaping operations. Other common
lubricants are talc, mineral oil, hydrogenated
vegetable oil (Sterotex, Lubritab), boric
acid, stearic acid, calcium stearate, glyceryl
palmitostearate (Precirol), polyethylene mon-
ostearates, glyceryl behenate (Compritol),
polyethylene glycol 6000, sodium lauryl sul-
phate, sodium benzoate, DL-Ieucine, adipic
acid, fumed silicon dioxide (Cab-O-Sil) and
silica hydrogel (Syloid) (Appendix Table 1.4).
7.6.8 ACTIVATORS
Many organisms used in soil inoculants
respond to external stimuli to perform their
benefiting functions. Inclusion of a small
250 Formulation of beneficial organisms applied to soil
amount of materials which can stimulate the
inoculant strains may pre-activate them for
action and increase the effectiveness of a pro-
duct. For example, Rhizobium reacts to compo-
nents released from the root of its compatible
host to initiate the nodulation process (Rolfe et
al., 1987), so inclusion of these components in
a formulation will ensure the inoculant Rhizo-
bium is pre-activated or is activated to nodu-
late roots as soon as it reaches the soil (Paau et
a!., 1990).
Biocontrol organisms (Table 7.1 in section
7.2.1), especially mycoparasites, also react to
the presence of their host and prepare them-
selves physiologically for action. Experiments
with Trichoderma and Gliocladium (Paau et al.,
1993) demonstrated that pre-exposure to an
extract of the targeted pathogen had profound
effects on their ability quickly to control dis-
eases caused by fast-growing pathogens such
as Pythium. Germination of macrospores of
Sporidesmium, a mycoparasite of Sclerotinia
sclerotia, also improved in the presence of a
sclerotiaI homogenate or extract (P. Adam,
USDA-ARS, Beltsville, Maryland, USA, perso-
nal communication).
7.6.9 FOOD SOURCES
To enhance the survival rate of organisms
introduced into soil, many formulators pro-
vide them with abundant food with the pro-
duct (section 5.3.4). The most commonly used
ingredients include, for example, wheat and
corn flour, molasses, wheat bran, wheat germ,
corn cob, soya meal and corn gluten. Many of
these have unanticipated side effects. The
high lignin content of corn cob may harm
some introduced organisms. Corn gluten has
also recently been shown to inhibit root
growth from germinating seeds (Christians,
1991; Christians et al., 1994) and hence may
retard growth of host plants. Each potential
food ingredient for a formulation must there-
fore be tested not only against the introduced
organisms, but also the host plants for which
the inoculant is intended.
7.6.10 COATING COMPOUNDS
To improve their surface properties, either for
better flowability or for delaying water imbi-
bition, many granules and powders are
often coated with selected compounds.
Water-soluble compounds, such as sucrose,
gelatin, polyvinylpyrrolidone, methyl cellu-
lose, hydroxypropylmethyl cellulose and
polyethylene glycol, improve flowability and
decrease dust formation without sacrificing
disintegration upon wetting. In contrast,
water-insoluble materials, such as maleic
acid co-polymer, methacrylic acid polymer
(Eudragit), shellac, cellulose acetate phthalate,
ethyl cellulose and zein, delay disintegration
of the product after application to soil. They
are normally used to target late-season
diseases or to act as a slow-release compon-
ent mixed with a more readily water-
disintegrated component to provide a contin-
ually functioning product.
7.7 SOME COMMON PROCESSING
METHODS
7.7.1 MIXING
The most common process in formulation is
mixing. While liquids can normally be mixed
easily by a magnetiC bar or a blade stirrer,
mixing of powders, slurry and dough-like
material is often challenging. Kitchen Aid
Hobart (Los Angeles, California, USA) manu-
factures many bowl-type mixers for dry
powder and wet dough, ranging from
research use (Kitchen Aid Pro-Line) to pilot
testing (Hobart, A200) to manufacturing.
High-shear mixers (e.g. Lodige), commonly
used in the pharmaceutical industry, are not
suitable for inoculant formulation.
7.7.2 DRYING
The most common drying processes used
directly on living organisms are freeze-and
spray-drying (sections 3.2.1, 3.2.2; Table 3.2
in section 3.2.1). While the former is expensive

and mostly limited to research use, the latter
is often used, especially for spore-forming
organisms. Both processes can dry initial fee-
der materials of up to virtually 100% moist-
ure, although suspenSions are commonly first
concentrated by centrifugation. Spray-drying
requires atomization using a pump and a noz-
zle to control the droplet size, so that powder
specification can be met. Once atomized, the
droplets encounter the drying air current
which controls evaporation rate and product
temperature. Co-current processes utilize a
mode by which drying air and droplets or
particles move through the drying chamber
in the same direction and are normally used
for organisms sensitive to heat. Counter-
current processes have drying air and
droplets or particles moving in opposite direc-
tions, and are often used for spore-forming
organisms able to withstand heat. For organ-
isms already in bulky carriers, spray-drying is
not feasible and can be replaced by fluid-bed
drying which is suitable for initial feed mate-
rials with less moisture. Niro (Columbia,
Maryland, USA) can custom make many
spray-dryers and fluid-bed dryers to use for
research, pilot, and commercial production.
Both Niro and Coating Place (Verona, Wis-
consin, USA) provide test and pilot runs on
a contract basis.
Microwave drying or dielectric drying often
harms living organisms because of the high
energy applied, and is not recommended by
the author.
The most economical method of drying is
atmospheric tray drying - a process by which
organisms and the carriers are air-dried
slowly for 16 h or longer (sections 3.2.1,
4.6.2). The drying process is simply evapora-
tion and can be controlled by the speed and
dryness of air moving over the trays. The slow
drying process is sometimes advantageous in
that it gives many organisms a chance to
slowly adjust physiologically to the drying
environment. Vacuum drying has some
advantages over atmospheric tray drying in
that it has less handling and is less labour
7.7.3 Size enlargement 251
intensive. It shortens the drying time, but the
removal of oxygen may not be desirable for
some organisms.
7.7.3 SIZE ENLARGEMENT
Granulating is a general term which includes
all types of size enlargement. The major rea-
sons for granulating are to improve ease of
handling, reduce dust, increase density,
improve product homogeneity (by avoiding
differential settling of the mixed components
of different density), improve uniform flow-
ability by optimizing particle size and shape,
and sometimes even to change the surface
properties of the particles. The following are
some methods commonly used.
7.7.3a Agglomeration Size is increased by
particle collision and adherence due to sur-
faces being wet, followed by redrying.
Agglomerates are soft, freely suspendable
and easily made in a rotating drum device
similar to a cement mixer. Surfaces can be
wetted with a dilute, weak binder (Appendix
Table 1.4) to improve the hardness of the
agglomerates. In a typical process, the particle
surfaces are wetted by spraying periodically
with the binder while the powder is continu-
ously rotating in a dish granulator, which is a
rotating pan fixed at an angle to allow parti-
cles to accumulate by gravity at one side of the
pan edge without overflowing. As the parti-
cles increase in size, agglomerates of a certain
size can be removed by screening and the
smaller particles recycled into the rotating
dish.
7.7.3b Induration As a variation of the above
process, small agglomerates are heated to
melt surface ingredients, which fuse together
upon cooling. Heating up to about two-thirds
of the melting point of the surface ingredients
is usually sufficient. To obtain a spherical pro-
duct, the induration process may also be car-
ried out in a rotating dish. Granular peat can
thus be enlarged to achieve bigger nuggets of
252 Formulation of beneficial organisms applied to soil
smooth surfaces. The smoother surface
ensures better flowability of the granules.
7.7.3c Compaction The size of powder is
enlarged to granule status by pressure applied
via either (i) a roller compactor to form a
large compressed sheet (smooth surfaced or
dimpled) which later can be broken into smal-
ler, granule sized particles; (ii) a die cavity
with a set of punches; or (iii) an extruder.
Briquetting is similar to roll compaction in
(i) except that a sheet is not formed first. The
rollers are paired with cups and individual
briquettes are formed directly. If the briquet-
ting process also includes an increase in
operational temperature to help fusing, the
process is called sentering.
Die-casting (ii) is often called tableting or
slugging. If the powder is wetted, it is called
pelletizing. The powder, often with a binder,
is pressed into shape and released from the
die as free-flowing granules or tablets.
Extrusion (iii) yields long noodles of
compressed material that require additional
processing to break into smaller granules.
The simplest method to do so is to allow the
noodles to dry to a certain degree and then
rotate them in a rotating drum. The length of
the granules is determined collectively by the
diameter of the noodles, the strength of
the binder used, and the moisture level of
the noodles during rotation. A more elegant
and more expensive way to make hard gran-
ules of low friability from extruded noodles is
to use a marumerizer (Luwa Bahnson, Green-
ville, South Carolina, USA). A marumerizer is
a horizontally rotating plate fitted inside a
stationary, cylindrical, smooth wall. The
plate has concentric groves. Noodle fragments
deposited in the centre of the plate are pushed
to the periphery, smoothing the corners in the
process by rolling over the concentric groves.
Once pushed to the edge at the smooth
cylindrical wall, the material is pushed
upward and forms a ring of continually mov-
ing material. Interparticulate friction, plus
that between the particles and the moving
base plate, spheronize and densify the
particles.
7.7.3d Gelation This includes processes in
which a fluid-state material is gelled either by
a drop in temperature (melt congealing) or by
a chemical reaction. The gelation of K-carra-
geenan from 42C to lower temperatures is a
good example of melt congealing, a process
generally undesirable for formulating living
organisms because of the initial high temperat-
ure involved. The gelation of alginate repres-
ents the chemical type of reaction and is by far
the preferred gelation process in entrapping
living organisms with other ingredients.
In a typical run, organism biomass and
other desired ingredients are suspended in
1-2% alginate solution. The suspension is
then pumped through a drainage system hav-
ing multiple small outlets (e.g. a bank of horse
needles) to create a continuous flow of dro-
plets into a gelling solution of succinate or
gluconate below, with constant agitation.
Alternatively, the alginate suspension can be
sprayed (atomized) into a gelling solution to
form micro-sized gel beads. The gelled beads
are then sieved from the gelling solution for
drying (sections 5.3.3, 7.5.3b; Table 5.2 in sec-
tion 5.3; Table 6.7 in section 6.8).
7.7.4 STERILIZATION
Sterilization of formulation components, due
to bulk, is often a resource- and time-consum-
ing process. Many inorganic components,
such as vermiculite, gypsum, perlite and
mineral oil, can be sterilized by dry heat at
high temperatures. Organic components are
conventionally sterilized by steam under
high pressure (autoclaving). Gamma-irradia-
tion (lsomedix, Libertyville, Illinois, USA) and
electron beam (Iotron Industries Canada;
Encinitas, California, USA) have also been
used successfully to sterilize bulk carrier
materials. Microwave processes are now
becoming more popular but are currently
still quite cost prohibitive.

7.8 RESEARCH REQUIREMENTS AND
FUTURE DIRECTIONS
In the development of a successful inoculant
product, the identification of an organism
with the desired beneficial action is often not
difficult. Reports of new isolates with specific
actions abound in the literature. To develop
these organisms, one must be able to produce
a commercially viable product. Formulation
of live organisms in commercial products,
however, continues to be a challenging and
often success-limiting step.
To meet this challenge, a thorough know-
ledge of the organism and the target host is
needed. It is important to study the organ-
ism's benefiting mechanism, its preferred
target or court of action, and its growth char-
acteristics, including nutritional require-
ments, dormancy (sporulation and sensitivity
to drought), reactivation, and metabolite pro-
duction, so that during the development pro-
cess the viability and efficacy of the organism
are not compromised. Equally important
when deciding on a commercially viable for-
mulation is understanding of the target host
and its conventional cultivation practice, as
well as information on the formulation pro-
cesses, raw materials and accessible applica-
tion machinery. Market acceptance is slow for
products requiring costly machinery which
growers do not already use routinely, or for
products requiring growers to substantially
alter their cultivation practice.
Researchers are often frustrated in that the
success of each formulation is highly depend-
ent on each individual organism to the degree
that there are strain-to-strain variations (sec-
tion 4.8). The lack of a generalization, or of a
specific pattern to strains - even within the
same species - requires a separate research
and development process for each organism
and adds tremendously to costs in product
development.
Products in the commercial market some-
times do not represent the best possible for-
mulation to deliver the organisms. Better
References 253
formulations often are available but are not
commercialized due to the limitation in cost
which the market will bear. High costs (d.
section 9.3.3) in the essential areas of raw
material, processing, transportation and sto-
rage invariably make many formulations
commercially unfeasible. To balance the
cost/benefit ratio of biological products
favourably for better market acceptance, mar-
keters should highlight the non-monetary
benefits such as public safety and the envir-
onmental friendliness of these products. The
importance of public educational programs
cannot be over-emphasized.
Research requirements and the future of
different types of microorganisms are corre-
lated in Chapter 10.
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Georgis, R. and Dunlop, D. B. (1994) Water dispers-
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Graham-Weiss, L. L., Bennett, M. L. and Paau, A. S.
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Johnston, W. R. (1962) Process for preparing viable
dry bacteria and molds. US Patent 3034968.
Kremer, R. J. and Peterson, H. L. (1982) Effect of
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Kremer, R. J. and Peterson, H. J. (1983) Effects of
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LeTacon, F., Jung, G., Mugnier, F., Michelet, P. and
Mauperir, P. (1984) Efficiency in a forest nurSery
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duced in a fermenter and entrapped in poly-
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Lewis, J. A. and Papavizas, G. C. (1985) Character-
istics of alginate pellets formulated with Tricho-
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571-9.
Marx, D. H. and Kenney, D. S. (1982) Production of
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Schenck), American Phytopathological Society,
St Paul, pp. 131-146.
Marx, D. H., Ruehle, J. L., Kenney, D. S. et al. (1982)
Commercial vegetative inoculation of Pisolithus
tinctorius and inoculation techniques for devel-
opment of ectomycorrhizae on container-grown
tree seedlings. Forest Sci. 28, 373-400.
McCabe, D. E., Martinell, B. J., Paau, A. S. and
Graham-Weiss, L. L. (1994) Production of micro-
bial field crop inoculants. US Patent 5288296.
Mihuta-Grimm, L. and Row, R. C. (1986) Tricho-
derma sp. as biocontrol agents of Rhizoctonia
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Mohammadi, 0. (1994) Commercial development
of Mycostop biofungicide, in Improving Plant
Productivity with Rhizosphere Bacteria (eds
M. H. Ryder, P. M. Stephens and G. D. Bowen),
Division of Soils, CSIRO, Australia, pp. 282-4.
Mohammadi, 0. and Lahdenpera, M. L. (1994)
Impact of application method on efficacy of
Mycostop biofungicide, in Improving Plant
Productivity with Rhizosphere Bacteria (eds M. H.
Ryder, P. M. Stephens and G. D. Bowen),
Division of Soils, CSIRO, Australia, pp. 279-81.
Nobbe, F. and Hiltner, L. (1896) Inoculation of the
soil for cultivating leguminous plants. US Patent
570813.
Paau, A. S. (1988) Formulation useful in applying
beneficial microorganisms to seeds. Trends Bio-
techno!. 6, 276-9.
Paau, A. S. (1989) Bacterial agricultural inoculants.
US Patent 4875921.
Paau, A. S., Bennett, M. L. and Graham-Weiss, L. L.
(1993) Production of enhanced biocontrol agents.
US Patent 5194258.
Paau, A. S., Bennett, M. L., Kurtenbach, C. J. and
Graham, L. L. (1990) Improvement of inoculant
efficiency by strain improvement and formula-
tion manipulations, in Nitrogen Fixation: Achieve-
ments and Objectives, Proceedings of the 8th
International Congress on Nitrogen Fixation, May
1990, Knoxville (eds P. M. Gresshoff, L. E. Roth,
G. Stacey and W. E. Newton), Chapman & Hall,
New York, pp. 617-624.
Ricard, J. J. L. (1987) Method of using immunizing
commensals. US Patent 4678669.
Rolfe, B. G., Redmond, J. W., Batley, M. and Djord-
jevic, M. A. (1987) Identification of flavones
which induce expression of Rhizobium or Bradyr-
hizobium legume-nodulating genes in legume
extracts. European Patent Application EP
245931.
Savoy, M. M. and Breitenbeck, G. A. (1988) Influence
of Various Carriers on Rhizosphere Colonization by
Inoculant Bradyrhizobia, Agronomy Abstracts,
American Society of Agronomy, Madison.
Smith, R. S. (1992) Legume inoculant formulation
and application. Can. f. Microbial. 38, 485-92.
Somasegaran, P. and Halliday, J. (1982) Dilution of
liquid Rhizobium cultures to increase production
capacity of inoculant plants. App!. Environ. Micro-
bial. 44, 330-3.
Strijdom, B. W. and van Rensburg, H. J. (1981)
Effect of steam sterilization and gamma irradia-
tion of peat on quality of Rhizobium inoculants.
Appl. Environ. Microbial. 41, 1344-7.
van Elsas, J. D. and van Overbeek, L. S. (1993)
Bacterial responses to soil stimuli, in Starvation
in Bacteria (ed. S. Kjelleberg), Plenum, New York,
pp.55-79.
Yang, S. S. and Ling, M. Y. (1989) Tetracycline
production with sweet potato residue by solid-
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biosynthesis of a microbial insecticide. US Patent
4133716.
APPLICATION OF MICROORGANISMS TO 8
SEEDS
Mark P. McQuilken, Peter Halmer and David J. Rhodes

CONTENTS
8.1 Introduction 255
8.2 Seed treatment processes and machinery 256
8.2.1 Organization of commercial seed treatment 257
8.2.2 Conventional and filmcoating techniques 257
8.2.3 Pelleting and coating techniques 260
8.2.4 Seed inoculation by encapsulation 262
8.2.5 Immersion techniques 263
8.3 Rhizobial inoculants 265
8.3.1 Preparation of broth cultures of rhizobia 266
8.3.2 Carriers 266
8.3.3 Stickers 268
8.3.4 Seed inoculation 269
8.4 Biological control agents 272
8.4.1 Actinomycetes 272
8.4.2 Bacteria 272
8.4.3 Fungi 273
8.5 Mycorrhizal inoculants 277
8.6 Research needs and future prospects 277
References 279

8.1 INTRODUCTION control of plant pathogens and, more recently,

Application of beneficial microorganisms to
seed for use in agriculture, forestry and horti-
culture has been under intensive investigation
for many years (Scott, 1989). Microorganisms
can be applied to perform specific functions,
notably nitrogen fixation, phosphate solubil-
ization, plant-growth promotion, biological
biological control of pests (recent reviews
include Paau, 1988; Taylor and Harman,
1990; Lewis, 1991; Smith 1992; Jones and
Lewis, 1993; Whipps and McQuilken, 1993;
Campbell, 1994; Rhodes and Powell, 1994).
Applying microorganisms to seed is an attract-
ive proposition because of the combination of
specific effect and limited environmental

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
256 Application of microorganisms to seeds
impact (see also section 5.3.1). In the familiar
adage, seed treatment has the potential to
deliver agents "in the right amount, at the
right place, and at the right time". With
increasing public awareness of the potential
environmental and health hazards of both
agrochemicals and fertilizers, and the
advances in biotechnology to improve the
performance of microbial products, applica-
tion of microorganisms to seeds is likely to
increase in the future.
The process of applying microorganisms to
seed presents a special set of technical consid-
erations. Obviously, sufficient numbers of the
inoculant must survive the process, and be
able to grow in the environment of germinat-
ing seed. However, there are likely to be many
crop situations where seed is to be inoculated
at some time and distance from the point of
sowing, rather than immediately before use.
In these situations, treated seed becomes in a
real sense a 'secondary formulation' of the
inoculant, and must have acceptable shelf-
life qualities. In practice, since seed must be
stored at low moisture levels, an inoculant
must be able to survive a period of low
water activity (section 4.2). Microorganisms
may also need to be applied in combination
with other active ingredients, such as fungi-
cides and insecticides. These factors raise
novel issues of strain selection and formula-
tion stability. Where microbial preparations
are stored for extended periods before appli-
cation, many manufacturers stipulate that
containers should not be left open. Obviously,
where optimum storage conditions vary from
these in the surrounding atmosphere, con-
tainers should be selected for minimum per-
meability to water and sometimes to oxygen.
This chapter reviews in detail our know-
ledge on the application of microorganisms
to seed, encompassing rhizobiaI inoculants,
biological control agents and mycorrhizal
fungi. Of these microorganisms, only rhizo-
bial inoculants have been extensively com-
mercialized for seed inoculation. Although
the literature on the isolation and evaluation
of microbial seed inoculants has grown ext-
ensively in recent years (see e.g. reviews by
Schippers et al., 1987; Weller, 1988; Lumsden
and Lewis, 1989; Renwick and Poole, 1989;
Harman, 1991; Campbell, 1994), relatively lit-
tle has been published on development of
seed inoculation methods per se. Research
papers often report use of the simplest inocu-
lation techniques, such as wetting seed just
before sowing, which are not always suitable
in commercial practice.
The chapter begins by describing the differ-
ent seed treatment processes and equipment,
and discusses how these processes are, or
might be, utilized to inoculate seed. Addition-
ally, new processes are considered, some still
at the research and development phase. Spe-
cific microorganisms are then reviewed as
case studies, with particular emphasis on rhi-
zobia and available inoculant formulations;
although the main focus here is on botanical
seeds, some examples are given concerning
the inoculation of potato tubers. All stages of
the process are considered where possible,
including production of microorganisms,
application to seeds and their subsequent
storage, and also efficacy after placement of
seeds in soil. An attempt has been made to
draw not only upon scientific literature, but
also patent literature, which contains much of
the current expertise. One difficulty in review-
ing this area, however, is that much of the
work is done by commercial companies, and
is not available for publication. Finally,
research needs and future prospects for
application of microorganisms to seeds are
summarized.
8.2 SEED TREATMENT PROCESSES AND
MACHINERY
Seed inoculation techniques are not yet
widely developed commercially, apart from
the great exception of the rhizobial inocula-
tion of legume seed. As future implementa-
tion of seed inoculation technology will
need to be integrated within the existing
 8.2.2 Conventional and filmcoating techniques
organizational framework of the seed indus-
try - comprising the stages of seed breeding,
production, processing, treatment, storage,
distribution and sowing - it is appropriate to
review briefly the key features of commercial
seed-treatment systems. For microbial inocu-
lation, these systems not only offer technical
options, but also impose certain constraints.
8.2.1 ORGANIZATION OF COMMERCIAL
SEED TREATMENT
Treatment of seeds before sowing to protect
against pests and diseases and to enhance
crop establishment and development is well
established in much of the world. For many
years, seed of most crops has been treated
with fungicides directed against seedborne,
soilborne and foliar pathogens (Maude,
1995), and for a few crops more recently
with insecticides as well. The way seed treat-
ment is organized, commercially and technic-
ally, differs considerably between crops and
between markets in different regions. A vari-
ety of equipment is used, ranging greatly in
terms of capacity, throughput and engineer-
ing design and sophistication. Values and
volumes of seed treated also cover a tremen-
dous range - from the so-called high-volume,
low-value agricultural crops at one extreme,
such as the small-grain cereals, to low-
volume, high-value horticultural and orna-
mental species at the other. Different types of
treatment are therefore appropriate for differ-
ent crops, and cost rather than performance or
biological efficacy may determine which treat-
ments are used (Taylor and Harman, 1990).
Relatively expensive and slow application
procedures and materials may be acceptable
for high-value crops such as small-seeded
vegetables, where maximum performance
potential is frequently required, but may not
be economically reasonable for agricultural
crops.
Differences also lie in where and who in
the industry is responsible for carrying out
the physical application of treatments. In the
257
more developed agricultural areas of the
world, much seed-treatment work is carried
out at specialist facilities by, or for, seed com-
panies or merchants. This applies particularly
to hybrid varieties, and to crops where spe-
cialist seed-processing technology is involved,
such as maize, cotton, sunflower, sugarbeet,
and many modern vegetable and flower vari-
eties. In contrast, for other crops - particularly
open-pollinated varieties of small-grain cer-
eals and legumes - a substantial proportion
of seed treatment is carried out near to the site
and time of sowing, using locally installed or
mobile treatment, and on seed saved by the
farmers themselves. Another seed treatment
situation involves outdoor crops grown from
transplants produced under cover, e.g. horti-
cultural brassicas, onions, celery, lettuce and
sugarbeet (in Japan).
It is of course vital that seed processing and
application of treatments do not diminish per-
formance potential or storability of seed. High
germinability and 'vigour' are important
quality parameters for seed, particularly for
crops sown to a stand or produced as trans-
plants, where as many seeds as possible
should establish uniform and healthy plants.
8.2.2 CONVENTIONAL AND FILMCOATING
TECHNIQUES
Seed-treatment techniques can be broadly
divided into two basic categories:
conventional treatment and thin filmcoating,
and
pelleting and coating.
8.2.2a Equipment and processes for treatment
and filmcoating In conventional treatment,
dry powders and (increasingly) solutions,
water-based wettable or water-dispersible
powders, or flowable concentrates (emulsion
or microencapsulated formulations) are appli-
ed directly to seed without further modifica-
tion. High-volume crops such as small-grain
cereals, legumes and oil seeds are usually
258 Application of microorganisms to seeds
treated in continuous-throughput machinery,
but batch machines are also available. Some
modern machines have very high capacities,
ca 5-25 t/h or more for fixed cereal-treatment
installations. Types of equipment have been
well reviewed, e.g. Jeffs and Tuppen (1986),
Clayton (1993) and Maude (1995). In all of
them, a key feature is to feed seed and formu-
lations at controlled, metered rates, while
the more sophisticated machines have fail-
safe controls to ensure this occurs correctly.
Often, drying of treated seed is unnecessary
because relatively little water is involved.
Various means are employed to ensure the
thorough mixing of seed and liquids, e.g.
spraying formulations onto a falling curtain
of seed, or stirring seed and liquid together
in a trough. Mechanical damage to seed must
be minimized, particularly in structurally
vulnerable seed such as maize. The dwell
time of seed in the machines can be less than
10 min.
By contrast, design of on-farm equipment is
much simpler - at the simplest, slurrying seed
with formulations or dusting with powders
just before sowing, in the 'planter-box' or
'hopper-box'. Dry materials do not adhere
well to seed, giving rise to poor, uneven load-
ing and dustiness.
Alternatively, for some crops filmcoating is
now preferred, particularly for horticultural
seed, and also for some pelleted species, not-
ably sugarbeet. Characteristically, a uniform,
dust-free, water-permeable, thin coating mem-
brane surrounds the seed. In commercial
practice the cosmetic quality standard of film-
coating is often high, achieving an even coat-
ing both over the seed surface and between
individual seeds.
In the filmcoating process, treatment
formulations are premixed with a binder,
adjuvants and pigments as an aqueous sus-
pension, then sprayed onto the moving seed
mass. The binder forms a film as the mixture
dries, so that the applied' additives are
stuck firmly to the seed surface. The complete
filmcoating mixture is usually prepared
only shortly before use, and may be kept
just for a few hours. Typical binders are
polymers of various types, such as deriva-
tized celluloses, polyvinyl acetate, alcohol or
pyrrolidone, and complex graft terpolymers,
based on starch, for example. Proprietary pig-
mented binder adjuvants are available from
specialist companies, in solid or water-based
forms.
Filmcoating formulations are applied onto
seed either in modified conventional or in
specialist equipment according to crop,
amount of seed to be treated, treatment for-
mulations, application rates and quality stan-
dard required. Conventional equipment is
appropriate where the amount of applied
liquid is relatively low, and where the seed
itself can satisfactorily absorb the added
moisture.. If necessary, a separate drying
stage can be added in the treatment process.
But where the amount of additives to be
applied to the seed is relatively large, or
where a higher standard of seed-to-seed dis-
tribution or of cosmetic finish is required,
relatively large amounts of liquid are usually
needed, and a different engineering approach
towards filmcoating is necessary.
In such specially engineered filmcoating
equipment, concurrent spraying and drying
takes place in an enclosed chamber, typically
at up to ca 35C, and seed is presented to the
spray system many times to build up the film
layer. It is thus possible, for instance, to
change the composition of the applied liquid
to give a multi-layered coat. Depending on
volume to be applied and spray rate, filmcoat-
ing runs usually take ca 15-100 min to com-
plete. Two basic design principles are widely
used commercially, both derived from batch
treatment equipment in the pharmaceutical
industry (Halmer, 1988).
In the spouted-bed system, seed is held in a
vertical cylindrical or inverted cone-shaped
vessel, and solutions are sprayed from the
bottom into a vigorous upward-moving
stream of air, which stirs and dries the
 8.2.2
seed mass. The system has relatively high
drying capacity, but can damage delicate
seed by mechanical attrition.
In 'side-vented' drums, solutions are sprayed
onto the surface of the seed mass, which is
stirred and mixed within a horizontally
inclined perforated rotating drum made
with baffles or riser blades. Drying air is
drawn continuously in a non-turbulent
stream across the drum and through the
seed. The drum is housed in a sealed cham-
ber, which in most designs is kept under
negative pressure.
In both types of machine, coating solutions
are sprayed by atomization through pneu-
matic or, more usually, hydraulic (air-spray)
guns, and e~tracted air is filtered to remove
seed and chemical dust. It is important to
minimize losses of material by spray-drying
onto the vessel wall, and blemishes in the
coating layer such as picking, peeling, crack-
ing or roughness. Liquid mixture composi-
tion, inlet and outlet pressure, flow rate,
relative humidity and temperature of the air,
rate of stirring and spraying, and spray-gun
placement are all key factors. Equipment is
commercially available to accommodate a
range of batch sizes, from ca 200 g to 50 kg
seed in spouted bed coaters, and up to ca
200 kg in drum coaters. Engineered to a high
standard, with safety cut-outs and fail-safe
features, the equipment is expensive and
needs careful optimization for successful
results with particular seed sizes and shapes,
and so at present in practice is suited only to
fixed-installation facilities. Throughputs of
several t/h are achieved in semi-automated
production lines, comprising machines work-
ing in series or in parallel.
8.2.2b Use for microbial inoculation The use of
conventional and filmcoating techniques for
inoculation has several implications. Bacteria
can be applied as vegetative cell cultures or
spores, and fungi as mycelial fragments, con-
idia, sclerotia or sexual spores. This variety of
Conventional and filmcoating techniques 259
cultural and cellular form does not in itself
present particular technical application prob-
lems, except that the inoculant needs to have
particles small enough to prevent gun block-
age during spray drying, and to avoid dusti-
ness and loss of cosmetic quality where that is
considered important. However, the microor-
ganism is subjected to considerable physio-
logical stress during seed treatment, so
natural resting structures such as spores tend
to survive best.
Although powder formulations may be
applied directly to seeds, as mentioned
above, this approach is associated with poor
adhesion. Preferably, and more likely, micro-
bial formulations need to be dispersed in aqu-
eous media for spraying or slurrying, which
thereby exposes the inoculant organism to
large and abrupt changes in water activity
(section 4.2). In the spray mixture, at a water
activity of about I, there is additional expo-
sure to other additives, and possibly also che-
mical pesticides - in a complete mixture that
may be prepared a few hours before use.
Microbes may then face physical abrasion
during spray droplet formation or in the tum-
bling seed mass. Then the inoculant is dried
within about an hour, as water activities are
reduced to levels suited to safe storage of the
seed (0.5 or less). If forced drying is needed,
there might also be exposure to relatively high
temperatures. Finally, if seed is being treated
well ahead of sowing time there may be a long
dry-storage period.
On the other hand, it may not be necessary
for more than a small proportion of the inocu-
lated microorganism population to survive,
providing that enough do - what may be
most important is that there is a certain mini-
mum level of viable cells per seed. The chal-
lenge lies in devising application techniques
to achieve reliable inoculation efficiencies,
and to minimize loss of inoculant viability
during processing and subsequent storage.
For example, in a model experimental inocu-
lation system of soybean with rhizobium, Bur-
ris (1994) investigated alternative filmcoating
260 Application of microorganisms to seeds
strategies. Most successful was a 'sandwich'
technique in which seeds were first coated
with 5% polyvinylpyrrolidone (PVP), fol-
lowed by peat-based rhizobium, and then
10-15% Sepiret (Seppic, France), both as per-
centage of seed weight. It is suggested that
using a peat carrier within the filmcoat layers
provided a suitable microenvironment for the
inoculant, buffering it against the stress of
coating conditions.
8.2.3 PELLETING AND CGA TING
TECHNIQUES
The main purpose of pelleting and coatingis
to change the handling characteristics of seed
by altering the shape, weight or size. Both
processes are commonly used for applying
chemical seed treatment formulations, and
both are, or could potentially be, used to
inoculate seeds with living microorganisms.
8.2.3a Crops pelleted or coated Pelleting makes
irregularly shaped seed round and smooth,
usually specifically for use with precision
drills, to mechanically singulate (prevent clus-
tering without obtaining standard spacing) or
space-plant in glasshouse or field sowings.
Pellet size tolerances are therefore normally
strictly specified, but differ for a given seed
species between markets, depending on the
particular drill settings used. By contrast,
seed coating - sometimes called encrusting -
applies relatively little material, still more or
less revealing the original seed shape. Struc-
turally, a coated seed can thus be regarded as
a 'partial' pellet. Indeed, the amount of mate-
rial added during coating may sometimes be
so low that the resulting product may be hard
to distinguish at first glance from filmcoated
seed. It is difficult, though, to define pelleting
and coating by an absolute degree of seed
weight increase, because this factor varies
markedly between species, depending on
natural seed shape, desired pellet size range
and materials used. Generally, however, pel-
leting increases seed weight by a factor of ca
2-50-fold, or more, compared with ca 0.1-2 for
coating.
By far the largest commercial use of pellet-
ing is for monogerm sugarbeet - almost all the
crop in Europe and Japan is grown from pel-
leted seed. Other species pelleted in commer-
cial quantity include carrot, celery, chicory
and endive, leek, lettuce, onion, pepper,
tomato and, to a lesser extent, some brassicas
and super-sweet corn varieties, as well as cer-
tain flower species, particularly those with
tiny seeds.
Seed coatings are used for a variety of pur-
poses, for example to upgrade the size range
of a seed batch, or to increase seed weight (e.g.
range grasses for air-seeding). Coatings are
also used commercially to apply chemicals to
horticultUl;al species and sugarbeet seed,
though this use is declining in favour of film-
coating. Most relevant to this chapter is the
well established use of coating techniques to
apply rhizobial inoculants to alfalfa (lucerne)
and other small-seeded legumes (section
8.3.4).
Pelleting and coating methods are technic-
ally applicable to seed of other crops, e.g.
small-grain cereals or oilseed rape, but at pre-
sent this is either not needed agronomically,
or cannot be justified on commercial or logist-
ical grounds.
8.2.3b Equipment and processes for pelleting and
coating Pelleting and coating involve essen-
tially the same processing steps, comprising
wet, drying and size-grading stages. Both pro-
cesses are usually on a batch or batch-contin-
uous basis. Typically, pellet batches are ca
250 g to 100 kg seed, though some commercial
systems operate by true continuous through-
put. In practice, pelleting has a slower
throughput than coating: more material is
added and addition is more gradual to control
final shape and size. Success depends on
application of a even base-coat layer, and
then on action of the tumbling mass to distri-
bute and mould blend materials as the pellet
grows around the seed. Because any particle

can serve as a nucleus during the process, it is
important beforehand to remove extraneous
matter of comparable size to seed. Both pro-
cesses are carried out commercially at special-
ist facilities, usually run on a secret basis. For
some seed species, notably sugarbeet, pellet-
ing is done semi-automatically in production
lines comprising several machines, but for
others it must be performed manually by
skilled operatives. Although much informa-
tion on the technology is proprietary, and
indeed involves a great deal of 'skill and art',
the general types of equipment, blend and
binder materials used have been reviewed
(e.g. Halmer, 1988; Taylor and Harman, 1990;
Appendix Table 1.4), and are covered in
patents.
Pelleting involves addition of materials in a
rotating horizontal or inclined pan or drum
(section 7.7.3a) to achieve the required control
of size. Seed is wetted, and then the powdered
blend is progressively added along with more
water by layering until the desired weight or
size increase is reached. Examples of materi-
als used include chalk, clays, diatomaceous
earths, perlite, sand, vermiculite, peat, and
wood fibres or other organic materials
(Appendix Tables 1.2-1.3); the water phase
also may contain dissolved or suspended bin-
ders or other slurried materials (Appendix
Table 1.4). Powder-size grades are important:
larger particles of some materials, e.g. above
en 100 pm, may not be incorporated into the
growing pellet but remain as free dust.
Throughout the size build-up stage, which
typically takes ca 20-200 min to complete, the
seed mass is continuously stirred mechanic-
ally to prevent formation of clumps, and the
agglomeration of 'empty' pellets. When large
quantities and longer times are involved, fric-
tional heating can slowly warm the mass by
up to ca 10C above ambient. Finally, the wet
pelleted seed is removed from the coating
pan, and dried at ca 30-50C depending on
the physiological tolerance of the species (e.g.
Table 7.5 in section 7.5.3), by forced air con-
vection in static or fluidized beds or rotating
8.2.3 Pelleting and coating techniques 261
perforated drums. Depending on the amount
of wet pellets and their moisture content -
which can be at least 50% on a fresh weight
basis - drying usually takes ca 30-300 min.
After size grading, any undersized pellets
can be returned to the process for rebuilding.
Coating can be done in a similar way to
pelleting, or in equipment of simpler design
where seed, water and coating material are
stirred together in a trough in a single addi-
tion stage. The commercial pre-inoculation of
small-seeded legumes with rhizobia uses this
approach (section 8.3.4.a). The time needed to
complete the wet and drying stages lies at the
lower end of the scale.
8.2.3c Use for microbial inoculation Chemical
seed treatment formulations are commonly
applied during commercial pelleting or coat-
ing, both as dry powders and aqueous slurries
or solutions. Pelleting permits applications to
be made at different points as layers are built
up. Active ingredients are placed either on the
seed surface (e.g. to control seedborne dis-
ease), or distant from it (e.g. to minimize phy-
totoxicity), and to separate different additives
from each other (e.g. if the formulations are
incompatible). In the simpler coating process,
however, treatment formulations are often
mixed throughout the blend.
Similar options can be used to apply
microbes to seed. As already discussed (sec-
tions 8.2.2 and 8.2.3a, b), inoculation subjects
an organism to a period of wetting - several
hours for pelleting, during which the microbes
lie in a water-saturated environment - fol-
lowed by periods of rapid drying and storage.
Nevertheless, seed pelleting and filmcoating
processes have been used successfully in
experimental studies to inoculate seeds, such
as with Pythium oligandrum (section 8.4.3c);
Trichoderma (Legro and Satter, 1995; Cliquet
and Scheffer, 1996; section 8.4.3f); Pseudomonas
putida (Shah-Smith and Burns, 1996, 1997; sec-
tion 8.4.2c); and also other bacterial genera e.g.
Streptomyces and Bacillus (P.H., unpublished
observations; section 8.4.2).
262 Application of microorganisms to seeds
8.2.4 SEED INOCULATION BY
ENCAPSULAnON
Gel-encapsulation technology has been
devised for delivery both of somatic embryos
(Redenbaugh et al., 1987) and microbe gran-
ules (sections 5.3.3, 7.5.3b; Fravel et al., 1985;
Bashan, 1986), and could perhaps be used to
deliver inoculated seed to soil.
One gel-encapsulation system, developed
for the formulation of biocontrol fungi, hard
alginate prill, (section 5.3.3; reviewed by
Lumsden and Lewis, 1989) could conceivably
be adapted to coat or pellet seeds. Digat (1991)
has patented a process to produce granules of
up to about 8 mm diameter, with a core con-
taining liquid-cultured bacteria and an outer
protective coating layer. The process was cap-
able of producing ca 2000 granules per hour
per injector. A variation on the technique was
devised to encapsulate seeds (soybean, pea,
maize, pelleted lettuce, sugarbeet and tomato
- a fairly round shape was a prerequisite)
with Pseudomonas or rhizobia inoculants (pro-
cess details in Table 8.1). Seeds were dropped
one at a time onto a hanging double meniscus
formed from two immiscible liquids, so as to
form around the seed an inner coating layer
containing bacterial culture mixed with algal
polysaccharide as a thickener, and an outer
envelope layer of sodium alginate and kaolin.
Capsules were immediately gelled by drop-
ping into calcium gluconate solution, then
dried and treated with a layer of bentonite
clay to aid handling. Freshly encapsulated
seeds had 107 - 108 living bacteria per seed,
but after only partial drying (1 h at 40 CC)
most recoverable titres fell ca lO-fold. There
was evidence of more bacterial death after
further drying to normal seed storage moist-
ure contents: recoverable titres of bacteria in
inoculated lettuce pellets fell from ca 108 to 105
c.f.u. per seed after drying to a residual
water content of 8-10%. The effect on seed

Table 8.1 Experimental seed encapsulation with bacterial inoculant (Digat, 1991)

Materials and equipment

Bacterial inoculant
Pseudomonas fluorescens-putida on Mishagi's liquid medium for 48 h at 26C, or Bradyrhizobium japonicum
on Yem liquid medium for 90 h at 28C, grown to stationary phase (ca 109 d.u./ml). Increase viscosity
to ca 80 c.p.s. by adding micronized, sterilized laminarin solution (9% w Iv). Larger seeds require higher
viscosity material. Adjust pH to 7
Envelope
Sterile aqueous 1.5% (w Iv) sodium alginate and 10% kaolin (w Iv), viscosity ca 350 c.p.s.
Seeds
Soya, pea, corn; or pelleted lettuce, sugar beet or tomato
Injector assembly
Inoculant layer is fed through a downward-pointing injector, and envelope material is simultaneously fed
through an outer concentric injector. Diameter of the central injection orifice depends on size of the seed
to be coated

Methods
1 Pump liquids simultaneously under pressure through the injector assembly to form a lower, denser
(envelope) meniscus, overlayed with an upper, lighter (inoculant) meniscus layer (see diagram, Digat,
1991)
2 Release a single seed over the double meniscus, so that as it falls under its own weight it is surrounded
by the two liquid layers
3 Allow the liquid-coated seed to fall into 0.1 M calcium gluconate, remove after 1 min and immediately
dry
4 Coat with bentonite clay

performance was not reported in this work,
and it will be important to evaluate in this
type of approach what effect the inoculant
coating has on germination and handling
properties of the seed.
8.2.5 IMMERSION TECHNIQUES
S.2.Sa Priming Seed-priming techniques for
the physiological enhancement of germina-
tion have been extensively researched and
developed over the past 25 years. Priming is
now in widespread commercial use as a tool
to help crop establishment over a range of
environmental conditions, especially for
horticultural crops, e.g. carrot, celery, leek,
lettuce, onion, pepper, tomato and some
flowers, and recently in some markets for
higher volume crops such as grasses and
sugarbeet. Some priming techniques also
have potential for the inoculation of seeds
(section 5.3.1).
All priming techniques incubate a batch
of hydrated seed at a predetermined water
activity for a time at a controlled temperature,
followed usually by drying (and treatment
and handling in the normal ways). Incubation
allows some of the germinative physiological
processes to occur, but germination itself is
not completed in any individual seeds - i.e.
embryo structures do not begin to emerge
from the seed coat. As a result, the subsequent
germination of the seed population is faster
and more synchronized. This response
increases the speed, uniformity and final
level of field emergence, particularly under
stress - such as cold, wet or a combination of
the two, supra-optimal germination tempera-
tures, or unstable soil structure prone to crust-
ing soon after sowing. Typically, enhanced
germination performance of dried, primed
seed is retained in storage for at least several
months, providing that suitable priming and
storage conditions are used.
There are three main large-scale priming
approaches, using different methods to regu-
late water potential.
8.2.5 Immersion techniques 263
In osmoconditioning, seeds are incubated in
an aerated solution of an osmoticum such
as polyethylene glycol, or an inorganic salt
such as potassium nitrate or phosphate,
using high liquid:seed ratios (e.g. 10:1) in
stirred bioreactors of various designs. At
the end of the process, seeds are rinsed
before further processing. Taylor and Har-
man (1990) and Gray (1994) provide access
to the very large literature on osmocondi-
tioning.
In the patented solid-matrix priming techni-
que, available commercially in the USA
(section 5.3.1), seed is mixed with an
approximately equivalent quantity of a fri-
able, non-clumping, inert material, e.g. a
carbonaceous, preferably ligneous shale or
coal, with an osmotic component at least
90% of the equilibrium water potential,
moistened sufficiently to equilibrate seeds
to the correct water content (Table 8.2; Tay-
lor et a!., 1988; Eastin, 1990). After incuba-
tion the extraneous solid material is sieved
off. A related variant technique, matricon-
ditioning, uses different inert materials in a
very similar way (Khan, 1992).
The third basic priming method also incu-
bates damp seed, but brings the seed
directly to a predetermined moisture con-
tent by various means, without using an
external matrix or osmotic agent to regulate
seed water potential. The patented drum
priming process is one version of this type
of treatment (Rowse, 1991, 1992).
Depending on species, priming may take ca
1-14 days or more, at 15-25C, with water
potentials between -0.5 and -2.0MPa. After
priming, seed is either pelleted or directly
dried, e.g. using fluidized-bed techniques.
Basic protocols are established for a species,
but often individual seed batches are pre-
checked to assess suitability for priming and
establish optimal conditions: these extra steps
take several days to complete.
In all priming-treatment approaches seed
must be regularly mixed and aerated to
264 Application of microorganisms to seeds
Table 8.2 Procedure for solid-matrix priming of seed (Taylor, 1990)

Optimal ratio of seed:solid matrix:water for effective priming at 15C

Parts by weight

Crop Matrix material Seed Matrix H 2 O*(%) Duratior/ (days)

Tomato Agro-Lit 1.0 1.5 95 6

Soft coal 1.0 1.5 95 6
Sphagnum moss 1.0 1.5 90 6
Carrot Agro-Lig 1.0 1.5 90 6
Onion Agro-Lig 1.0 2.0 80 6
Lettuce Agro-Lig 1.0 2.0 85 1
Cucumber Agro-Lig 1.0 1.5 60 6

Calibration
Pre-test a small sample of the seed at a range of water amounts and temperatures, according to experience
(guide conditions are shown in Table). Establish conditions that just prevent radicle emergence of seed
Methods (small-scale)
1 Wet seed with about 25% of its weight of water
2 Coat wet seeds with predetermined amounts of dry, flowable, particulate solid matrix material with
thiram at 0.2% w Iv
3 Add remainder of predetermined amount of water. Mix. Incubate in aerated conditions, but so as to
minimize evaporation, e.g. by standing in closed metal container with a ventilation hole
4 After incubation, mechanically separate matrix material from seed. Rinse seed. Blot dry.
Based on dry weight of matrix component.
t Leonardite ligneous shale. Total percentage organic 84%, < 1% nitrogen. Typically 90% < 200 mesh.
t Total percentage organic 90%, < 1% nitrogen.

ensure efficient and even gaseous exchange
and avoid local heating in the seed mass.
These are of course similar concerns to those
met in the large-scale production of microbial
cultures.
The matrix and damp-seed priming ap-
proaches are particularly suited to inoculation
of seeds. Both are akin to solid-substrate
fermentation, and allow the populations of
microbes to colonize the seed coat itself.
Several such biopriming studies have been
reported. Indeed, solid-matrix priming was
originally conceived partly to encourage
colonization of seeds after inoculation with a
bioprotectant slurry. The technique has been
used experimentally with Enterobacter cloacae
and Trichoderma spp. (sections 8.4.2b, 8.4.3f;
Harman and Taylor, 1988; Harman, 1991).
Callan et al. (1990, 1991) also used this
approach to inoculate super-sweet corn vari-
eties with Pseudomonas, and Legro and Satter
(1995) experimentally used a commercial bio-
priming technique to inoculate tomato seed
with Trichoderma, both to control Pythium.
8.2.5b Hydration Seed humidifaction is a
physiological enhancement technique particu-
larly suited to large-seeded species such as
beans and soybean that are highly susceptible
to imbibitional damage. The purpose is to
raise seed moisture contents by a relatively
small degree, and hydration levels reached
are much lower than in priming. Recently,
Szafirowska and Khan (1995) effectively trea-
ted snap bean seeds with Bacillus subtilis
(Kodiak, section 8.4.2a; Table 5.1 in section
5.2), applied before humidifaction.
8.2.5c Fluid drilling Fluid drilling is a precon-
ditioning technique related to osmocondition-

ing, which delivers pregerminated seedlings
to the soil, and which can also be used to
introduce inoculants to germinated seeds (sec-
tion 5.3.1). The technique allows a seed batch
to complete germination in an aerated liquid
medium (but without high concentrations of
osmotic agents) then, after removal of any
ungerminated seeds by density separation,
directly sows the seedlings by extrusion in a
viscous nutrient gel which may also contain
fungicides (Pill, 1991). The technique is parti-
cularly applicable to small-seeded vegetables.
Since success depends on timely production of
the germinated seed suspension, it is effect-
ively an on-farm technique. It requires reliable
sowing equipment for precision seeding and
optimized seed-bed conditions to ensure
development of the young seedlings, which
are very vulnerable to desiccation damage at
this stage. Although fluid drilling has been
used by some specialist horticultural growers,
e.g. for celery and tomato establishment, it has
not met with widespread commercial success,
for both practical and logistical reasons.
The fluid-drilling suspension can be used to
apply microbial inoculants to the germinated
seeds (section 5.3.1). This has the potential
advantage that the microbes do not have to
be dried, and may have the opportunity
directly to colonize the growing seedling
root before delivery to the soil. Conway
(1986) successfully used a modified hydro-
xyethyl cellulose gel to deliver sclerotia of
Laetisaria arvalis to control Rhizoctonia damp-
ing-off of pepper seedlings and Southern
blight (Sclerotium rolfsii) in apple seedlings.
Also, gel solutions have been investigated to
inoculate soybeans with rhizobia (Berg et al.,
1989); to inoculate pea and radish seeds with
Trichoderma spp. (section 8.4.3f; Hadar et aI.,
1984; Mihuta-Grimm and Rowe, 1986), and to
apply mycorrhiza (section 8.5; Hayman et aI.,
1981).
8.2.5d Steeping and heat Several physical
treatment techniques could theoretically be
combined with seed inoculation.
8.3 Rhizobial inoculants 265
Prolonged seed steeps with water-dispersed
fungicides to penetrate seed tissues and
control deep-seated seedborne pathogens.
Most notably, thiram soaking is commer-
cially used in the UK to treat vegetable
and sugarbeet seed.
Soaking with antibiotics, such as streptomy-
cin, to control seedborne bacteria. However,
these can be phytotoxic; also commercial
use is proscribed in many countries because
of the importance of antibiotics in medicine.
Hot water or aerated steam treatments, e.g.
for ca 30 min at 50-60C, are used commer-
cially for some small-seeded species, e.g.
flowers and celery. Success depends on
finding conditions lethal to the pathogen
with minimal damage to seed quality.
Further details of these techniques are
given by Maude (1995). Though it is possible
to envisage such treatment techniques being
used as a preliminary sterilization step before
the application of beneficial microorganisms,
no research seems to have been reported on
these lines.
8.3 RHIZOBIAL INOCULANTS
Symbiotic rhizobia fix atmospheric nitrogen
and indirectly increase soil fertility when in
association with a host leguminous plant
(Postgate, 1978; Eaglesham, 1989). The rhizo-
bia infect the root hairs of the host, causing
nodules to form. Within these nodules, the
bacteria obtain food and energy from their
host, and in turn fix nitrogen for plant growth.
There are many different types and strains of
rhizobia, most of which are specific to a parti-
cular legume. Most soils contain a population
of rhizobia, but these are often ineffective
strains which are unable to form active nitro-
gen-fixing nodules. Successful nodulation
depends on the presence of rhizobia, which
are able to form effective nitrogen-fixing
nodules on the host, and to compete success-
fully with the indigenous rhizobial popula-
tion. Inoculation of seed with beneficial
266 Application of microorganisms to seeds
strains of rhizobia, either before or at the time
of sowing, is an established and widespread
commercial practice for ensuring that effect-
ive nitrogen-fixing nodules are formed
(section 7.1). It demonstrates that the seed
application approach can work very success-
fully, at least for the leguminous plant-
rhizobia combination. Special considerations
involved in establishing microorganisms in
soil are similar to those faced by organisms
applied in separate formulations and are ela-
borated in sections 7.4 and 7.6.1
The process of seed inoculation with rhizo-
bia is principally concerned with growing a
broth culture of a viable strain, mixing the
broth with a suitable carrier and applying
this inoculant to seed employing an adhesive.
8.3.1 PREPARATION OF BROTH CULTURES OF
RHIZOBIA
To produce broth cultures of rhizobia prior to
their incorporation into a carrier, simple glass
vessels (e.g. round-bottomed flasks) are
required for small production and suitable
fermenters for scale-up (Burton, 1981; Soma-
segaran, 1991). The ingredients of the liquid
culture medium must be cheap and readily
available to minimize the costs of production.
Burton (1979) has listed several media. Var-
ious industrial by-products have also been
exploited for producing a range of rhizobial
species. For example, after 48 h incubation,
Bissonette et al. (1986) obtained 1010 viable
cells of Rhizobium meliloti per millilitre of
whey (a by-product of the cheese industry)
supplemented with yeast extract and phos-
phate. Similarly, high viable cell counts of
Rhizobium leguminosarum bv. viceae were
obtained in 200 1 capacity industrial fermen-
ters, using yeast extract as the sole carbon and
nitrogen source (Meade et al., 1985). However,
care must be taken in selecting the concentra-
tion of yeast extract as a number of workers
have demonstrated deleterious effects of con-
centrations between 3.5 and 10 gil on viable
cell counts, nodulating ability and subsequent
nitrogen fixation (Staphorst and Strijdom,
1972). Storage of washed, aqueous suspen-
sions and post-application problems are dis-
cussed in section 7.5.1.
8.3.2 CARRIERS
Broth cultures of rhizobia, usually containing
at least 109 cells/ml, are mixed with a suitable
solid carrier which serves several functions
through to product use (Smith, 1992; section
7.6.1). The carrier materials (Appendix Tables
1.2 and 1.3) form the major portion of the
inoculant, facilitating packaging, application
and use. They must have a high water-hold-
ing capacity, permit gas exchange, provide a
nutritive medium for growth of rhizobia,
and enhance survival during distribution
and when inoculated onto seed (Roughley
and Pulsford, 1982). Carriers for seed applica-
tion are usually in a powder form. Burton
(1967) has recommended that the particles of
the carrier must pass through a 0.25-mm
sieve, whereas Strijdom and Deschodt (1976)
have suggested that 50% pass through a
0.075-mm sieve. In general, better seed adhe-
sion is obtained with a finer particle size.
8.3.2a Peat Peat is usually the preferred car-
rier worldwide for rhizobia inoculated onto
seed (Strijdom and Deschodt, 1976; Burton,
1982). The type of peat affects the number of
rhizobia cells which develop, and their sub-
sequent survival during storage. Its suitability
can also vary depending on source and batch
(Roughley and Vincent, 1967; Somasegaran,
1991). Notably, the chemical analysis of a
peat may not always confirm its quality as a
suitable carrier. The choice of an acceptable
carrier peat can be made only on actual tests
of its suitability for growth and survival of
particular strains of rhizobia. Once selected
and harvested, peat should be screened to
remove debris, milled, then dried with forced
air. Drying temperature should not exceed
100C as higher temperatures can degrade
peat and release toxic substances, which can

also restrict subsequent growth and survival
of rhizobia (Roughley and Puisford, 1982; sec-
tion 7.6.1). Most peats are too acidic to use as
carriers without prior neutralization. Finely
powdered CaC03 is the most satisfactory neu-
tralizing agent, and is usually added to the
peat before milling (Roughley and Pulsford,
1982).
Two methods are currently used to produce
seed inoculants with peat carriers. The main
factor distinguishing the two methods is
whether or not the peat is presterilized. In
the USA, the non-sterile peat method is well
developed (Nethery, 1991). Neutralized peat
is blended with a broth culture of rhizobia in
mixers, then incubated in large metal trays for
4-5 weeks to allow population increase, fol-
lowed by milling to break up clumps prior to
packaging (Burton, 1979; Smith, 1987). In the
presterilization method, neutralized peat is
usually packaged in thin polythene bags
before sterilization by gamma irradiation
(Thompson, 1980; Day, 1991). The rhizobial
culture is injected aseptically into the bags of
sterile peat and the puncture securely sealed
with an irradiated plastic adhesive label, after
which the contents are thoroughly mixed by
manual pummelling. Bags are then incubated
in rooms, usually at 28 DC for 7-10 days, to
allow multiplication before final quality
control procedures. A modification of this
presterilized method is used to produce
the commercial NPPL HiStick Rhizobium
inoculants (MicroBio, Cambridge, UK) (Day,
1991).
Seed inoculants produced with sterilized
peat support more rhizobia which live longer.
They also contain few, if any, contaminants
and provide the option for extending culture
production by diluting the broth without
sacrificing the final quality of the inoculant
(Somasegaran, 1985). However, they have
some disadvantages, including higher pro-
duction costs, increased labour, necessity for
a sterilizing unit and the use of aseptic proce-
dures during addition of the broth culture.
Also, sterilization by steam or by gamma irra-
8.3.2 Carriers 267
diation often causes peat to make toxic by-
products, and to undergo structural and com-
positional changes unfavourable for sub-
sequent growth and survival of rhizobia
(Parker and Vincent, 1981; Strijdom and van
Rensburg, 1981; Mulligan and Cooper, 1985).
8.3.2b Alternatives to peat Although peat is
generally considered to be the best carrier
identified to date, many countries, particu-
larly those in the tropics and subtropics,
have no deposits or reliable sources of peat.
A number of alternatives have been evaluated
with varying degrees of success (Table 8.3).
Crawford and Berryhill (1983) studied the
survival of Rhizobium phaseoli in coal-based
inoculants applied to seed. Although peat
was by far the best carrier, the eight coal-
based inoculants promoted the survival of
R. phaseoli on pinto bean seeds to provide
more than 104 viable rhizobia per seed after
4 weeks. In a similar experiment, Kremer and
Peterson (1982) compared the survival and
effectiveness of R. phaseoli applied (section
7.5.1d) to seeds in peat and oil-based inocu-
lants: the oil-based inoculant promoted better
survival, gave more nodules, and increased
both shoot dry weights and total nitrogen
content. Clays can also be used (Table 8.3).
For example, Dormal (Research Seeds, USA)
and Nitragin Gold (LiphaTech, USA) are two
commercial clay-based pre-inoculants (section
8.3.4a; Smith, 1992). Vermiculite has a multi-
lamellate structure which provides aeration
and space for microbial proliferation. When
supplemented with moisture and a nutrient
source, it was used to culture a range of
Rhizobium spp., producing populations of
108-109 / g. The final vermiculite-based inocu-
lants were stable and had good seed-sticking
properties. Agracetus (USA) developed a
vermiculite-based Gold-Coat rhizobium
inoculant by direct fermentation in a point-
of-sale container. Polyacrylamide, alginate
and a mixture of xanthan and carob gum
have also been suitable as inoculant carriers
(Jung et al., 1982).
268 Application of microorganisms to seeds
Table 8.3 Alternative carrier materials to peat for Rhizobium spp. inoculated on seed

Carrier material Reference

Cellulose Pugashetti et al., 1971

Charcoal-bentonite Bhatnagar et al., 1982
Clays Nethery, 1991
Coal Dube et al., 1980; Crawford and Berryhill, 1983
Coal-bentonite-lucerne Deschodt and Strijdom, 1976
Filter mud Philpotts, 1976; Anyango et al., 1985
Lignite Kandasamy and Prasad, 1971
Mineral soils Chao and Alexander, 1984
Polyacrylamide Dommergues et al., 1979; Jung et al., 1982
Vegetable oils Kremer and Peterson, 1983
Vermiculite Sparrow and Ham, 1983
Vermiculite and additives Graham-Weiss et al., 1987

8.3.2c Survival and quality standards Although
growth of rhizobia in the carrier is beneficial,
viability of the bacteria over a long time per-
iod is the most important factor. Survival of
rhizobia in peat-based inoculants depends on
type of peat, its pH (section 7.6.4), method of
sterilization, and temperature during incuba-
tion, storage and distribution (Date, 1970;
Mulligan and Cooper, 1985). High tempera-
tures above 30 OC should be avoided as they
can cause desiccation of the bacteria. Con-
sequently, the use of rhizobial inoculants in
tropical areas can be severely limited (Soma-
segaran et al., 1984). Wolf and Hoflich (1986)
have shown that the growth rate of rhizobia in
peat was increased by adding molasses, but
emphasized that storage of the inoculant at
cool temperatures was most important if via-
bility of the bacteria was to be maintained for
long periods. MicroBio recommend that their
NPPL HiStick inoculants are protected from
temperatures above 25C.
Rhizobial inoculants should contain
enough viable rhizobia to ensure that effective
nitrogen-fixing nodules are formed, and that
the inoculated rhizobia can compete success-
fully with the indigenous soil population. At
present, there is insufficient knowledge and
understanding of the responses of rhizobia
and its appropriate host to select an inoculant
standard that would achieve successful
nodulation by different strains under all con-
ditions. However, artificial standards for
levels of viable rhizobia in seed inoculants
have been established in many countries,
including Australia, Canada, France, The
Netherlands, New Zealand and South Africa
(Smith, 1992). In France, for example, alfalfa
inoculants must provide 106 viable Bradyrhi-
zobium japonicum per seed at sowing to satisfy
the registration procedure (Catroux, 1991).
These inoculants must also increase yield in
field trials conducted for a minimum of two
consecutive years in different geographical
areas of France. Although some countries do
not have formal mandatory standards to
cover inoculants, in practice quality levels
may be set by markets in areas of use. For
example, MicroBio, a UK company, ensures
that inoculants contain at least 2 x 109 cells!
g, and that this level is maintained for at least
18 months (personal communication).
8.3.3 STICKERS
Stickers are important in ensuring that the
rhizobial inoculant adheres to seed (see also
section 5.3.1; Appendix Table 1.6). They also
protect the rhizobia from desiccation and
therefore contribute to the shelf-life and effi-
cacy of the inoculant. Much work has evalu-
ated the effectiveness of a wide range of

potential stickers. For example, Elegba and
Rennie (1984) compared 12 commercial and
non-commercial adhesives to optimize inocu-
lation of soybeans with a commercial, seed-
applied, powdered, peat-based inoculant of
R. japonicum (Nitragin, USA). Gum arabic
(40% w Iv) and carboxymethylcellulose
(4% w Iv) were excellent stickers, binding
over 800 mg inoculant per seed and protecting
the bacteria from desiccation. Readily avail-
able wallpaper glue (10% w Iv) bound almost
900 mg inoculant per seed, whereas two pro-
prietary adhesives, Nitrigum and Nitracoat
(Nitragin), bound 453 and 541 mg inoculant
per seed, respectively. Gum arabic, carboxy-
methylcellulose and wallpaper glue gave a
high level of rhizobial survival. Despite
poorer adhesive properties, Nitrigum, Nitra-
coat and Pelgel (Nitragin) also gave good rhi-
zobial survival. Water, sugar, corn syrup,
honey and evaporated milk were poor stick-
ers and did not protect the rhizobia. However,
all of the stickers tested resulted in more than
105 viable rhizobia per seed which, under the
Fertilizers Act of Canada, is sufficient to guar-
antee adequate nodulation and N 2 fixation.
The use of Nitracoat, Nutrigum, gum arabic
and wallpaper glue in the field gave more
than 100 nodules per plant, and also increased
yield (Elegba and Rennie, 1984). Proprietary
wallpaper glues containing fungicides should
be avoided.
Copolymers have also been tested as stick-
ers (Williams, 1991, 1994). PVP/VA-S-630 is a
60:40 (w /w) polyvinylpyrrolidone/vinyl acet-
ate copolymer from GAF (Great Britain). It has
a high molecular weight and forms stable
emulsions in water. Incorporation of PVP /
VA-S-630 (10% w /w suspension) into peat-
based rhizobial inoculants had no detrimental
effect on rhizobial numbers during long-term
storage (Agricultural Genetics, Cambridge,
personal communication, 1994). Adhesion of
rhizobia to legume seeds was also improved
when seeds were slurried with inoculants con-
taining the adhesive copolymer compared
with water-soluble PVP and gum arabic.
8.3.4 Seed inoculation 269
Further improvements to satisfactory levels
of more than 105 rhizobia per soybean seed
and survival in the carrier for at least 5 months
at room temperature were obtained by mixing
the inoculants containing PVP-VA-S-630 with
an additional aqueous suspension (10-
20% w /w) of the copolymer before slurrying.
The coated seeds were dusted with powdered
clays, e.g. calcium montmorillonite (Surrey
Powder), to improve their appearance. Pig-
mented dyes (e.g. Rhodamine B500 and Blue
23123) were also incorporated into the coats
for identification. Both powdered clays and
pigmented dyes did not harm survival and
efficacy of rhizobia. Field trials using lucerne
seeds coated with R. meliloti and Blue 23123
gave 39-59% increase in weight of plant tops.
Similar trials with soybean seeds coated with
B. japonicum and Rhodamine B500 resulted in
increased seed yield, nodule number and
weight compared to uninoculated seeds. This
seed-coating method was superior to a three-
step method (P. M. Williams, Microbio, per-
sonal communication) of (1) coating seeds
with a mixture of sodium caseinate and finely
ground limestone (CaC03), then heat-drying;
(2) mixing with a slurry of a rhizobium culture
in a solution of PVP in water; and (3) adding a
kaolin-lime mixture to absorb excess moist-
ure. Another adhesive copolymer which
improved survival and subsequent adhesion
of rhizobiaI inoculants to seed is Aatara 430.
This copolymer is a vinylpyrrolidone/styrene
emulsion, also from GAP.
A number of proprietary stickers are com-
mercially available. These are obtainable
either as a separate container (e.g. Nitracoat;
LiphaTech, USA), or in the same package as
the inoculant but separated into two bags (e.g.
Grip from Titre, Canada), or premixed with
the inoculant in the same bag (e.g. HPPL
HiStick from MicroBio).
8.3.4 SEED INOCULATION
8.3.4a Pre-inoculation Substantial propor-
tions of small-seeded legume crops are
270 Application of microorganisms to seeds
grown from seed pre-inoculated with rhizo-
bial inoculant. It is convenient to the grower
to buy this seed ready to use. Most of the
alfalfa seed sown in the USA, and a large pro-
portion of clover and trefoil seed, for example,
is treated by seed-processing companies in this
form, and thus is often inoculated up to several
months before it is sown.
Pre-inoculation of seed was initially
undertaken using a process known as seed
impregnation (Brockwell, 1982). The seed
was moistened with a suspension of rhizobia
and then subjected to a vacuum. When the
vacuum was withdrawn, bacteria were sup-
posedly drawn into the seed through the
micropyle and scratches on the seed coat.
The process was disappointing in that the
vacuum was effective in drawing rhizobia
within the seed coat of only a small percent-
age of treated seed, and that few rhizobia
retained viability for any length of time dur-
ing storage. Seed impregnation of maize with
endophytes transformed with the toxin gene
of Bacillus thuringiensis was not commercially
successful (section 3.5.2).
The development of commercial lime
(CaC03) seed coating offered better prospects
than other methods for acceptable pre-
inoculation of seed. The method was devel-
oped in New Zealand to improve seedling
Table 8.4 On-farm pelleting of seed with lime (from Brockwell, 1982)
Materials
Gum arabic
Water
Inoculant
Seed
small (e.g. white clover)
medium (e.g. subterranean clover)
large (e.g. vetch)
very large (e.g. soybean)
Fine lime (CaC03 , ground limestone)
Methods
1 Dissolve gum in hot water. Cool. Chill overnight in a refrigerator. Add inoculant and mix thoroughly
2 Add gum-inoculant mixture to seed and mix until seed is evenly coated
3 Add lime all at once and mix rapidly for 1-1.5 min only. Remove excess lime, if necessary, by sieving
establishment by providing a more favourable
microenvironment when legume seeds were
sown in low-fertility, acid soils (Lowther and
McDonald, 1973). It basically involves apply-
ing the peat-based rhizobial inoculant as a
slurry to the seed using an adhesive, and cov-
ering the inoculated seed with a pellet of
finely ground lime, using seed-coating techni-
ques (section 8.2.3; details in Table 8.4). The
lime neutralizes acid soil around the seed, and
improves survival of applied rhizobia prior to
root infection.
A number of commercial lime-coated seeds
are currently available. Examples include
Guardcoat (Grow Tec, Alberta, Canada), Jet-
cote (Canadian Seed Coaters, Brompton,
Canada) and Rhizo-Kote (CelPril Industries,
Manteca, California, USA). All of these seed
coatings contain a peat-based inoculum, an
organic nutrient for the rhizobia, an unspeci-
fied polymeric sticker, and lime. A number of
beneficial effects have been reported following
their use. For example, seedling emergence
was increased by an average of 42% when
commercial lime-coated alfalfa seed was
sown in neutral soils in the midwestern USA
(CelPril, 1979). Thompson and Stout (1993)
reported improved seedling emergence and
increased root nodulation from using com-
merciaI lime seed coatings of alfalfa on a trial
Quantity
60 g
200ml
Manufacturers' recommendations
5 kg
10 kg
20 kg
40 kg
4 kg

site with low nitrate levels. However, Jones
and Lewis (1993) tested various commercially
prepared seed samples inoculated with rhizo-
bia, with very variable results. Samples were
often not as effective as seed freshly inoculated
with rhizobia immediately before sowing.
Since a high inoculant mortality rate is
observed in many samples (Brockwell, 1982),
length and conditions of storage by the farmer
after purchase are often the most critical fac-
tors affecting their field efficacy. Storage for
too long is likely to reduce rhizobial viability
to below a minimum threshold level (Brock-
well et al., 1975), resulting in reduced efficacy.
In some markets, pre-inoculated seed
batches are routinely tested by statutory
authorities. In Canada, for example, a most
probable number assessment is made using a
bioassay, based on the ability of a serially
diluted extract to induce nodules on aseptic
test legume seedlings or, alternatively, using a
colony immunoblotting method (Anon.,
1991). In the State of Wisconsin method
(USA), 35 two-seed replicates are planted in
tubes, and the number of individual seedlings
that nodulate is assessed against a 90% thresh-
old standard.
8.3.4b On-farm application at sowing Direct
application of inoculants to seed by the
grower immediately before sowing is the
most common method of applying rhizobia
to large-seeded legumes. One of the most
popular grower-applied methods is to pour
dry inoculant preparation directly onto seed
in the hopper-box and mix gently before sow-
ing. Commonly known as the dry method, it
is particularly convenient to the grower. How-
ever, there are reports that it may be the least
effective method of application as a result of
poor seed adhesion and retention (Smith,
1992). Much inoculum may also fall to the
bottom of the seed hopper. Use of seed drills
worked by either air pressure or air suction
may also reduce inoculum retention on seed.
Seed adhesion and retention of a number of
inoculants applied by the dry method has
8.3.4 Seed inoculation 271
been improved by the introduction of novel
stickers. For example, the NPPL HiStick pea-
nut inoculant (MicroBio), available in the
USA, contains a patented sticker (copolymer
of vinylpyrrolidone and vinyl acetate) which
ensures maximum adhesion of the inoculant
to the seed surface and is said to protect the
rhizobia after inoculation.
Slurry inoculation is a better method of
applying peat-based inoculants to seed, as it
increases the amount of inoculant adhering to
the seed. Basically, the inoculant is suspended
in water, with or without a sticker, and mixed
with the seed. The inoculant adheres to the
seed and upon drying remains on the seed,
especially if an adhesive is used.
On-farm 'pelleting' of legume seed is a
more complex method of inoculation. Techn-
ically more akin to a coating process (section
8.2.3), it involves mixing the inoculant with a
sticker (e.g. gum arabic), applying this mix-
ture as a slurry to the seed, then coating with a
finely ground material such as lime or phos-
phate/dolomite (details in Table 8.4). There is
no need for a drying step. Batches of pelleted
seed are best made in motor-driven rotating
drums, although a bucket or even a rotating
tyre will suffice. Advantages of using pelleted
seed include protection of the rhizobia against
toxic substances contained in the seed coat of
some legume seeds and against unfavourable
physical and chemical factors in soil, and
improved survival of the rhizobia on the
seed. However, on-farm pelleting has not
been widely accepted, mainly because of the
time required for this multi-step method.
A fourth, but simple, method of on-farm
seed inoculation is to mix commercial liquid
formulations with the seed before sowing, e.g.
Liqui-Prep (Research Seeds, USA) and Cell-
Tech (Lipha Tech, USA) (Smith, 1992). Con-
venient to use, these formulations provide
both good seed coverage and good adhesion.
However, since they have no substantial par-
ticle matrix, they are unlikely to assist the
survival of rhizobia on seed during environ-
mental stress (Smith, 1992).
272 Application of microorganisms to seeds
8.4 BIOLOGICAL CONTROL AGENTS
Application of biological control agents to
seed is typically to protect the seed and emer-
ging seedlings from effects of soilborne and
seedborne plant pathogens. Plant growth-
promoting rhizobacteria are also applied by
this route. Use of biological seed treatments to
control insects has received comparatively
little attention, although two examples are
discussed below. Most successful biological
control agents applied to seed must grow
and colonize the rhizosphere in order to pro-
tect the plant (Rhodes, 1993; Deacon, 1994;
section 7.2), although some early seedling dis-
eases may be effectively controlled with lim-
ited colonization beyond the spermosphere.
Release of biological control agent from the
formulation, and provision of the require-
ments for growth, are therefore of paramount
importance.
8.4.1 ACTINOMYCETES
The commercial product Mycostop (Kemira
Agro Oy, Finland; Table 5.1 in section 5.2),
based on the actinomycete Streptomyces griseo-
viridis strain K61, is formulated as a dry pow-
der of density 500 kg/m 3 , containing 108
c.f.u./g. The powder is dusted onto seeds at
5-8 g/kg seeds for control of soilborne and
seedborne fungal pathogens in vegetables,
ornamentals and herbs (Tomlin, 1994). The
formulation remains viable for 6 months at
8C or for 12 months at -12 GC, and must be
stored in unopened containers. After opening,
the product loses viability within a few days.
8.4.2 BACTERIA
The level of difficulty associated with form-
ulation of bacteria as seed treatments varies
widely between genera. In general, bacteria
which produce spores survive dehydration
better than those which do not (Potts, 1994).
The ability to survive in a dehydrated state is
an important characteristic of microorganisms
applied to seed, since it greatly increases the
usable range of formulation technology (Paau,
1988). In particular, the ability to retain viabi-
lity of cells at low water activity extends shelf-
life and allows the formulation to be applied
by the seed merchant or commercial seed trea-
ter, rather than on-farm.
8.4.2a Bacillus subtilis Seed treatment with
B. subtilis to stimulate plant growth and to
protect against soilborne pathogens has been
a subject of research interest for over 20 years
(Merriman et al., 1974; section 5.2.2). Recently,
a product (Kodiak; Table 5.1 in section 5.2)
based on B. subtilis strain GB03, a variant of
the strain A13 described by Merriman et al.
(1974), was introduced (Gustafson, Plano,
USA) as an inoculant for cotton, peanut and
beans to suppress the Rhizoctonia and Fusar-
ium root disease complex (Mahaffee and Back-
man, 1993). Another version of the A13 strain,
Quantum-4000, used to be marketed as a
peanut seed treatment, and strain GB07 is
also now available as a cotton seed inoculant,
Epic. Applied as a seed treatment, the bacteria
colonize the root system, competing with
root-invading pathogens (Tomlin, 1994). The
bacterium produces resistant endospores tol-
erant to heat and desiccation, and whose pro-
duction is controlled by fermentation
conditions (Anon., 1993). Spores are concen-
trated and dried into a powder. The products
may be applied as a water-based slurry or a
hopper-box treatment in several alternative
formulations at the rate of 104 - 105 spores
per seed, and are compatible with a number
of chemical seed treatments. One formulation
of Kodiak is intended for use on-farm as an
over-treatment on pre-treated seed. Another,
a co-formulation with fungicides, is currently
used to treat a substantial quantity of cotton
seed in the USA, and is applied either as a
slurry with lime directly after the acid delint-
ing process, or on-farm (Gustafson, personal
communication). In a recent experimental
study (Szafirowska and Khan, 1995), the
emergence of snap bean seed was greatly

improved if inoculation with Kodiak was fol-
lowed by a short period of seed humidifica-
tion. This treatment might allow proliferation
of the bacterium on the seed surface and will
also reduce the likelihood of imbibitional
damage to the seed after sowing, with the
consequent leakage of metabolites. More pro-
ducts are described in section 5.2.2.
8.4.2b Enterobacter cloacae This enteric bac-
terium has potential for control of Pythium
seed rot. It does not form spores, or other
structures able to survive dehydration. Stu-
dies have used simple inoculation techniques
successfully, e.g. application to cotton seeds
with methyl cellulose sticker (Nelson, 1988).
Harman and Taylor (1988) applied a liquid
culture of the bacterium to cucumber and
tomato seeds, together with a fungicide, by
solid-matrix priming. Bituminous coal (pH
6.6) was a more effective matrix material
than an organic leonardite shale (pH 4.1) -
which may reflect the better growth of the
organism at pH levels above 7, a point
returned to in section 8.4.3f.
8.4.2c Pseudomonas spp. These have been
under investigation for several years as poten-
tial biological control agents to control soil-
borne plant pathogens. In common with
E. cloacae, these non-spore-forming Gram-
negative bacteria pose particular challenges
for formulation as seed treatments. Kloepper
and Schroth (1981) studied the use of various
gums to maintain viability of plant growth-
promoting rhizobacteria during the dehydra-
tion process, presumably by forming a
protective layer around the cells. Dustable
powder formulations with gums and talc
were prepared and dusted onto potato seed-
pieces. The highest viability was obtained
with 20% xanthan gum. However, 90% viabil-
ity was lost during several months of storage
at 4C. The difficulty of obtaining an ade-
quate shelf-life with formulations of Pseudo-
monas fluorescens was also experienced by
Ecogen (USA), who were forced to withdraw
8.4.3 Fungi 273
the peat-based granular product Dagger G for
this reason (Lisansky and Coombs, 1993).
A commercial peat inoculum based on Bur-
kholderia (Pseudomonas) cepacia (Blue Circle;
section 5.2.1 and Table 5.1 in that section)
contains 5 x 108 viable cells/ g and can be
applied to seed as a planter-box treatment or
as liquid inoculum, either at planting or in
advance. The application rate is approx-
imately 106 cells per seed.
Recently, cells of P. putida 40RNF were
applied to sugarbeet seeds using two pro-
cesses: incorporation into filmcoats sprayed
onto pre-pelleted seeds, and incorporation
into the pellet material prior to pelleting
(Shah-Smith and Burns, 1996, 1997). Survival
of cells during the two processes was poor,
with only 7.1% surviving filmcoating and less
than 0.2% surviving seed pelleting. Neverthe-
less, cell viability and biocontrol efficacy of
40RNF against Pythium ultimum damping-off
were maintained for 24 weeks when stored at
18-20C (Shah-Smith and Burns 1997).
8.4.2d Serratia entomophila This entomo-
pathogenic bacterium is supplied to order
and applied as a soil treatment for control of
the grass grub (Costelytra zealandica) in New
Zealand. Preliminary investigations suggest
that it may have potential for application as
a seed treatment on ryegrass. Seeds were
coated with a lime-based formulation at a
rate of 1.5 x 106 to 7.5 X 109 c.f.u./g (Sadler
et al., 1992). Pearson and Jackson (1992)
reported that the shelf-life of the bacterium
could be doubled by mixing with a commer-
cial polyacrylamide, Alcosorb AB3. In this
formulation, 50% of the bacteria survived 11
weeks of storage.
8.4.3 FUNGI
8.4.3a Chaetomium globosum This has demon-
strated potential for control of soilborne plant
pathogens and seedborne Alternaria raphani
and Alternaria brassicicola when applied as a
seed treatment. Vannacci and Harman (1987)
274 Application of microorganisms to seeds
formulated the fungus by suspending asco-
pores in a solution of methyl cellulose to a
concentration of 107_10 8 spores/ ml. This was
applied to crucifer seeds at the rate of 1 ml
suspension to 220 seeds. Gordon-Lennox et al.
(1987) inoculated sugarbeet seeds by coating
them in methyl cellulose and dusting with
ascospores. Applied in this way, the fungus
remained viable for more than 2 years.
8.4.3b Gliocladium virens This is used in a
soil-applied granular formulation for control
of damping-off and root-rot pathogens,
including Pythium and Rhizoctonia (Chapters
5 and 7). Ristaino et al. (1994) formulated the
fungus in both bran prills and a vermiculite-
bran formulation. Its use as a seed treatment
has also been investigated. Mukhopadhyay et
al. (1992) coated seeds of chickpea, soybean
and lentil in conidial suspensions of the fun-
gus using various stickers: methyl cellulose,
carboxymethylcellulose, polyvinyl alcohol,
Polysulf, Laponite, Polytran and Pelgel. Stick-
ers were found to be beneficial only for
smooth-coated seeds.
Burgess and Hepworth (1996) treated sun-
flower seeds with an isolate of G. virens by
soaking them in a conidial suspension of the
fungus for at least 2 h. The crude seed treat-
ment was effective in controlling Sclerotinia
minor in both glasshouse and field experi-
ments.
8.4.3c Pythium oligandrum This occurs in soil
and gives promising biological control for a
range of damping-off plant pathogens (Al-
Hamdani et al., 1983; Vesely and Hejdanek,
1984; Martin and Hancock, 1987; Walther
and Gindrat, 1987; McQuilken et al., 1990b;
Whipps et al., 1993). The mycoparasite pro-
duces thick-walled oospores which can be
readily bulk-produced for application to
seeds. The inherent stability of the oospores
is an exceptionally valuable commercial attri-
bute.
Experiments have shown that P. oligandrum
can be successfully applied to seeds by both
pelleting and filmcoating. High yields of oos-
pore biomass of P. oligandrum were obtained
by liquid fermentation (details, Table 8.5).
Air-dried oospore preparations of biomass
were then applied to seeds of cress and
sugarbeet by two commercial processes
(McQuilken et al., 1990b), either into seed pel-
lets dried at 30 or 45C, or coated onto seed
surfaces using a filmcoating binder system
(section 8.2.3c) with drying at 25C. Approx-
imately 104 oospores per seed were recovered
from both seed types, achieving 75-95% of the
target dose. Oospores survived the processes,
which included physical abrasion and ex-
treme temperatures, giving levels of germina-
tion (9-19%) similar to those found before
processing. Mycelia of P. oligandrum grew
from all pelleted and filmcoated seeds when
plated on im agar medium after storage at 18-
22C for 16 weeks. In an earlier study, oos-
pores of the same isolate of P. oligandrum were
incorporated into a vermiculite-based carrier
which was then used to form commercial pel-
lets around seeds of carrot, cress and sugar-
beet (Lutchmeah and Cooke, 1985). Samples
of these seeds still produced mycelia of the
biological control agent when plated on agar
after 6 years' storage at room temperature
(18-22C; M. P. M., unpublished results).
Both the EB3-pelleted and filmcoated seed
treatments reduced damping-off of cress
caused by Pythium ultimum and Rhizoctonia
solani in artificially infested sand and potting
compost. In general, pelleting of P. oligandrum
gave better control than filmcoating. The
mycoparasite also reduced damping-off of
sugarbeet in soil naturally infested with Apha-
nomyces cochlioides and Pythium spp. Control
was equivalent to that obtained with hymex-
azol fungicide seed-coating treatments, and
was related to the natural infectivity levels of
A. cochlioides in the soil. At high infectivity
levels, neither hymexazol seed coatings
nor P. oligandrum treatments gave control
(McQuilken et al., 199Gb). P. oligandrum was
not rhizosphere-competent on cress or
sugarbeet which, together with the high
 8.4.3 Fungi 275
Table 8.5 Bulk production of oospores of Pythium oligandrum in liquid culture, and application to seeds of
cress and sugar beet (McQuilken et aI., 1990a,b)

Ingredients Apparatus
Cornmeal agar (CMA) 1-1 Roux bottle
Cane molasses (United Molasses, Dagenham, UK) Buchner flask with funnel
Distilled water Whatman No.1 filter paper
Cress and sugar beet Laminar flow cabinet
Autoclave

Methods
1 Maintain P. oligandrum on CMA at 25C
2 Add 200 ml of liquid cane molasses (30 gil distilled water) to a series of 1-1 Roux bottles, autoclave at
120C and 103.4 kPa for 15 min, and leave to cool
3 Inoculate bottles aseptically, each with three 20-mm diameter CMA discs cut from a 3-day culture of P.
oligandrum, and incubate on their sides at 25C for 21 days.
4 Harvest oospore biomass by vacuum filtration (Buchner flask with funnel) onto filter paper, wash in
three changes of distilled water, and air-dry overnight in a laminar flow cabinet at room temperature.
Each bottle produces ea 200-250 mg (dry weight) of oospore biomas containing ea 1.4-1.8 x lOs
oosporeslmg
5 Mix cress (1 kg) and sugar beet (100000) seeds with ea 4.0 and 4.5 g oospore biomass to achieve 35 x 103
or 50 x 103 oosporeslseed, respectively

temperature optimum for maximum growth
of P oligandrum (McQuilken et al., 1992;
Whipps et al., 1993), must limit the use of
the mycoparasite for long-term disease con-
trol of root-invading pathogens in temperate
regions. A search for rhizosphere-competent
and more active isolates which function in a
greater range of environments may be of
value (Whipps et ai., 1993).
8.4.3d Penicillium oxalicum Kaiser and Han-
nan (1984) reported that a seed treatment
of chickpea with P. oxalicum consistently
enhanced emergence and increased yields
under field and glasshouse conditions. Seeds
were dusted with dry conidiospores or coated
with a suspension in 1.6% methyl cellulose.
8.4.3e Talaromyces flavus This is a potential
biological control agent of a range of plant
pathogens including R. solani, Sclerotinia
sclerotiorum and Verticillium dahliae (Marois et
ai., 1982; Fravel et ai., 1986; Adams, 1990).
Approaches to formulation include applica-
tion as a dust to potato seedpieces and encap-
sulation in alginate with a bran nutrient base
(Papavizas et al., 1987; methods, Table 5.2 in
section 5.3.3 and Table 6.7 in section 6.8).
Ground soil-oatmeal preparations of T. flavus
have also been incorporated into seed pellets
of Chinese aster and tomato for control of
damping-off pathogens, using a commercial
split-pill process (Nagtzaam and Bollen,
1994). The biological control agent survived
17 years' storage at room temperature in the
pelleting material which contained quartz
flour and a polymer binder.
8.4.3f Trichoderma spp. These have received
significant attention for many years as biolog-
ical control agents with potential to control a
range of plant pathogens. A number of for-
mulation approaches have been investigated
(see also section 5.3.1). Beagle-Ristaino and
Papavizas (1985) applied the fungus to potato
seedpieces either as dry chlamydospores or as
fermenter biomass mixed with Pyrax ABB.
Suspensions of spores, produced on agar,
were diluted in distilled water and added
to Pyrax ABB (pulverized pyrophyllite,
276 Application of microorganisms to seeds
anhydrous sodium silicate, pH 7.0), to a con-
centration of 106 spores/g. Hadar et al. (1983)
evaluated three gel materials for application
to pea seeds, and found that inoculation using
hydroxyethyl cellulose performed best in
Pythium-infested soil. Mihuta-Grimm and
Rowe (1986) concluded that fluid drilling of
natural isolates from Ohio locations was most
effective in controlling R. solani on radish in
all soil types tested, compared to coating dry
seed using methyl cellulose, or drenching the
soil with a suspension.
Spores of Trichoderma take 12-14 h to germ-
inate after inoculated seeds have been planted,
but in this time seed-rotting fungi or competi-
tive microflora can become established. For
example, propagules of Pythium spp. germ-
inate rapidly, and begin to infect seeds within
4-6h after planting (review by Harman, 1991).
Prior colonization of the seed with Trichoderma
spp. by solid-matrix priming greatly enhanced
protection against P. ultimum and Fusarium
graminearum, presumably because the inocu-
lant was given a competitive advantage (sec-
tion 5.3.1; method, Table 8.2 in section 8.2).
There is evidence that inoculation methods
can be advantageously manipulated by favo-
uring the optimal acidic growth conditions for
Trichoderma spp. (pH 4-5) (section 5.3.5.).
Nelson et al. (1988) applied conidia to seeds
in a suspension of methyl cellulose, together
with various substrates which promoted bio-
control activity - the addition of organic acids
to Trichoderma koningii, and of polysaccharides
and polyhydroxyl alcohols to Trichoderma har-
zianum. The solid-matrix priming was more
effective with Trichoderma using Agro-Lig, a
lignaceous shale with a pH of 4.1 as the
matrix, than with bituminous coal at pH 6.6;
the converse was true with E. cloacae, which
grows well at pH levels of ca 7 (Harman and
Taylor, 1988).
In a different approach, Taylor et al. (1991)
double-coated the fungus onto cucumber
seeds, first with a slurry of Trichoderma, then
a slurried mixture of solid particulate (muck
soil or Agro-Lig) plus binder (FeIgel or Polyox
N-lO). The resultant thin 0.1 mm) uninter-
rupted layer over the seed surface was con-
sidered sufficient to slow infection by Pythium
by about 6 h, and biocontrol was substantially
improved over that obtained by a simple
slurry.
Recently, Inbar et al. (1996) coated seeds of
lettuce and cucumber in a conidial suspension
of T. harzianllm using 10% (w Iv) Pelgel (Nitra-
gin). The seed treatments reduced sclerotinia
wilt of cucumber and lettuce in artificially
infested soil. Experiments have also shown
that Trichoderma spp. can be successfully
applied to cucumber and radish seeds by an
industrial filmcoating process (Cliquet and
Scheffer, 1996). Spore suspensions were
mixed with a 0.05% vinyl acetate sticker and
coated onto seeds using a fluidized bed-
bottom spray coater. The filmcoated seed
treatments reduced damping-off caused by
P. ultimum and R. solani in an artificially
infested peat-based soil.
A limitation to the efficacy of fungi applied
to seed is that they typically do not colonize
the rhizosphere as effectively as do bacteria
(Lewis, 1991). Various attempts have been
made to overcome this limitation.
8.4.3g Verticillium biguttatllm This has shown
promise when applied to seed tubers for
control of black scurf (R. solani) on potatoes.
Jager and Velvis (1986) harvested conidios-
pores from sporulating mycelium on agar,
resuspended in 0.5% carboxymethylcellulose,
adjusted the pH to 6.4 then inoculated them
on seed tubers (section 5.3.1).
8.4.3h Metarhizium anisopliae This entomo-
pathogenic fungus is used in several countries
to control foliar-feeding and soil-dwelling
insect pests. It is typically applied as sprays,
foliar dusts or granules (Chapter 4; Rhodes,
1993). However, Rath (1992) applied conidios-
pores to ryegrass seeds in a methyl cellulose
suspension up to a concentration of 5 kg fun-
gal biomass per 15 kg seed without loss of
germination.

8.5 MYCORRHIZAL INOCULANTS
The role of mycorrhizas in improving phos-
phate uptake and plant growth is now widely
recognized (Stribley, 1989). Vesicular-arbus-
cular endomycorrhizas (VAM), the most com-
mon type, are formed in all agricultural crops
except sugarbeet and brassicas. The symbiotic
fungi improve plant productivity under cer-
tain conditions, especially where soil phos-
phate is a limiting factor, and they may have
potential of assuring plant production with a
minimum input of fertilizers (Gianinazzi et aI.,
1989). Advances in biotechnology have pro-
moted experimentation in artificial inocula-
tion of plants with VAM. Trials have mainly
involved broadcasting and raking-in inocula
over plant-growing substrates rather than
application of inocula to seed before sowing.
Since large-scale production of VAM in axenic
culture is not yet possible, inocula have been
produced in pot cultures or in small field
plots on plants grown under carefully con-
trolled conditions, to avoid contamination by
plant pathogens (Mitchell, 1993). Such inocula
have comprised infected roots or spores and
hyphae trapped in soil, peat and clay carriers
(Warner et al., 1985; Dehne and Backhaus,
1986; Gianinazzi and Gianinazzi-Pearson,
1986; Gianinazzi et al., 1989). Native Plant-
s(USA) have incorporated inoculum into
Nutri-Link, a commercial inorganic carrier
(Wood, 1987). It is based on a patented tech-
nique for large-scale production of spores
from VAM cultures, which are bound to a
clay support and sold in a dried form (section
7.5.3a).
Introduction of VAM into plant-growing
media on a large scale has tended to use
inoculum at excessively high and impractical
rates in order to guarantee rapid infection by
the introduced fungi (Powell, 1984; Hall,
1988). Application of inocula directly to seed
may reduce the amounts of inocula required,
but research on this has been limited. Experi-
mental inoculation has involved coating
spores, mycelium and infected root frag-
8.6 Research needs and future prospects 277
ments, wet-sieved from soil, onto seeds
using methyl cellulose as sticker. This coating
method produced reasonable mycorrhizal
infection in six plant species sown in pots
(Hayman and Mosse, 1977), onion and tomato
sown in pots (Gaunt, 1978), and citrus sown in
nurseries (Hattingh and Gerdemann, 1975).
Other experimental inoculation of seed with
VAM has involved fluid drilling soil inocula
suspended in methyl cellulose (Hayman et al.,
1981).
Ectomycorrhizas have also been delivered
on seed to a limited extent. For example, bas-
idiospores of Rhizogen luteolus were coated
onto Pinus radiata seeds by covering them
with a spore suspension in a rotating screw-
top drum (Theodorou and Benson, 1983).
Excess suspension was drained, then the
seeds were spread on plastic sheets to air-
dry. Coated seeds increased the mycorrhizal
infection of seedlings grown under nursery
conditions.
8.6 RESEARCH NEEDS AND FUTURE
PROSPECTS
This chapter helps to focus the factors that
limit inoculation of seeds for delivery of
microorganisms to plants. These limitations
should be a target of research, and the factors
should be duly considered in forecasting the
future for seed inoculation. General research
on further development of seed-applied rhi-
zobial inoculants is likely to concentrate on
increasing nitrogen fixation by improving
nodulation. Nodulation has been improved
in particular when rhizobia were combined
with plant growth-promoting rhizobacteria
or biological control agents (Grimes and
Mount, 1984; Handelsman and Halverson,
1989). Such dual inoculation of seed with rhi-
zobia and other beneficial microorganisms, in
combination with fungicide seed treatments,
warrants further investigations. Applying rhi-
zobia to seed in combination with other
microorganisms and fungicides may require
the generation of fungicide-tolerant strains
278 Application of microorganisms to seeds
of the symbiotic fungi, and multi-layered
seed-coating technology in conjunction with
formulation could be used to apply these
materials separately from each other.
Advances in molecular biology and in the
understanding of genetic control of nitrogen
fixation \'\fill provide an opportunity for
genetic engineering of improved strains of
rhizobia with enhanced nitrogen fixation effi-
ciency, increased competitive ability for nodu-
lation of a given host, and adaptation to
specific environments Oones and Lewis, 1993
and references therein). The application of
such superior strains to seed may require
improved carriers in formulations to provide
protection from desiccation and to extend
shelf-life. Investment in these strains may just-
ify more costly seed treatment.
Most biological seed treatments rely for
their success on the inherent biological prop-
erties of the organism (including its ability not
only to survive desiccation and a period of
storage in sufficient numbers, but also to mul-
tiply in the spermosphere and colonize the
root) rather than on the properties of the for-
mulation and the seed inoculation technique
alone. The microbial ecology of the rhizo-
sphere environment, and population
dynamics in soil and along developing roots,
are extremely complex but still poorly under-
stood. Renewed research emphasis on this
area has been stimulated recently by concern
about the release of natural or genetically
engineered organisms into the environment
(Kluepfel, 1993). This effort should lead to a
better understanding on such matters as the
developing patterns of colonization along the
seedling root from the inoculum on the seed,
how different strains respond to the presence
of root exudates, and the relationship between
density of initial inoculum and final coloniza-
tion for different microorganisms. Strategies
for strain selection - based on criteria of colo-
nizing, on competitive ability, or on suiting
particular environmental conditions - have
been extensively reviewed (e.g. Weller, 1988;
Renwick and Poole 1989; Harman, 1991). All
these research areas are linked to improving
seed delivery systems and to showing how
additives on the seed could facilitate expan-
sion of beneficial organism populations.
Formulation remains a major technological
barrier to commercialization, especially for
non-spore-forming bacterial species (chapters
5 and 7). In general, there is less concern about
inoculum viability with fungal spores and
spore-forming bacteria such as B. subtilis
than for Gram-negative bacteria, since spores
are more easily formulated. Formulation of
Gram-negative bacteria is likely to be similar
to that of rhizobia, which are also sensitive to
drying and heat (Weller, 1988). Improvement
of their survival during application to seeds
and in storage remains a major research tar-
get, even to the extent of searching for strains
better equipped for survival.
In the straightforward technical sense,
liquid formulations can be physically handled
through most of the seed application techno-
logy discussed in this Chapter, i.e. conven-
tional seed treatment using slurries with
optional binders, pelleting, coating, filmcoat-
ing and biopriming. However, solid formula-
tions are usually preferred for microbial
inoculants, to stabilize bacterial strains with
a protective carrier such as clay, peat or cellu-
lose, and are preferable for the production of
spore-producing or very slow-growing fungi
(e.g. Lumsden and Lewis, 1989; Paau et al.,
1991; Campbell, 1994; chapters 5 and 7). Such
solid formulations can also be successfully
handled through a variety of seed-treatment
procedures, providing that for pelleting, coat-
ing and filmcoating the particle grade is kept
suitably fine. Coarse powder material is liable
to be lost as dust from treated or coated seeds,
and would cause problems in the mixing and
spraying of blends or suspensions.
By its nature, most work on formulation
and seed application closely involves agro-
chemical, biotechnology or seed-treatment
companies, and tends not to be published.
Seed applications used in most research
reported in the scientific literature are based

on simple techniques, such as coating in
methyl cellulose or application in peat, or
simply dipping or soaking seed in microbial
cultures just before sowing, which in many
cases fail to confer adequate shelf-life on
organisms lacking specialized survival struc-
tures. Indeed, often storage on inoculated
seed is not the subject of investigation. Reluc- .
tance to publish handicaps progress. More
directly comparative studies on storage and
efficacy of seed treated with microbes in dif-
ferent ways should evolve and include
batches of seed treated by commercially con-
fidential processes, but these ought to be par-
alleled by fully described laboratory processes
- or commercial variations - to allow compar-
ison of specific treatment factors such as tem-
perature.
Opinions differ on how widely microbes
will be commercially delivered on seed in
future, and how long it will take to happen.
As yet, few products have been marketed
successfully. Trade-named formulations of
Trichoderma, Pseudomonas, Streptomyces griseo-
viridis and Bacillus subtilis strains for treating
seeds are the prime examples (Table 5.1 in
section 5.2). One likely trend is the co-formu-
lation of biologicals with conventional chem-
ical pesticides, as already seen with some
rhizobial inoculants and the Kodiak formula-
tions of B. subtilis. Also, as biological control
systems become established, it is foreseeable
that quick and simple analytical tests or bioas-
says, analogous to the nodulation tests for
Rhizobium spp./ will be necessary to ensure
that strain identity and biological activity are
retained, and to satisfy the customer of the
quality of the product. This quality issue is
gaining prominence in the general area of
application by seed treatment (Halmer,
1994)/ and will probably be of some impor-
tance where the grower is buying pre-inocu-
lated seed, probably at a premium price.
Seed inoculants will need to be integrated
with established seed treatment practices,
which will tend to dictate the type of formula-
tion and method of delivery for particular
References 279
crops. Application techniques such as coating
or biopriming, for example, could be consid-
ered for use with low-or medium-volume
crops, but agronomic crops are likely to be
treated by conventional or simple on-farm
methods. In the final analysis, whether bio-
logical treatments are viable commercially
depends on logistical and economic factors.
As Taylor and Harman (1990) point out/
seeds are treated to enhance profitability,
and even though their biological performance
might be very good, they might not be tested
if treatment is not cost-effective or is too time-
consuming to perform. Economics are further
elaborated in Chapter 10/ together with other
formulation aspects pertinent to seed treat-
ment.
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PART FOUR

ORGANISMS WITH A POWER OF SEARCH

Entomopathogenic nematode juveniles search or lie in wait for target insects.

They attack via both the gut and outer surface of an insect. In storage their
dormancy is shallower than that of the survival stages of most microorganisms
and they must be formulated to retain the power of movement.
FORMULATION OF 9
ENTOMOPATHOGENIC NEMATODES
Ramon Georgis and Harry K. Kaya

CONTENTS
9.1 Introduction 289
9.2 Mass production 290
9.3 Formulation development 291
9.3.1 Background 291
9.3.2 Desiccated nematodes 294
9.3.3 Manufacturing cost 295
9.3.4 Market acceptance 295
9.4 Considerations for nematode formulation 298
9.4.1 Nematode quality 298
9.4.2 Application method 299
9.5 Future directions 300
9.5.1 Nematodes 300
9.5.2 Formulations 300
9.5.3 Application technology 300
9.5.4 Shelf-life 305
9.6 Conclusions 305
References 306

9.1 INTRODUCTION because they kill a wide range of insect spe-

cies, especially under laboratory conditions.
Entomopathogenic nematodes in the families
Although the laboratory host range is exten-
Steinernematidae and Heterorhabditidae
sive because behavioural and ecological
have been known since 1929 and 1975, res-
barriers are non-existent, in the field the
pectively (Glaser and Fox, 1930; Poinar, 1975;
nematodes' host range is much more
Gaugler and Kaya, 1990), but they became
restricted (Kaya and Gaugler, 1993).
commercially available only during the past
The biologies of Steinernema and Heterorhab-
decade (Georgis, 1992). Since their discovery,
ditis have many similarities. Only the third-
tests have indicated that these nematodes
stage infective juvenile (dauer larva) can
have the potential to control insect pests
survive outside an insect host and move

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
290 Formulation of entomopathogenic nematodes
from one insect to another. The infective juve-
nile carries the symbiotic bacterium in its
intestine (Xenorhabdus for steinernematid or
Photorhabdus for heterorhabditid). It releases
the bacterial cells after entering the insect's
haemocoel by way of natural openings (spira-
cles, mouth, anus) and, with Heterorhabditis,
directly through the soft cuticle of certain
insects. The bacteria proliferate, kill the insect
host (usually in 24-72 h) and render its inter-
ior favourable for nematode development. For
Heterorhabditis, the entering infective juvenile
develops into a hermaphroditic female, and
hence only one nematode is needed for pro-
geny production; for Steinernema, at least two
nematodes must enter and grow in an insect
since the infective juvenile develops into a
male or female (amphimictic) and the mated
female is required for progeny production.
One or more generations develop until the
host-derived nutrients are depleted. At this
time, the nematodes synchronously become
third-stage infective juveniles that leave the
cadaver to forage for new hosts by detecting
insect excretory products (Kaya and Gaugler,
1993). At temperatures ranging from 18-28 C,
the life cycle is completed in 6-18 days,
depending on the host insect and nematode
species (Poinar, 1990).
Breakthroughs in production and formula-
tion have made commercialization of
nematode products possible. Major accom-
plishments are:
in vitro production of nematodes in numbers
sufficient for field applications, at a cost
generally competitive with chemical pesti-
cides (Georgis, 1992);
consistent production of high-quality (i.e.
viable, pathogenic) nematodes;
development of nematode formulations that
provide a shelf-life sufficient for storage
and transport to the site of use;
formulations that make application rapid and
simple.
Nematode-based products that fulfil these
requirements are presently available (Georgis
and Manweiler, 1994) and improved versions
are being actively pursued.
Application strategies providing control
levels comparable to those of standard chemi-
cal insecticides have been developed through
extensive field research programs (Georgis
and Gaugler, 1991). Selective control of pest
species that spares their natural enemies can
be achieved through an understanding of fac-
tors that limit the nematodes (e.g. desiccation,
ultraviolet light, temperature extremes),
knowing the ecology of the target insect (e.g.
developmental stage, interaction with host
plant) and application methodology (e.g.
spot application, bait formulation, dispersion
through drip-irrigation systems).
The formulation of entomopathogenic
nematodes differs in one major respect com-
pared with standard chemical insecticides.
The difference is the maintenance of biologi-
cal activity during storage and application of
the nematode (Georgis, 1990). To avoid loss of
biological activity, the most logical approach
is to understand the physiological chemistry,
ecology and behaviour of the nematode. Once
most of these parameters are defined, selec-
tion of the formulation type and its ingredi-
ents can be undertaken. This chapter outlines
recent efforts in the formulation and applica-
tion technology associated with the use of
steinernematids and heterorhabditids.
9.2 MASS PRODUCTION
Rudolph Glaser was the first to successfully
devise a culture method for an entomopatho-
genic nematode (Steinernema) on artificial
medium (Glaser, 1931). However, Glaser was
unaware of the need for the symbiotic bacter-
ium which kills the insect host by septicaemia,
supplies nutrients for the nematode, and pro-
duces antibiotics to suppress secondary inva-
ders. The absence of the bacterium resulted in
the nematode's failure to become a biological
control agent of the Japanese beetle (Gaugler
et al., 1992). Subsequent rearing efforts with
other entomopathogenic nematode species

were made primarily with insect hosts (Dutky
et al., 1964). This method is still widely
employed in research laboratories and by
some in cottage industry. However, in vivo
production costs are far too high for large-
scale commercial production.
A major breakthrough in mass production
was made by Bedding (1984) who developed
a monoxenic semi-solid culture technique that
achieved much higher and more consistent
yields. Production costs were further reduced
by a semi-automated harvesting process. This
technique has been the choice for a number of
decentralized societies, such as China, and
small commercial operations (Georgis, 1990).
In scaled-up production by the Bedding
technique, costs fall with increasing scale uE
to an output level of approximately 10x10 2
infective juveniles/month. Above this point,
labour costs become significant. Production
by monoxenic liquid fermentation costs less
than other methods, and these costs decrease
more rapidly up to a capacity of approx-
imately 50x101 infective juveniles/month
(Friedman, 1990).
At present Steinernema carpocapsae, Steiner-
nema jeltiae, Steinernema giaseri and Steinernema
riobravis can be consistently and efficiently
produced in 7500-80000 litre fermenters
with a yield capacity as high as 150000 infect-
ive juveniles/ml (Georgis and Manweiler,
1994). Because of distinct physiological differ-
ences among nematode strains and species
(Akhurst and Boemare, 1990; Poinar, 1990),
the growing medium varies significantly. In
general, it contains water, an emulsifier, a
yeast source, a vegetable oil and a protein
source (Georgis, 1992). Other important con-
siderations for achieving consistently high-
quality yields are optimum aeration, shear
sensitivity, and the stability of the phase I
bacterium and its interaction with the nema-
tode (Friedman, 1990). The bacterium exists in
two phases, I and II. Phase I produces anti-
biotics and supports greater nematode pro-
duction in vivo and in vitro than phase II
(Akhurst and Boemare, 1990).
9.3.1 Background 291
9.3 FORMULAnON DEVELOPMENT
9.3.1 BACKGROUND
The first attempts at formulating entomo-
pathogenic nematodes were initiated in 1979
(Table 9.1), but at best, shelf-life was about 1
month. Infective juveniles carried on moist
substrates such as sponge, vermiculite and
peat require continuous refrigeration to main-
tain their viability (Georgis, 1990). Immobil-
ization of the nematodes in a matrix increased
shelf-life, but desiccation of the nematodes,
which reduces energy utilization, has been
the most effective means of extending their
shelf-life (Table 9.1). As more basic studies
on the nematodes were conducted, distinct
biochemical, behavioural and morphological
differences among steinernematid and
heterorhabditid species were documented
(Table 9.2). For example, an important
physiological adaptation is to have an appro-
priate lipid composition that enables the
nematodes to regulate membrane function
and to adjust for environmental extremes
(Yamaoka et al., 1978). The relatively high
lipid content of these nematodes suggests
that they are adapted to survive prolonged
periods of environmental stress (Selvan et ai.,
1993b).
The selection of formulation type, ingredi-
ent, packaging size and storage conditions
cannot be undertaken unless the oxygen,
moisture and temperature requirements for
each species are defined. Thus, a better under-
standing of the biochemistry, physiology and
behaviour of the infective nematodes will lead
to the development of formulations that are
more stable and easier to use. Clearly, the
evolution from moist to partially desiccated
formulations points to the advances that
have been made in understanding the nema-
todes (Table 9.1).
Nematode metabolism is temperature-
dependent, with warm temperatures in-
creasing the rate of lipid reserve used but
decreasing the time that the infective juveniles
remain viable and pathogenic. An important
292 Formulation of entomopathogenic nematodes

Table 9.1 Historical review of the development of nematode-based formulations (Georgis, 1990) with
shelf-life comparison data based on 106 Steinernema carpocapsae per 1.2 I container*

Formulation strategy Nematode Oxygen used/l0 6 Shelf-life

Period of nematode status nematodes/day t (months)!

1979-1984 Placement on moist substrate Active 3.2ml 0.5-1.1

such as vermiculite,
polyether-polyurethane
sponge,peatorcedar
shavings
1985 Placement on absorbent Low motion 1.2
activated charcoal
1987-1990 Placement on moist gel Immobilized a.67ml 2.6
materials such as calcium
alginate, xanthene, or
polyacrylamide
1988 Placement on or between two Partially a.38ml 2.2
layers of absorbent clays desiccated
1993 Encasement in 10-20 mm Partially 0.21 ml 5.1
diameter water dispersible desiccated
granule consisting of silica,
clays, cellulose compounds,
lignins and starches
* Standard-size container was used to generate meaningful comparison between formulations (Georgis, unpublished).
t Calculated 3 days post-formulation.
:j: Based on > 90% nematode viability and unchanged pathogenicity from the day of formulation.

factor in temperature tolerance and adapta-
tion to extreme environmental conditions is
lipid saturation (Selvan et al., 1993b). Steiner-
nematids retain a relatively high level of
saturated fatty acids that could explain, in
part, their greater shelf-life capability as
compared to heterorhabditids (Table 9.3). By
desiccating the infective juveniles, the lipid
reserves are conserved (lipid reserves will
still be used at a low rate) and less oxygen is
required. In addition, the infective juveniles
possess greater ability to tolerate higher tem-
peratures than other developmental stages
(Womersley, 1990).

Table 9.2 General morphological, behavioural and biochemical characteristics of certain infective stages
of steinernematids and heterorhabditids (Poinar, 1990; Grewal et aI., 1994a, b; Selvan et aI., 1993a, b)

Nematode Length Width Activity Lipid

species (/-lm) (/-lm) Movement (0C)* content (%)
S. carpocapsae 558 25 Ambusher 8-32 32.4
S. feltiae 849 26 Intermediate 8-30 34.8
S. glaseri 1130 43 Cruiser 10-37 36.1
S. scapterisci 572 24 Ambusher 12-35 33.4
S. riobravis 622 27 Intermediate 10-39
H. bacteriophora 588 23 Cruiser 12-32 37.8
H. megidis 768 29 Cruiser 10-35 36.3
*Temperature range in which infective juveniles can penetrate into the haemocoel of Galleria mellonella larva.

Table 9.3 Comparison shelf-life data and ease of use of 250 x 106 steinernematid or heterorhabditid
nematodes formulated on calcium alginate in 4.0-1 containers
Nematode species Calcium alginate (%)
S. carpocapsae 20
H. bacteriophora 20
35
S. glaseri 20
35
* > 90% nematode viability and unchanged nematode pathogenicity from the day of formulation.
t Time required to dissolute calcium alginate with water and sodium citrate; range is due to water quality.
Differential water absorption by different
species of entomopathogenic nematodes
appears to affect their desiccation tolerance.
A slow rate of water loss or uptake is essential
for nematode survival during desiccation and
rehydration. This maintains the structural
integrity of the membranes (Preston and Bird,
1987). Steinernematids have better desiccation
survival capabilities because they can regulate
the slow drying and rehydration compared to
heterorhabditids (Selvan et al., 1993a).
Entomopathogenic nematodes may adopt
various energy utilization strategies to maxi-
mize their survival in the environment. Some
nematode species are active foragers or crui-
sers (e.g. Heterorhabditis bacteriophora, Hetero-
rhabditis megidis, S. glaseri) and expend a
significant amount of energy to find their
host; others are sit-and-wait foragers or
ambushers (5. carpocapsae, Steinernema scapter-
isci) and do not expend much energy. In terms
of energy expended, Baldwin (1964) proposed
that oxidation of 1 mg of lipid, protein, or
carbohydrate yielded 39.3, 23.7 and 17.4J
energy, respectively. Considering these
values, estimated energy content of a single
infective juvenile of H. bacteriophora, H. megi-
dis, S. glaseri, S. feltiae, S. carpocapsae, and S.
scapterisci was 0.038, 0.042, 0.123, 0.065, 0.030
and 0.029 J, respectively (Selvan et a!., 1993a).
Even though infective juveniles of S. carpocap~
sae and H. bacteriophora contained similar
amounts of energy, S. carpocapsae lived longer
(16-week ambusher) than H. bacteriophora (7-
week cruiser) when these nematodes were
9.3.1 Background 293
Shelf-life* at 25C Preparation t time (min)
3 months 20-30
1.5 weeks 20-30
1.2 months 50-70
2 weeks 20-30
3.1 months 50-80
held at 25C in water (Selvan et a!., 1993a).
In another study, Selvan et al. (1993b)
demonstrated that S. glaseri (cruiser) con-
tained three times the energy of H. bacterio-
phora and survived for 24 weeks (Klein, 1990),
whereas H. bacteriophora (cruiser) survived
only 3 weeks.
Active movement of the infective juveniles
that results in expended energy may be
responsible for the differential mortality.
H. bacteriophora infective juveniles were active
in water (Gaugler and Campbell, 1991),
whereas S. carpocapsae remained inactive
with a typical J-shaped posture. These beha-
vioural differences may explain why S. carpo-
capsae-based products have a room
temperature shelf-life of 5 months, while het-
erorhabditid-based products require continu-
ous refrigeration for equivalent shelf-life
(Georgis et a!., 1995). The infective juveniles
of S. glaseri have a high oxygen consumption
rate because of their continuous movement
and large size. Consequently, products based
on this nematode have a limited room tem-
perature shelf-life compared to S. carpocapsae-
based products (Table 9.3).
The foraging strategy of the nematodes dic-
tates whether a given formulation can be used
(Table 9.2). In laboratory bioassays, both S.
carpocapsae and S. scapterisci nictate (nema-
todes stand on their tails and wave) 50-80%
of the time, whereas H. bacteriophora and
S. glaseri spend 70-90% of their time moving
on a sandi agar plate. When searching for a
host in nature, S. glaseri moves through soil,
294 Formulation of entomopathogenic nematodes
whereas S. carpocapsae often remains station-
ary on the soil surface (Kaya and Gaugler,
1993). Because of their tendency to be station-
ary, S. carpocapsae infective juveniles can be
immobilized in the 20% calcium alginate for-
mulation which can be dissolved within
30 min wit~ sodium citrate (Table 9.3). In con-
trast, S. glaseri and H. bacteriophora are active
foragers and can be immobilized only in a
35% calcium alginate formulation, which
requires twice the amount of time to dissolve.
At 20% calcium alginate, S. glaseri and H. bac-
teriophora actively migrate out of the alginate
within 1-2 weeks, resulting in their death.
9.3.2 DESICCATED NEMATODES
A significant advance in formulation was
made with the development of the water-
dispersible granule (WDG) in which the
infective juveniles are partially desiccated
during the formulation process (Tables 9.1
and 9.4). The infective juveniles are encased
in 1O-20-mm diameter granules consisting of
silica, clays, cellulose compounds, lignins and
starches (Table 9.5). Nematode droplets, each
with approximately 40000 infective juveniles,
are mixed with the formulation ingredients on
rotating pans, creating 1O-20-mm granules
(Figs 9.1 and 9.2; section 7.7.3a). Each granule
contains a 'soft centre' of the nematode sus-
pension surrounded by the dry ingredients.
The moisture level in each granule can be
adjusted, depending on the nematode species,
at 35-45%. This can be done by calculating the
residence time and the amount of formulation
ingredients that are delivered to the pans to
create the desirable hardness of the granules.
Depending on the nematode species, granules
collected from the pans are then exposed to
optimum temperatures resulting in partial
desiccation by the gradual absorption of
water from the nematode (see also section
7.5.3b).
The desired shelf-life can be met as long as
the moisture level of >90% RH can be main-
tained for the partially desiccated nematodes
in the granules. Prior to formulation, a fung-
icide is added to the nematode suspension to
restrict contaminants in the granules to a low

Table 9.4 Comparison between two formulations of Steinernema carpocapsae, based on 250 x 106
nematodes per container

Characteristics Water dispersible granule Calcium alginate

Product description 250 X 106 nematodes in 680 g 250 X 106 nematodes trapped
granules (40 000 nematodes per in a gel matrix and coated on a
granule) mesh screen
Nematode status Partially desiccated Immobilized
Container size 1.21 4.0 I
Shelf-life* 5-6 months at 4-25C, 2 months at 3-4 months at 4-25C, 0.5
30C, 6 days at 36 C months at 30C, 1.0 at 36 C
Ease of use Dissolve immediately in water. 20-30 min preparation steps.
Ready to use. Easy to measure Once diluted the entire product
must be used
Product size: coverage Comparable to chemical products Adequate only in greenhouse
ratio and adequate for use in various and home-garden markets
markets
Disposal Minimal product disposal Burden to dispose of screens
Usage range Various nematode species are Suitable for few nematode
compatible with WDG species
Cost Low for ingredients and manufacture Higher than WDG
* > 90% nematode viability and unchanged pathogenicity from the day of formulation.

Table 9.5 Major ingredients of water-dispersible granule formulation
Ingredient
Diatomaceous earth
Hydroxyethyl cellulose
Amorphous silica
Fumed hydrophobic silica
Lignosulphonate
Modified starch
Attapulgite clay
Fungicide
Wetting agent
level (Table 9.5). The ingredients that sur-
round the nematodes are porous, allowing
the infective juveniles to receive oxygen that
is needed to maintain the low rate of respira-
tion. To provide sufficient oxygen and main-
tain the moisture level for the nematodes,
granules are packaged in containers that
have lids with small holes covered with spe-
cial film. The film is gas permeable and main-
tains the desired moisture level in the product
(for bags used to store fungi, see section 4.7.1).
9.3.3 MANUFACTURING COST
Minimum mass-rearing and formulation costs
are required before nematode-based products
can become competitive with chemical insect-
icides. The processing cost of the WDG prod-
uct is less than that for the calcium alginate
product (Georgis and Dunlop, 1994). It does
not require the custom-made equipment
needed for processing of the calcium algin-
ate-based product. Tons of granules can be
made efficiently in a short period of time,
and the ingredients of the WDG product are
cheaper. Therefore the profit margins from
WDG products are higher than from calcium
alginate-based products (R. G., unpublished
data).
9.3.4 MARKET ACCEPTANCE
Limited shelf-life and difficulties of mixing
and applying nematode-based formulations
9.3.4 Market acceptance 295
Function
Absorbent
Binder
Absorbent
Absorbent
Dispersant and binder
Binder
Absorbent
Control contaminants
Facilitate mixing in water
developed between 1980 and 1990 (Table 9.1)
have hindered their market potential. The
introduction of the calcium alginate formula-
tion, which is based on immobilizing the
nematode, was a major breakthrough in for-
mulation development. It was the first formu-
lation with a shelf-life of up to 3--4 months
(250x 109 nematodes per 4.0-1 container) at
room temperature under ideal conditions
(Georgis and Dunlop, 1994). As a result, a
number of agrochemicals companies intro-
duced the formulation into various market
segments including turfgrass, greenhouse,
berries, and home and garden. Unfortunately,
it took 20-30 min to extract the nematodes
from the calcium alginate before they could
be applied through standard application
equipment (Table 9.3; Fig. 9.2). Time of
extraction for application limited the market
penetration, especially when large-scale
treatment was required. Furthermore, in
large-scale applications the disposal of the
framing material (i.e. plastic screen) that
held the calcium alginate was burdensome
and the number of containers required to
treat large areas was considered impractical
(Fig. 9.2).
For all the above reasons, research in the
early 1990s focused on developing more
compact product configurations and simpler
mixing and application for further market
penetration. These efforts led to develop-
ment of a WDG product that allowed the
infective juveniles to enter a partial
296 Formulation of entomopathogenic nematodes

(a)

Figure 9.1 Morphological difference between active (a) and desiccated (b) Steinernema carpocapsae
infective juvenile (558 {LID long, 25 {LID wide).
 9.3.4 Market acceptance 297

(b) . . . . .

Figure 9.2 Steinernema carpocapsae infective juvenile (a) formulated in water-dispersible granules (40000
nematodes per 10-20mm diameter granule) or (b) trapped into calcium alginate coated on a mesh screen
(250 x 106 nematodes per 1.2 m screen). Scale in inches.
298 Formulation of entomopathogenic nematodes
desiccation state, enhancing survival and
preserving pathogenicity for up to 5-6
months at 25C and providing better toler-
ance of nematodes at 36 C (Table 9.4).
The product is saleable and easy to apply
without the time-consuming preparation
steps (Table 9.4).
Inducing nematodes to enter a partial desic-
cation state means that less oxygen is
required. Thus, a higher nematode population
per litre of container space was attained
(Table 9.4). To obtain a 5-6-month shelf-life,
a 1.2-1 container is adequate for 250x106
desiccated S. carpocapsae in the WDGs. In con-
trast, a 4.0-1 container is needed for the same
number of immobilized nematodes in calcium
alginate with a shorter shelf-life.
The introduction of the WDG formulation
has captured 90% of the total market available
for nematode-based products (R. G., unpub-
lished data). Furthermore, the market size of
the nematode-based products has increased
by 52% in 1995 since the introduction of the
WOG formulation in 1994 (R. G., unpublished
data).
9.4 CONSIDERATIONS FOR NEMATODE
FORMULATION
9.4.1 NEMATODE QUALITY
The successful market acceptance of nema-
tode-based products depends heavily on
their stability during shipping and storage,
as well as their ease of use and consistent
performance under field conditions. Stability
refers to maintenance of nematode quality
throughout all stages of the production pro-
cess (Table 9.6). The first step in standardiza-
tion is aimed at producing reliable and
consistent nematodes. Inoculum batches
from in vivo cultures are produced from
stocks of nematode strains that are stored by
cryopreservation (Popiel and Vasquez, 1991)
to minimize variation in nematode pathogen-
icity among in vitro production lots. Sub-
sequent steps are focused on maintaining the
viability and pathogenicity of the nematodes
immediately after they are harvested from the
fermenter until the product is applied by the
end-user. To assure this process, LTso (lethal
time needed to kill 50% of the test insects)

Table 9.6 Steps required to develop stable and reliable nematode-based formulation

Steps Requirements Benefits

Nematode inoculum Cryopreserved nematode originated
for in vitro from in vivo production
production
In vitro production Optimum operation conditions and
contamination-free system
Storage Maintain nematode suspension for
< 2 months in large aerated tanks at
5-10 dc. Use of antimicrobials
Formulation Standardized ingredients. Sterilized
raw materials. Effective sanitation.
Optimum operation conditions.
Antimicrobials
Packaging Maintain adequate water activity
moisture level and oxygen for
partially desiccated nematodes
Shipping Refrigerated or not in a 2-week
period
Product use Following label instructions
Consistent production of high
quantity and quality of nematodes
Production of virulent nematodes
with high lipid content
Maintain nematode virulence and
lipid level
Provide optimum conditions to
induce partial desiccation of the
nematodes. Produces ease of use
and reliable formulation
Maintain nematode viability and
virulence
Quality products to distributors
Effective insect control

performance standards and lipid contents of
infective juveniles have been determined, and
are used to measure product stability (for
quality standards for seeds inoculated with
microbials, see section 8.3.2c).
Another aspect of product assurance is tim-
ing production according to market needs.
Most production is accomplished from Janu-
ary to March for products needed from May
to August. For August to December markets,
nematodes are produced from March to June.
This production spans just 6 months. How-
ever, such considerations are dependent on
the nematode species, storage requirements,
market forecast and distribution channels.
To assure stable products, nematodes are
stored at 5-10C in large, aerated tanks and
are formulated within 4 weeks of harvest
(Georgis, 1992). Shelf-life of infective juveniles
is a function of stored energy and the rate of
its utilization. Lipid is a major energy reserve
for infective juveniles (Selvan et al., 1993b) and
initial lipid level appears to have a direct
impact on shelf-life. The rate of utilization of
stored energy depends on many factors such
as temperature, environmental stress, and
nematode activity and behaviour. These fac-
tors have a direct effect on energy bum-rate
and, therefore, affect the shelf-life of nema-
tode-based products (Selvan et al., 1993a). As
discussed previously, the ideal formulations
are those with nematodes that have entered
into a partially desiccated or anhydrobiotic
state (Georgis and Dunlop, 1994).
9.4.2 APPLICATION METHOD
A spray volume of 75Q-18901/ha is usually
required for most nematode species to reach
the target soil insect. Infective juveniles can be
applied to the target zone with nearly all com-
mercially available ground and aerial spray
equipment (Table 2.2 in section 2.2.2). These
include ground equipment such as small pres-
surized sprayers, mist blowers and electro-
static sprayers, as well as the traditional
sprayers used in aerial application via heli-
9.4.2 Application method 299
copters (Georgis, 1992). Moreover, nematodes
are commonly applied using drip and sprink-
ler irrigation systems. Pressures of up to
1068 kPa have no detrimental effect on the
infective juveniles. They can pass easily
through sprayer screens with openings as
small as 100 pm in diameter. However, clog-
ging of nozzles may occur with some formu-
lations. Therefore it is important to adopt a
quality assurance program so that fine parti-
culate ingredients are suspended and can be
dispensed through the sprayer (section
2.2.2b).
Control strategies usually involve an inun-
dative application of nematodes against a par-
ticular insect pest, with short-term control
being the primary goal. Another approach is
an inoculative release that relies on long-term
nematode persistence and recycling to main-
tain control of the target pest. Most inocula-
tive releases have been discouraging. A
notable exception has been the establishment
of S. scapterisci that has given some degree of
biological control of mole cricket species in
Florida (Parkman et al., 1994).
As with chemical insecticides, spraying
nematodes directly onto the soil surface is
the most commonly used application. This
broadcast method is quick, simple and pro-
vides good coverage. However, in most cases
high nematode concentrations (2.5-7.5x109 /
ha) are needed to ensure that sufficient num-
bers of infective juveniles will contact the tar-
get pests. In some situations, infective
juveniles have demonstrated control potential
when applied at planting time (Georgis and
Manweiler, 1994). However, when the target
insect stage does not appear until 4-6 weeks
after planting (e.g. the com rootworm, Diabro-
tica spp.; and the crucifer flea beetle, Phyllo-
treta cruciferae), insect damage was not
prevented by the nematodes (Georgis and
Manweiler, 1994).
Use of entomopathogenic nematodes to
control foliage feeding insects presents a con-
siderable challenge because of rapid desicca-
tion, ultraviolet light (sections 3.4.4, 4.3.3) and
300 Formulation of entomopathogenic nematodes
difficulty in establishing attraction gradients
(Glazer, 1992; Bedding 1993). Yet, under the
right conditions (i.e. moisture, early morning
or evening application) S. carpocapsae was
effective against the beet armyworm (Spodop-
tera exigua) on chrysanthemums, demonstrat-
ing that nematodes can be used against
certain foliage-feeding insects. In addition,
where the foliage creates a cryptic habitat
(e.g. leaf rollers, buds, dense canopies, leaf
mining), the potential for control is increased.
The addition of antidesiccants and sunscreens
(sections 4.3.4, 6.5; Table 3.16 in section 3.4.4;
Table 4.4 in section 4.3; Appendix Tables 1.4,
1.9-1.12) to nematodes has improved the level
of insect control, but usually economic control
has not been achieved (Kaya, 1993). The
development of sophisticated products and
delivery systems may extend the range of
potential targets and the level of control. In
general, everything must be done to ensure
that nematodes enter the host in the relatively
short period before they desiccate or are
damaged by ultraviolet light (Bedding, 1993).
9.5 FUTURE DIRECTIONS
9.5.1 NEMATODES
New nematode isolates should be collected to
increase the genetic base of material for the
development of new products (Gaugler, 1988,
1993). Research in molecular and classical
genetics is needed to improve the persistence
of the nematodes in products and after appli-
cation. In addition, further research on phy-
siology and desiccation processes of the
nematodes currently being marketed is
necessary to help increase their predictable
shelf-life. Finally, an understanding of the
environmental impact of nematode-based
products is needed.
9.5.2 FORMULATIONS
All commercial formulations are developed to
maintain product stability during storage and
transportation, current formulations being
mixed with water and sprayed against the
target pest. Future research needs to address
whether nematodes can be formulated in
granular, capsule and bait pellets, and be
applied by aircraft and standard applicators.
Formulations include attracting insects to a
nematode trap or bait, and developing comb-
inations of nematodes and other insect control
agents (Table 9.7). For example, Connick et al.
(1993) developed a granular formulation
called pesta (sections 5.4, 6.8). This formula-
tion is based on a cohesive dough made of
wheat flour, fillers and other additives (Table
6.8 in section 6.8). The formulation allows
more precise placement, e.g. application to
soil through the plant canopy, and subsurface
application of nematodes without water.
Further research is needed to determine the
effects of temperature and moisture content in
the pesta on the viability, movement and
pathogenicity of nematodes. The practicality
and economics of the commercial scale-up
should also be investigated.
9.5.3 APPLICATION TECHNOLOGY
Successful control of several pests has been
achieved by using nematodes in combination
with chemical or other biological insecticides.
Because nematodes are compatible with many
control measures (Table 9.8), numerous
opportunities exist for including nematodes
in programmes relying on chemical pest-
icides. Of primary importance to the feasibil-
ity of nematodes as part of an integrated
strategy is whether mortality caused by nema-
todes and other control measures (e.g. insecti-
cides) is additive or synergistic. Cost-benefit
ratios, including both direct and indirect costs
and benefits, will determine whether the
addition of nematodes to an integrated pest
management system is commercially justifi-
able. Indirect benefits, such as reduced
pesticide use and resultant decreased envir-
onmental contamination and resistance devel-
opment in insects, are sometimes difficult to
 9.5.3 Application technology 301
Table 9.7 Application technologies and associated target insects for steinernematid and heterorhabditid
nematodes (Kaya, 1993; Georgis and Manweiler, 1994)

Form of application Carrier Target insects Test site Application strategy

Capsules Calcium Melanoplus sp. Soil surface Nematodes migrate from
alginate' Agrotis ipsilon In soil at capsule to infect target
Diabrotica planting insects
virgifera virgifera
Capsules in traps Calcium Solenopsis invicta Laboratory Workers carry capsule to
alginate t the brood
Liquid baits Sucrose Vespula sp. Laboratory Partially desiccated
Lepidopterous Laboratory nematodes rehydrate in
larvae insect's gut causing
insect infection
Pellet baits Bran Melanoplus sp Soil surface Nematodes migrate from
Agrotis ipsilon pellets or insects feeding
Scapteriscus on nematode-
vicinus impregnated pellets,
causing insect infection
Granules (pesta) Flour, filler, Soil insects Soil surface or Nematodes migrate from
additives in soil granules to infect target
insect
Spray Water Soil insects Soil surface, Various surfactants or
injection sunscreen additives to
Foliar insects Leaves, improve nematode
trunks penetration in soil or
Boring insects Fruit, trunks nematode persistence in
the environment
Traps Moist pad Musca domestica Laboratory Nematodes attach to and
with Blattella Apartments infect attracted
attractants germanica insect
Traps Water Cosmopolites Banana, stump Adult weevil becomes
sordidus surfaces infected after being
attracted to nematode-
treated pseudostems and
corms
Sound traps Pads, sand Scapteriscus vicinus Golf courses Females disperse
nematodes to other areas
after being attracted to
male-sound traps
Cardboard bands Water, Cydia pomonella Tree trunks Nematode-impregnated
additives bands to catch and infect
pupae
With or without bran
t With liquid bait.
302 Formulation of entomopathogenic nematodes
Table 9.8 Chemicals compatible* with Steinernema carpocapsae (Georgis and Poinar, 1994)

Compounds Chemical class Trade name

Tank-mix
Biopesticides Azatin Margo-san
Bacillus thuringiensis M-One, Dipel
Fatty acids Safer soap
Insect growth regulators Diflubenzuron Dimilin
Fenoxycarb Logic
Kinoprene Enstar
Methoprene Apex
Insecticides Acephate Orthene
Bifenthrin Talstar
Carbaryl Sevin
Cyfluthrin Tempo
Diazinon Knox-out
Endosulfan Thiodan
Esfenvalerate Asana
Etridiazole Terrazole
Isofenphos Oftanol
Malathion Cythion
Methidathion Supracide
Trichlorfon Dylox
Fungicides Benomyl Benlate
Bromine-chlorine Agribrom
Chlorothalonil Daconil
Copper hydroxide Kocide
Fosetyl-Al Aliette
Iprodione Chipco 26019; (= Rovral)
Metalaxyl Subdue; (= Ridomil)
Oryzalin Surflan
Oxazoidinedione Ornalin
Pentachloronitrobenzene Terraclor
Thiophanate-methyl Zyban
Triademefon Bayleton
Herbicides Chlorthal dimethyl Dacthal
Glyphosate Roundup
Use one week after nematodes
Miticides Dienochlor Pentac
Fertilizers Most fertilizers are compatible with
nematodes
Insecticides Bendiocarb Turcam
Chlorpyrifos Dursban
Fungicides Anilazine Dyrene
Dimethyl benzyl ammonium chloride Physan 20
Fenarimol Rubigan
Mercurous chloride Calo-Clor
Use 2 weeks after nematodes
Insecticides Ethoprop Mocap
Isazophos Triumph; (= Miral)
Nematicides Fenamiphos Nemacur
Laboratory bioassays. Weeks needed to assure that the survival and the pathogenicity of the nematodes are not
affected.

quantify in monetary terms, although they
can be very significant (Georgis, 1990).
Attracting insects to a trap or bait with
chemical insecticide has been a successful
concept for insect control (Kaya, 1993). In
some instances, nematodes can replace the
chemical insecticide to make a biologically
based trap. This approach has received con-
siderable attention because insect pests either
not normally in the nematodes' habitat, or
difficult to kill, can be attracted to a nematode
source. For example, traps with nematodes
have been tested against adults of holometa-
bolous insects such as the house fly (Musca
domestica) banana weevil (Cosmopolites sordi-
dus) and yellowjacket (Vespula spp.); larval
stages of a holometabolous insect, the black
cutworm (Agrotis ipsilon); also immature and/
or adult stages of hemimetabolous insects
including grasshoppers (Melanoplus spp.),
tawny mole cricket (Scapteriscus vicinus) and
the German cockroach (Blattella germanica).
The trap may contain baits such as a food
arrestant or a sex attractant, or an attractive
food source (section 3.4.3; Table 3.14 in section
3.4.3) for the insect pest. It may also attract the
insect to a sound source or may serve as a
harborage or pupation site for the pest. With
some pests, the trap also serves as a mating
and oviposition site. One of the key elements
of the traps is that they must be moist to
ensure nematode survival.
Steinernema carpocapsae shows particular
promise for control of nymphs and adults of
the German cockroach that are attracted to
traps in buildings (Appel et al. 1993). Infective
juveniles placed on pads and enclosed in a
moisture-retaining trap significantly reduced
German cockroach sticky trap catches in
infested apartments. Results were comparable
with the standard insecticide trap containing
hydramethyluon as a food bait. The nematode
trap design apparently maximizes the sit-
and-wait behaviour of the nictating infective
juveniles of S. carpocapsae.
With the banana weevil, banana pseudos-
terns and residual corms are naturally moist
9.5.3 Application technology 303
and favour survival of S. carpocapsae. How-
ever, the adult banana weevil is not very sus-
ceptible to nematode infection because the
major route of entry - the large spiracles of
the first abdominal segment - is covered by
the tightly fitting elytra (Treverrow and Bed-
ding, 1993). Nematode entry occurs only after
several days of contact with banana weevil
adults. Accordingly, the successful use of
S. carpocapsae is dependent upon the adult
weevils being in continual contact, which is
assured by attracting the weevils to a bait
source (Le. a piece of pseudostem or a resi-
dual corm infested with nematodes). The
adult weevils are thigmotactic, and once they
are in a pseudostem or corm, they tend to
remain in the baited trap. The presence of
nematodes does not deter the weevil adults
from using the bait traps (Treverrow and Bed-
ding, 1993).
The use of sound traps to attract adult mole
crickets to a nematode source has been suc-
cessfully tested in the field (Parkman and
Frank, 1993). Adult mole crickets, especially
females, are attracted by the songs of males.
Emitters producing synthetic sound that sti-
mulates the song of the males have been
developed to attract mole crickets. The flying
mole crickets land on a funnel and fall
through a wire mesh tube into a bucket con-
taining moist sand or foam with S. scapterisci,
or directly onto the soil surface infested with
nematodes. The mole crickets in the bucket
are released a day later, whereas those that
fall onto the soil surface disperse immedi-
ately. The disadvantage of the bucket system
is that there is no means of escape for the mole
crickets, but this problem should be easily
overcome through modification of trap
design. Seven of 22 sites using the sand and
two of three using a foam method resulted in
successful establishment of the nematode.
Unfortunately, the direct soil method was
used only at one site and the results were
negative. This sound method takes advantage
of the sit-and-wait behaviour of S. scapterisci
to attach on and infect the insect, and the
304 Formulation of entomopathogenic nematodes
insects' behaviour to disperse the nematode to
establish new foci of infection in an inoculat-
ive release program (Parkman et ai., 1994).
Formulations that combine nematodes with
chemical pesticides or other biological control
agents, with or without traps, may increase the
overall efficacy against an insect pest. How-
ever, since each control agent has different
requirements for stability, developing such a
formulation will require extensive research. A
simpler approach is to tank mix the control
agents prior to application. Ishibashi and
Takii (1993) demonstrated that larvae of the
turnip moth, Agrotis segetum, were difficult to
kill with the foliar application of oxamyl or
S. carpocapsae alone. By combining the chemi-
cal and nematode, 95-100% larval mortality
was recorded compared with 70-78% mortal-
ity with S. carpocapsae alone, and 45-55%
mortality with oxamyl alone. Although the
behaviour of the nematode was not examined
in this test, the enhanced larval mortality by
the chemical-nematode combination was
probably due to the increased nictating beha-
viour of the nematode. In other tests, syn-
ergistic effects of chemical insecticides and
s. carpocapsae were observed against the scar-
abaeid larva, Anomala schoenfeldti (Hatsukade,
1990). The insecticides may have served as
stressers to enhance infection by the nema-
tode. However, not all pesticides are compati-
ble with nematodes (Ishibashi and Takii,
1993), and each should be examined for com-
patibility.
The combination of entomopathogenic
nematodes with other biological control
agents may also enhance their efficacy against
insect pests. Barbercheck and Kaya (1991)
showed that when the larval beet armyworm,
S. exigua, was in continual contact with the
entomopathogenic fungus Beauveria bassiana
and then exposed to Heterorhabditis bacterio-
phora, the combination produced higher mor-
tality than either pathogen alone. The
increased mortality was additive. In contrast,
the combination of B. bassiana and s. carpocap-
sae did not produce an additive effect.
Because H. bacteriophora tends to have an
active searching behaviour, it moves and
infects the host, resulting in higher host mor-
tality than S. carpocapsae which sits and waits.
Accordingly, on the basis of these laboratory
observations it seems that the combined soil
application of the fungus and H. bacteriophora
should provide equal or better reduction of a
susceptible pest than either pathogen alone.
The combination of nematodes with Bacillus
thuringiensis or entomopathogenic viruses
(Kaya and Burlando, 1989) may also offer the
possibility of obtaining control of a single pest
species. Another approach is to combine the
pathogens to target different insect species, so
that B. thuringiensis or the virus applied to
foliage infects the above-ground insect pest,
whereas the nematode applied to soil infects
the soil-inhabiting pest. For example, the
combination of B. thuringiensis and H. bacter-
iophora was effective against the larvae of the
cabbage looper Trichoplusia ni feeding on cab-
bage leaves, and the larvae of the scarabaeid
Cyclocephala hirta in soil (Kaya et ai., 1995). The
bacillus killed the cabbage looper larvae,
and the heterorhabditid killed the scarabaeid
larvae.
The combination of two nematode species
with different searching strategies in one for-
mulation to control one or two susceptible
insect pest species in a soil habitat appears
feasible (Kaya et al., 1993). In the following
example, the pest species occupied different
parts of the soil habitat so that the sit-and-
wait forager infected the pest near the soil
surface, and the actively searching forager
infected the subterranean pest. Thus tank mix-
ing of S. carpocapsae and H. bacteriophora was
successful against larvae of the black cutworm
(A. ipsilon) and the black vine weevil (Otior-
hynchus sulcatus), with S. carpocapsae infecting
the cutworm and H. bacteriophora infecting the
vine weevil. S. carpocapsae alone was effective
against the soil-surface-feeding black cut-
worm, but was ineffective against the subter-
ranean black vine weevil; and vice versa for H.
bacteriophora. Two nematode species with simi-

lar searching strategies, for example S. carpo-
capsae and S. scapterisci, both sit-and-wait for-
agers (Grewal et aI., 1994a), may be combined
against black cutworm and mole crickets (Scap-
teriscus spp.) occurring in the same habitat. S.
carpocapsae is highly infectious to the cutworm
(Capinera et aI., 1988; Levine and Oloumi-
Sadeghi, 1992), but less so to the mole crickets,
whereas S. scapterisci is adapted to infect mole
crickets and is not very effective against lepi-
dopteran hosts (Nguyen and Smart, 1991).
9.5.4 SHELF-LIFE
One area of importance is the use of know-
ledge gained from the study of plant and ani-
mal parasitic nematodes to enhance the
desiccation tolerance of entomopathogenic
nematodes. Trehalose has been found to pro-
tect purified proteins against denaturation
caused by desiccation (Crowe et aI., 1987). As
a result, these anhydrobiotic nematodes have
the ability to survive cycles of disruption
while dehydrated and to repair damage
quickly upon recovery (Barrett, 1991). The
dried nematodes can restore ionic and meta-
bolic gradients rapidly during rehydration to
prevent lethal effects. However, attempts at
total removal of free water from entomo-
pathogenic nematode cells to induce them
into complete dehydration have caused
membrane damage, preventing the goal of
exceeding a 1-year room temperature shelf-
life. Therefore, current formulations are based
on the removal of some free water from the
cells to induce partial desiccation and reduce
metabolism, in order to achieve up to a 6-
month room temperature shelf-life.
To avoid damaging the membranes and to
maintain viability of partially desiccated
nematodes, a relative humidity of > 90% is
required in the package and formulations
(e.g. WOG). Under these conditions, micro-
biocides and preservatives (sections 3.3.3b,
4.6.6b; Appendix Table 1.8) are usually used
in most formulations to prevent fungal and
bacterial growth. The long-term effect of
9.6 Conclusions 305
these additives on nematode viability and
pathogenicity, the half-life of the additives in
the formulation, and the optimum time of
adding them during the manufacturing pro-
cess should be observed carefully. Advanced
research on nematode physiology and genetic
manipulation, the optimum procedures and
conditions of inducing entomopathogenic
nematodes into anhydrobiosis, and ways to
enhance the trehalose level in the nematodes
are required to extend the shelf-life of current
nematode-based products. Along these lines,
the shelf-life of H. bacteriophora at 23C was
enhanced from 2 months to nearly 6 months
(Bedding, 1993). Survival stages of micro-
organisms usually benefit from a greater
degree of dryness than nematode juveniles
(sections 4.6.2, 7.7.2).
9.6 CONCLUSIONS
Successful commercialization of products
containing entomopathogenic nematodes is
well under way, and many more products
are actively being developed. Commercial
technologies for production, quality control,
formulation, transportation, storage and spe-
cialized forms of application exist today, and
are being rapidly improved. The current polit-
ical atmosphere favouring a reduction of the
use of chemical pesticides and promoting
alternative control methods is conducive to
the introduction and widespread use of
nematode-based insect control products. In
response to these trends, the pesticide indus-
try has reacted in several ways, including the
formation of joint ventures for the introduc-
tion of nematode products into the market,
and the funding of research.
Much effort needs to be directed towards
further optimizing more stable formulations
that are simpler to use and have longer than a
1-year shelf-life. New nematode isolates
should be collected to increase the genetic
base of material available for the creation of
new products and control programmes.
Advanced research on nematode physiology
306 Formulation of entomopathogenic nematodes
and biochemistry is necessary to help increase
the predictable shelf-life, the development of
more novel nematode applications, the inte-
gration of nematodes into existing pest con-
trol programs, and the understanding of the
environmental impact of nematode-based for-
mulations.
The quality of commercial nematode pro-
ducts is critical if entomopathogenic nema-
todes are to realize their full potential as
biological insecticides. The stability and ease
of use of the WDG formulation and the excel-
lent-quality nematodes grown in liquid cul-
ture are significant steps towards this goal.
Commercial products have been developed
to maintain product stability during storage
and transportation, and juveniles are released
for application as a spray in water against the
target pest. Granules, capsules and bait pellets
that can be applied by aircraft, and standard
granular applicators to protect and/or release
nematodes in the soil, are also desirable and
worthy of further investigation. The develop-
ment of baited traps may extend the market
potential to insects (e.g. cockroaches, flies)
that are difficult or impractical to control
with current products. Genetic engineering
may be the solution for the development of
an optimum formulation (longer than I-year
shelf-life, tolerant to temperatures above
30C, with minimum oxygen and moisture
requirements) by inducing the nematodes to
enter into a full anhydrobiotic state without
loss of pathogenicity.
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PART FIVE

THE FUTURE

Technical formulation to improve organism efficiency is only one objective;

commercial products must be developed that users can afford and that they
will want to use, despite the inherent difficulties. The trend is to research
formulation in the perspective of commerce as well as science. The limits of
formulation are now obvious - research is still vital, but must be directed to
exploit organisms within these limits.
TRENDS IN FORMULATION OF 10
MICROORGANISMS AND FUTURE
RESEARCH REQUIREMENTS
H. Denis Burges and Keith A. Jones

CONTENTS
10.1 Introduction 311
10.2 Consequences of active ingredients being live or
protein in nature 312
10.3 Consequences of active ingredients being particulate 313
lOA Commercialization and costs 316
10.5 Production, standardization and quality control 320
10.6 Storage 321
10.7 Application and aftercare of organisms 322
10.8 Limits of improvement by formulation 326
10.9 Basic research 328
References 331

10.1 INTRODUCTION for pest and disease control made it abund-

In this book, key features of organisms and
targets for their application have been com-
bined to divide the subject of formulation into
nine areas, each served by a chapter. Recur-
rent themes in these chapters show general
trends in the improvement of formulation,
and combine to give a good indication of its
future. Early research papers investigating
potential new organisms usually spoke airily
of promising results, often like love affairs
between scientists and their science. Further
investigations in the harsh reality of field use
antly obvious that progress in attempts to
better the natural performance of the organ-
isms required eventual commercialization of
well-formulated products, because most
efforts to make organisms available by gov-
ernment and communal projects petered out.
Therefore from the earliest stages of investiga-
tion the final goal, i.e. potential commercial
products, should be kept firmly in view dur-
ing research design. Although, initially, for-
mulation was neglected, it has since been
taken seriously in all subject areas and is
well advanced in some. The feedback

Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments. Edited by H. D. Burges.
Published in 1998 by Kluwer Academic Publishers, Dordrecht ISBN 0 412 62520 2.
312 Trends in formulation of microorganisms and future research requirements
mechanism from researchers, industrial pro-
ducers and users to policy makers is poor, a
shortcoming that we seek to rectify. There
have been some solid achievements with
microbials. Some effective, well tried, com-
mercial Bacillus thuringiensis products are
competitive, with conventional chemical
insecticide alternatives. Other workable com-
mercial products have been developed for a
variety of other types of organism, such as
rhizobia, plant disease inhibitors (section 5.2
in Chapter 5; Table 5.1 in section 5.2), and
weed control organisms (section 6.1). More
organisms have potential for a competitive
position, e.g. Metarhizium flavoviride conidia
in oil formulations, applied topically in
laboratory assays, are - weight for weight -
as active as the most potent chemicals (Bate-
man et al., 1992) and field application is effect-
ive. Real industrial achievements, as well as a
feeling of unfulfilled potential expressed in
each chapter in turn, show that the role of
microbials and nematodes - not only in pest,
disease and weed control but also as self-
replicating fertilizers - should steadily
increase, subject to realistic limitations and
specializations. The organisms have the great
benefits of being relatively safe and very
environmentally friendly in comparison with
chemical pesticides. Throughout the book, it
is fully recognized that formulation must play
a key role in future development. Thus during
the recent decade, the proportion of published
research on microbials devoted to formulation
has been comparable with that on chemicals
(Dent, 1997); while research by industry has
increased, application research has decreased.
The present chapter analyses formulation
procedures during manufacture, storage and
use in relation to commercialization and its
costs. Some problems encountered with pro-
ducts, their causes and solutions are summar-
ized in Table 10.1. The different areas
described in the preceding chapters have
reached different degrees of development,
while each has made particular advances
with certain materials and methods. Opport-
unities for cross-fertilization of information
are explored. Suggestions are made for future
short- and mid-term research on immediate
problems, also for basic research in the long
term. Firstly, we will assess the impact of two
key features common to all the organisms:
their live state and their particulate nature.
10.2 CONSEQUENCES OF ACTIVE
INGREDIENTS BEING LIVE OR PROTEIN IN
NATURE
The state of being live or proteinous has pro-
found effects on the sequence of processes
from manufacture to action at the target. The
consequences of these effects must be
assessed by comparison with alternative
materials, i.e. chemical pesticides, herbicides
and chemical fertilizers. The responses to
these consequences include a number of
trends in formulation, listed below.
The relative delicacy of organisms resulted in
increased protection at all stages of produc-
tion, storage and use (sections 4.3.2, 3.4,
6.5).
The need to consider' ecologies of both the
organism and its target resulted in more
formulation objectives.
The delayed and relatively slow action of
organisms created the need to advance
and speed action after application (section
4.3.2).
While the ability of some organisms to
replicate, spread and move may reduce the
impact of some of the above consequences, it
increases the need for formulation even more.
These trends build up to more functions for
formulation and a great variety of industrial
products (e.g. Table 5.1 in section 5.2) and
tank mixes (e.g. Table 3.15 in section 3.4.3).
They result in more specialization of pro-
ducts, each with a limited market. Life may
be relatively tenuous in products based on the
vegetative forms of organisms, on the more
delicate types of spores, and on nematodes. In
contrast, some organisms form tough survival
 10.3 Consequences of organisms being particulate
stages. The trends in formulation described
above are discussed below in the context of
research and the future.
10.3 CONSEQUENCES OF ORGANISMS
BEING PARTICULATE
The particulate nature of organisms has little
impact on products applied dry, although
there is a need to prevent clumping and cak-
ing of powders. However, with liquids it is
important. Compared to sprays of chemicals
in solution, or very finely dispersed in emul-
sions, organisms are relatively large and tend
to settle rapidly. This necessitates extensive
use of additives to improve suspension of
the particles both in the formulated product
in store and when diluted in the spray tank.
After dilution, agitation is often needed, e.g.
by an electrically driven agitator with Bacillus
thuringiensis. Also, organisms may be main-
tained in suspension by trapping them in oil
emulsions so that the buoyant oil counteracts
the relatively high density of the organisms,
creating a net particulate density near that of
water (sections 2.3.2a and b, Table 6.6 in sec-
tion 6.6). Care must be taken to avoid inver-
sion, which thickens the suspension and
makes it difficult to pump into spray boom
systems.
Spray strategy differs according to whether
many organisms are needed in order to have
the desired effect on the target, or whether a
single organism will suffice (Chapple and
Bateman, 1997). With the requirement for
many organisms to form a lethal dose, formu-
lation to achieve even distribution is often
essential. Volume tends to be reduced to eco-
nomise on additives. With ultra-low volume
(ULV) application the particulate nature of
organisms increases viscosity, which limits
the numbers of organisms contained in a
droplet (Appendix II; Appendix Tables 11.7
and 11.8, Fig. 11.7). A single droplet of the
most potent peroral insect pathogens, such
as some baculoviruses, easily holds a lethal
dose, but with less potent organisms the
313
insect would need to eat a deposit containing
more than one droplet. The action of the toxin
of B. thuringiensis may stop the insect feeding
before it encounters another droplet, while the
deposit may have deteriorated before feeding
is resumed, allowing the insect to survive.
Thus, with this organism it is necessary to
maximize the concentration of ULV sprays to
try to make each droplet lethal (section 3.3.3a),
a requirement made more difficult as viscos-
ity of particulate suspensions rapidly in-
creases with increasing concentration,
eventually impairing spray performance if
too high (section 11.3).
With many species a single microorganism
is effective, so a different strategy is needed.
For example, a single spore of a fungal hyper-
parasite will attack all the downy mildew
within reach of its germ tube. The most effi-
cient application of these species has one
spore per drop, with drops evenly spaced
over foliage. If they are to be successful in
arable agriculture and orchards, conventional
low-volume (LV) to high-volume (HV)
sprayers available on farms must usually be
used at standard volumes. For particulates
this would lead to excessive wastage of addi-
tives included to improve spray characteris-
tics, because many drops would not contain
organisms. The smaller the drop, or the wider
the drop size range, or the larger the spray
volume, the greater the probability that a
given drop will not contain an organism and
will be wasted. To reduce wastage, nozzle
settings, pressures and volumes need adjust-
ment in conjunction with formulation, which
involves time-consuming recalibration of
equipment (Chapple and Bateman, 1997;
Appendix Tables 11.4 to II.6; Fig 11.7). Wastage
can be reduced by applying the principle of
controlled-droplet application (CDA): choos-
ing droplet size to suit the control organism
and target pest/vegetation (section 2.2.2b).
CDA for locust control uses 50-1 DO-Jim
drops, but a mycoherbicide may need
250 Jim. This minimizes empty or inadequate
small drops, which may not impact on the
 C".)
Table 10.1 Formulation and application approaches for organisms (adapted from Bohmont, 1990) ......
oj::.

Phase/stage Problem Cause Impact Solution

1. Shelf storage
Storage of formulated product
up to 2 years at room
temperatures (up to 40+ 0c)
2. Mixing in spray tank
Normally product is mixed and
sprayed. Mix should not be left
to stand for a long time;
occasionally occurs due to
unforeseen weather, equipment
breakdowns, etc.
Degradation Inactivation by high/low pH or
of organism by additives. Can be caused by
growth of contaminant microbes
or natural ageing of organism
Unwanted growth/activity of
organisms
Settling/caking Liquids: particulate microbes
and additives settle during
storage and can cake to form a
solid mass difficult to resuspend.
Solids: caking
Foaming Excess or vigorous agitation.
Emulsifier wetter or dispersant.
Soft water
Suspension settling Organisms and particles settle if
large and heavy, or if agitation
poor or stopped. Wettable
powder may form dry balls
Incompatibility Inactivation/antagonism or
synergism between organism
and additives. Chemical
reactions may occur between
product and chemical pesticides
or fertilizers, which may gel,
curdle or separate products
Reduced/loss of activity
Heterogenous mixture not
suspended evenly in carrier,
causing erratic/poor
application, blocked nozzles
and poor results. Lumps
rather than fine powder
Foam (trapped air bubbles):
can affect mixing, calibration
and distribution; can cause
overflow and loss of
organisms
Heterogenous mixtures
produce erratic control,
plugged screens and/or
nozzles
Possible reduction or increase
in activity of product
Selection of appropriate
microbial strain or production
methodology e.g. for heat
tolerance. Choose formulation
materials based on long-term
tests. Physical (e.g.
encapsulation) or chemical (e.g.
buffering) means control
contaminants by pH,
antimicrobial additives or drying
below 7% moisture content
Dry, entrap physically, control
presence of nutrients or pH
Grind product to fine powder.
Add thinners and/ or wetters.
Form emulsion. Free-flow
additives, e.g. silica, to prevent
powders caking. Formulate as
granules
Avoid excess surfactant/correct
choice of surfactant. Use
antifoam/defoam to break
foam/prevent formation
Use wetter for initial suspension
and thickener suspender (e.g.
clays, chlorides, gums) to aid
suspension and resuspension
Check compatibilities and labels.
Test combinations in laboratory
or field before mixing, including
feeding of target on peroral
organism. If incompatible, try
feeding stimulant, protectants,
etc.

3. Application
Time between discharge from
sprayer and contact with target
4. Spray
Droplets/product in contact with
target
Degradation Breakdown of product due to
high pH of water carrier,
chlorine in water or long
standing
Drift Wind, speed of application
vehicle, spray pressure, nozzle
design; small droplets
Coverage Droplet cover can be reduced by
'beading' of individual drops on
target surface. Mainly due to
surface tension, waxy-oily target
surfaces (e.g. leaf or insect
cuticle) and hairy or pubescent
leaves
Adherence poor Surface tension of droplets and
target/leaf angle cause 'bounce'
and allow run-off. Powder may
not adhere to leaf. Rain, sprinkler
irrigation and wind may remove
organisms
Degradation High temperature and humidity,
leaf surface wetness, sun, UV
radiation, allelochemicals,
competitive microbes, host
defences inactivate organism
Attractiveness to / Odour, taste, absence of
compatibility with seisiochemical reduces
target, e.g. insect acceptance, or repels feeding or
contact of target
Inactivation or breakdown of
product may curb effect,
depending on susceptibility
of microbe, pH and time in
spray water
Product missing target
wasted
Reduced contact area lowers
control levels for insecticides,
fungicides and herbicides
Lack of adherence causes
physical loss and reduces
performance of organisms
Reduce organism activity,
sometimes rapidly
Reduce performance
Check pH of water and
susceptibility of organism. If
problematic, adjust pH with
approved additive or use water
after standing
Optimize equipment/
formulation (e.g. nozzle
orientation, rotary nozzles).
Thickeners to reduce number of
very fine droplets. Anti-
evaporants; oil carrier. For
applications to water bodies,
replace powders by granules,
slow release pellets/briquettes
Lower surface tension with
wetter so that droplets spread
and penetrate between hairs. Use
oil carrier, which has same effect
Use sticker to adhere organism to
target surface. Change carrier
Protect with suncreens,
stabilizers, stickers,
phagostimulants, chemical
neutralizers, buffers, synergists
and encapsulation
Add attractant, phagostimulant,
seisiochemical, inhibitor; remove
repellent
VJ
......
U1
316 Trends in formulation of microorganisms and future research requirements
target anyway. It also minimizes large drops,
which contain a gross overdose and involve
most wastage because of their large volume.
ULV minimizes spray volume, usually with
oil as carrier, and improves cover of foliage
canopies in favourable situations. With high-
potency organisms, this risks many small,
empty droplets which could be countered by
increasing organism concentration (i.e.
dosage). The increase could be considered as
wasting active ingredient, but would improve
survival time of a lethal dosage. It would raise
costs, which ultimately must be balanced
against the improvements discussed above.
10.4 COMMERCIALIZAnON AND COSTS
The earliest planning in the investigation of an
organism must anticipate the ultimate object-
ive. This has to meet commercial as well as
scientific requirements and is usually a realis-
tic series of durable, profitable, well-formu-
lated commercial products (e.g. section 5.2
and Table 5.1 in that section). Research and
development should be sufficient to ensure
that these products are reliable, because a sin-
gle bad result will have a disproportionate
adverse effect on a good trade reputation
built up on reliable operation. An analysis of
drop-outs of commercial products will help to
design success. The main reasons for drop-
outs at the prototype and early marketing
stages have been: market too small; product
not competitive; deterioration in storage;
growth of contaminants; poor or inconsistent
field results; difficulty of application; inade-
quate development; and lack of user educa-
tion. Many drop-outs have been due to
inadequate formulation. Arguably, commer-
cialization often has been too early and with-
out enough investment, so commercial
expectations have been too high.
Of these reasons, one of the most important
for a commercial product drop-out is small
market size. Multi-national agrochemicals
companies will not undertake development
for small markets. Even small companies
must anticipate a minimum critical earning
power. If this cannot be expected from pro-
ducts based on a single organism, a range of
products must be planned. These should be
based around finite expertise and industrial
facility, so that research, development and
capital costs fall with each successive product.
The most successful biotechnology companies
have been of moderate size - large enough to
mount a significant effort, while being flexible
enough to develop and market a range of
products, preferably complementing each
other. Complementary products may have
common formulation technology. Multi-
purpose formulation may extend the market
range of a product, e.g. granules to control
mosquito species of different feeding habit
(section 3.6.4). This is particularly appropriate
for integrated pest management (IPM) sys-
tems, a major outlet for microbials. Dent
(1997) reviewed research on microorganisms
in relation to IPM, in particular the interac-
tions between these organisms and other IPM
components. Work has been done by many
independent researchers, involved in many
different pest/crop situations, all too often
without any clear idea of how the IPM sys-
tems would be implemented and by whom.
Dent concluded that no general principles
evolved, although Jones (1994) and Jones et
al. (1996) summarized approaches that should
be taken to promote beneficial microorgan-
isms in IPM systems. There are obvious
opportunities for formulation to improve
compatibilities between IPM components,
and for manufacturers to list, describe and
advise on systems in which their products
can be used. The most effective ploy will be
to assemble a range of compatible, comple-
mentary, formulated products to cover the
whole of a grower's needs at one marketing
point (Anon., 1997). Only when effective pro-
ducts are easily available to farmers will they
be widely adopted in IPM systems.
Another important reason for products
becoming uncompetitive is high cost. Formu-
lation costs money for the manufacturer, but

saves it for the user. For example, doubling
the effective post-application life of an organ-
ism by better formulation may be far more
than repaid by halving the number of applica-
tions. In six types of formulated product
costed by Lisansky et al. (1993), technical
B. thuringiensis material accounted for 46-
92% of total material costs; non-aqueous car-
rier 2-14%; and additives 2-20%. Even the
most costly formulation additives in these
products, which were in an oil-based flowable
(e.g. Table 3.10 in section 3.3.3), amounted to
only 20% of material costs, while the user
would save 100% on product not needed in
the unnecessary second application, a net sav-
ing to the user of 80% compared with an
unformulated product.
If the product accounted for only 60% of
total user ULV operational costs (Fig. 3.1 in
section 3.3.3),20% of this for the most expens-
ive additive component would be 12%. In two
further examples, formulation might double
the survival time of organisms during storage,
or it might double the effective activity of the
organisms. With the latter, the saving for
the user is then the value of the dosage less
the cost of formulation. Realistically, the cost
of formulation is better viewed as a propor-
tion of the total cost of manufacture, covering
material storage and logistics, energy input,
manufacturing time and labour, including
time for cleaning machinery, as well as the
cost of materials.
In the final analysis, the success of a pro-
duct depends largely on the competitive man-
ufacturing cost of the fully scaled-up,
formulated product. Media and formulation
ingredients affect the ease of handling and
processing, and hence process cost. Use of
more expensive ingredients to avoid pro-
blems, such as flocculation or residual nutri-
ents in an aqueous product that would
encourage growth of contaminants, may cost
much less than the resulting saving in process
cost. A major expense is toxicity testing and
other tests for registration, which must be
conducted on the final formulated product.
lOA Commercialization and costs 317
Consequently, work at the research stage
should be projected to cover the final produc-
tion facility, formulation and other ingredi-
ents for use in the ultimate product for sale,
to avoid expensive re-tests at a late stage.
Most markets for microbial products are
small. Formulation and other product
changes that may be needed to meet the
requirements of additional small markets can
be prohibitive if safety tests have to be re-
done, so lobbying to streamline legislation to
reduce the difficulty of accommodating small
markets is urgently required.
Organisms are commonly deemed relat-
ively expensive to produce. This may not be
so compared with some chemicals if the costs
of new specialist chemical plant and safety
requirements are considered, which however
are likely to be 'one off' costs offset by econ-
omies of large-scale chemical production.
Some organisms are expensive to develop,
particularly if they have been genetically tai-
lored for a specific formulation (e.g. fungicide
resistance for co-formulation with a plant pro-
tection fungicide, or ability to utilize a specific
but unusual formulation nutrient, such as sal-
icylate), or developed for superior nitrifying
or competitive ability in soil. Development
costs too can be compared to the huge sum
total of research into 'failed' chemicals. The
trend for new chemical pesticides is toward
more expensive, more specific chemicals,
used at low concentrations. Seen in cost per-
spective, expensive organisms can stand con-
siderable formulation costs. Seen in another
way, to add to the cost of an already expen-
sive technical material reduces the chances of
it being competitive. Formulations chosen for
production are often scientifically not opti-
mal, but the best at a cost the market can
stand.
There are too few cost analyses in the
research literature. Papers on comprehensive
cost analysis would be valuable, and should
include costs of manufacture as well as the
highly variable costs of storage, transport,
marketing and application. Even the cost of
318 Trends in formulation of microorganisms and future research requirements
new additives is rarely defined; often, one
must rely on comparison between the sale
prices of additives to judge their cost impact.
Cost analyses should emphasize applica-
tion costs incurred by the user, including
time, which is often not costed effectively.
For example, much time spent preparing
sprays before application is saved by the
new water-dispersible granules, a trend in
formulation likely to increase in future. As a
further development these granules can be
made from relatively large particles in which
additives are held in close juxtaposition to the
organisms (section 3.7.2b, third trend). Not
only does this improve efficiency and reduce
the dosage required by the user, but it also
means that the amount of additives needed
(except possibly wetters and stickers) is inde-
pendent of the spray volume in use. If the user
has to make more additions to tank mixes, the
extra amounts increase to prohibitively costly
levels for LV and HV sprays, commonly
resulting in additives being diluted too much
to be effective. Unless they are clearly advert-
ised, new economies of time and dosage may
not be costed by users.
Technical reasons account for a number of
product drop-outs failing to reach market
acceptability, including poor user friendliness
(Gazzoni, 1993), product deterioration and
inconsistent field performance (sections 10.5
and 10.6). All these features can be improved
by appropriate formulation. A user may shy
away from a microbial product that is difficult
or unfamiliar to use. At least when alternative
additives are available, the most user-friendly
can be chosen, e.g. wetters (section 10.7). For-
mulation to limit contaminants and other
causes of deterioration in store (e.g. sections
3.3.3, 4.6.4 to 4.6.6 and 7.6.2 to 7.6.4), or
inconsistent performance in the field (e.g. sec-
tions 4.3.2 to 4.3.6, 3.4.4. and 3.4.5), must be
well studied in reliability trials before product
launch. With organisms that do not have a
specialized survival stage, survival is limited
during the more challenging formulation pro-
cesses. For instance, poor survival of rhizobia
and Gram-negative bacteria during seed
inoculation and subsequent storage is a bar-
rier to commercialization of pre-inoculated
seed (sections 8.3 and 8.4).
Strain variation is a striking feature, evident
with remarkable regularity in Chapters 3-7.
At an early stage, the key requirements for
the successful commercialization of a particu-
lar organism must be defined and strain
search continued until adequate strains are
found to meet these requirements. Anon.
(1997) points out that this is the easy stage
compared with the later problems of bringing
a product to market. Initially, strains should
be challenged by simple research and, if they
are found wanting, the search can continue
before expensive, time-consuming studies on
one strain are involved. It must be emphas-
ized that different strains vary in their reac-
tions even to formulation, among many other
factors, so the formulation technology should
be tried with candidate strains at an early
stage. Generalizations cannot be made; is the
best choice the most potent strain, or a more
productive or faster-acting strain? Would it be
better to use a more robust strain that is easier
to produce or store? The eventual choice will
depend on the target, mode of action and
strategy of use. Tested strains must be num-
bered carefully and, if strains are taken from a
collection, accession numbers must be
recorded together with previous numbers in
other collections.
To reduce the chance of product failure,
commercialization must depend on an ade-
quate base of knowledge and experience.
This is typically obtained by academia and
government-based research until success
appears likely and cooperation by industry is
attractive. International programmes are of
enormous value because they provide laborat-
ory and field data in depth. For example, the
LUBILOSA programme for grasshopper and
locust controlled to a breakthrough in formu-
lation technology of the fungal insect patho-
gens, using ULV sprays in oil (section 4.3.1). It
bridged the gap between fundamental studies

and field use, which progressed stepwise in
scale, from hand-held to vehicle-mounted and
finally to aerial application, towards a market-
able, formulated product.
Smaller amounts of international funding
are also valuable. For example, microbial herb-
icides is a subject area in the early stages of
development, with two tentative commercial
products that have periodically come into and
out of production (section 6.1); the interna-
tional COST 816 programme funds meetings
and committees to coordinate information
exchange and research. It has secured an
agreement to channel research toward four
weed species, which has brought the develop-
ment of at least one new commercial product
much closer. Limited experience has revealed
great differences between an insect and a
weed plant as targets for fungal pathogens.
Weeds have a vigorous defence mechanism,
so leaves may be killed while the plant sur-
vives with new growth. Leaf wetness seems to
be more critical than the relative humidity
around insects (G. A. Matthews, Imperial
College, Silwood Park, UK, personal com-
munication).
Having attracted industry, academic pro-
grammes in the broadest sense should con-
tinue to support it and liaise closely to
investigate problems that arise early in com-
mercialization, and later to ensure continuity.
For example, joint research by Thailand's
Department of Agriculture and the Natural
Resources Institute, UK demonstrated the
potential of the Spodoptera exigua nuclear poly-
hedrosis virus as an effective insecticide on
several crops. This has resulted in local, as
well as international companies registering
products for use in Thailand. Cooperative
research is being established between workers
formulating microbial products and the food,
seed and cosmetics industries. As in the food
industry, the purity of additives should be
standardized, including a watch for supple-
ments put in commodities purchased retail for
additive experiments, e.g. improvers in bak-
ing flour, food emulsifiers and substances
10.4 Commercialization and costs 319
added to fuel oil and aviation spirit to
improve performance.
Sound user education is vital. Essential fea-
tures during application of microbials tend to
differ from those for chemicals. Important
instructions vital for successful use of micro-
bials must be given prominence. For instance,
with most microbiaIs, once opened a pack
must be securely resealed to prevent unused
material absorbing moisture and deteriorat-
ing. Alternatively, 'one-use' packages that do
not have to be opened, but simply dissolve
when placed in water, might be used. Short-
term storage limits need emphasis. Some
instructions such as 'agitate the spray tank
regularly to prevent settling' may be so
important as to warrant prominent overprint-
ing across a label. Such emphasis of the key
instructions should be backed up by generally
clear labels and also comprehensive leaflets
for each product. It is not uncommon, parti-
cularly in developing countries, for packaged
products to be split into smaller volumes and
sold without instructions. This has significant
implications for safety with chemical pro-
ducts, but with beneficial microorganisms
the main concerns would be wrong use and
failure. Extensionists need to emphasize the
importance of adherence to label instructions.
To counter a risk that users will not include
recommended additives in tank mixes, twin
packs of product and adjuvant can be sup-
plied for mixing at application (e.g. section
6.2). Formulation needs to be developed and
coordinated into these educational activities
by industry, extension services and research
organizations; no one group can do it alone.
Formulation problems with some micro-
bials may not be solvable to the conventional
commercial standards of a durable on-shelf
product, i.e. preservation for long periods
may not be reliable without risk of damage
to the organisms. Such weak areas should be
recognized and dealt with by early decisions.
Thus Metarhizium flavoviride conidia can be
stored long-term in stable dry powder at low
temperature (section 4.6.3) and formulated
320 Trends in formulation of microorganisms and future research requirements
locally as less-stable sprays in the short term
(section 4.6.5). As with the microbial herbicide
DeVine, and the relatively delicate Serratia
bacteria in Invade for control of pasture
scarab beetles, delivery to custom order may
be adopted. This approach has been success-
ful with other biologicals such as predators,
parasites and silage inocula, even to the extent
of organizing special distribution chains. For-
mulation can still be helpful in this system,
particularly to improve application and logist-
ical planning, even though the system has the
advantages of avoiding storage by middle
men and warehousing.
10.5 PRODUCTION, STANDARDIZATION
AND QUALITY CONTROL
There has been a tendency to optimize pro-
duction for a high yield of numbers of organ-
isms. However, batches of organisms leaving
the manufacturing facility have varied in per-
formance, which depends on the initial organ-
ism quality in terms of organism size and
physiological state. Aspects of performance
include percentage germination (e.g. sections
4.6.2 and 6.5); appresoria formation (section
6.5 and Table 6.2 in section 6.5); shelf-life
(e.g. sections 4.6 and 6.5); lag time from appli-
cation to start of activity (section 4.6.7); and
eventual effectiveness (sections 4.6.7 and 6.5).
In a recent trend the quality of newly pro-
duced organisms in respect to all five aspects
has been improved by research on harvest
and formulation (e.g. sections 3.2.1, 3.2.2 and
4.7.1 to 4.7.3), and more improvements should
be made in future. The importance of quality
control cannot be understated; however, once
accepted it should not present a problem.
Work is also needed to decide how to mea-
sure and standardize the initial condition of
organisms. This initial condition is vital when
setting up experiments and when manu-
facturing commercial products. A fungal
conidium, for example, starts to change
immediately after it is formed and may need
prompt protection by formulation, so ideally
formulation should start during the harvest of
a conidium. All aspects of performance of
conidia, nematodes and vegetative organisms,
such as rhizobia, depend on initial organism
size and finite energy store. Size varies with
moisture content, therefore size measure-
ments are useful only if made at a stated
moisture content or relative humidity. Even
so, size or weight may be the best measure of
initial organism quality in conjunction with a
viability measure, such as percentage germ-
ination, plus an activity measure, such as
potency.
Other qualities of an organism relate to spe-
cific functions, e.g. storage. Durability of an
organism depends not only on its initial finite
energy store, but also on the rate at which this
is dissipated. Energy use can be measured by
the rate of respiration. Too little research
attention has been paid to the study of
respiration. Periodic experimental measure-
ments during the course of storage until unac-
ceptable deterioration takes place should give
a value for available energy reserves. This
might allow prediction of storage capability
in different conditions of temperature, etc.,
from initial organism weight and respiration
rate, which in turn might provide a basis for
product standardization. Oxygen consump-
tion of infective nematode juveniles is roughly
inversely proportional to storage life (Table
9.1 in section 9.3.1). Much might be gained
by reference to methods of studying perform-
ance in the seed industry, also by reference to
the use of respiration rate and moisture cont-
ent to indicate storage quality of cereals in the
stored products industry. Models, based on
the critical parameters that determine the sto-
rage life of seeds, predict storage life quite
accurately over a range of conditions (Ellis,
1988; Roberts and Ellis, 1989). Similar predict-
ive equations for fungal conidia may be feas-
ible.
Some organisms enter a state of dormancy,
which prevents their energy use being
switched on and off in strict response to
moisture content and temperature. Dormancy

has not been studied in many of the organ-
isms considered in this book, although fer-
mentors are well aware of the need to adjust
the nutrient balance in media to synchronize
maturation or sporulation of the organisms in
a fermentation, and understand the notion of
ripeness of a spore. There may be important
discoveries to be made about durability and
post-storage performance in relation to
degrees of dormancy. The rate of respiration
might be an indication of the degree of inert-
ness or the state of dormancy of organisms
and spores. Occluded viruses grown in
insects are often harvested before insect
death to minimize contaminating bacteria,
resulting in a proportion of partly formed or
unripe occlusion bodies with possible uncer-
tain storage qualities and possible reduced
environmental persistence; this problem may
be overcome by post-harvest, low-tempera-
ture incubation before storage.
A number of organism features could be
measured for quality control and formulation
requirements. These include germination
capaCity, potency, organism size or weight,
respiration rate and appearance, e.g. phase
brightness and wall thickness. Quality is a
complex concept. For instance, a reduction in
the percentage germination of conidia of some
fungi is accompanied by a slowing of germ-
ination (section 4.6.7) and perhaps other
losses of quality, possibly causing a greater
than expected fall in pest control power. This
might be predictable by modelling (see
above). Standardization, stated on product
labels, could be based on a selection of the
above features. Standardization of B. thurin-
giensis products was pioneered in the 1960s,
and has since played an important role in its
success. It should be an important trend in the
future of many organisms, accompanying
improved formulation practices.
10.6 STORAGE
Storage of powders is improved by low moist-
ure content (section 2.2.1), but the discovery
10.6 Storage 321
that low moisture content of conidia of Metar-
hizium spp., even in oil, dramatically length-
ened shelf-life is a recent breakthrough
(sections 4.6.3, 4.6.5). More such systematic
research on moisture content is required on
other fungi (including antagonists of plant
pathogens and herbicidal pathogens), on
vegetative bacteria, baculoviruses and - with
less expected improvement - on some spore-
forming bacteria. Dry products have to be
kept dry during storage, necessitating moist-
ure-proof packaging and formulation with
desiccants. Deterioration of fungal conidia
began immediately after harvest if the moist-
ure content was unfavourable, making
documentation and early control of spore
condition important in experiments. It is
speculated that deterioration of unpurified
baculovirus may be due to auto-intoxication
by fats from insect cadavers harvested with
the virus polyhedra; this may be influenced
by product moisture content (section 3.2.2); in
future, moisture content should be measured
as an essential datum in all work on storage of
organisms.
Durability in store has had research prior-
ity, but now there is a trend to delve beyond
just measuring survival time to more subtle
aspects of organism quality (sections 10.5,
4.7.3, 4.8.2, 4.8.3). Even with dry storage of
spores, organisms do not become totally
inert and respiration does not completely
shut down, although the bacterial spore
becomes virtually inactive. The fermentation
schedule and growth media influence the
physiological state of the organisms and
hence their durability, for example by control-
ling the depth of dormancy. The oxygen
requirements of dormant microorganisms
are generally unknown - several microorgan-
isms (e.g. fungi and, more obviously, viruses)
can be stored for long periods under vacuum
when freeze-dried. Drying below 5% moisture
content mayor may not be desirable, al-
though researchers have stopped experiment-
ing below this level because it is difficult to
reach in practice. If further study shows
322 Trends in formulation of microorganisms and future research requirements
additional drying to be desirable, thorough
drying may be continued during storage by
incorporation of powerful desiccants. Resp-
iration in store leads to accumulation of CO2
and possibly anaerobiosis if containers are
sealed. With some organisms this improves
stability; with others it is harmful (section
4.6.4) and some ventilation of packs or select-
ive permeability of packaging material is
needed.
Some organisms, notably the commercial
entomophilic nematodes, respond to formula-
tion and moderate drying. They should be
dry - not too dry - to extend storage life
(section 9.3.2), but this approach may
change as knowledge of nematode physiology
improves.
Some organisms store best in water, even
preventing the growth of contaminants when
in concentrated suspension (section 7.5.1c).
However, on an industrial scale contaminants
would be a risk, e.g. explosion of containers
due to gas production and possible presence
of human pathogens. Preservative techno-
logy, developed for B. thuringiensis (section
3.3.3b) and for other organisms (section 7.6.3)
formulated in water for application reasons,
could be applied experimentally. In develop-
ing countries lactic acid fermentation is a com-
mon preservation technique for foodstuffs.
This suppresses food-spoilage bacteria, prob-
ably by reduction in pH, by nutrient deple-
tion, or by production of hydrogen peroxide,
carbon dioxide and antibiotics (Jones et al.,
1993b). This approach may prove suitable for
preventing growth of contaminants in some
formulations. Bacterial spores are remarkably
inactive and insensitive to spore concentra-
tions, but other organisms, particularly in the
vegetative form, may be less so (section 4.6.6).
Even with spores there may be suppression of
subsequent growth through production of
antibacterial agents by other microbes
(D. Grzywacz, Natural Resources Institute,
UK, personal communication). Such interac-
tions should be tested species by species.
Harmful compounds may leach out of the
organisms. For example, certain combinations
of amino acids may induce germination of
bacterial spores, which become active and
deteriorate. Some leached compounds may
be toxic to the organisms. Additives which
were beneficial in dilute suspensions of
organisms did not prevent deterioration in
slurries (section 4.6.6), the level of concentra-
tion necessary in a stored commercial pro-
duct. This illustrates the importance of
matching experimental details as near as pos-
sible to the likely scenario in practice. Preser-
vatives that lower water availability by means
of high osmotic pressure may be useful with
some organisms (section 3.3.3b). They may
also act later as humectant or nutrient formu-
lation ingredients. Some contaminant organ-
isms, whilst not affecting storage, may be
antagonistic to beneficial microorganisms in
the host, e.g., dual infection with some bacu-
loviruses (section 3.4.6).
Many additives have been tried for prolon-
gation of storage and some have proved bene-
ficial (section 4.6.6). More work is needed
with mixtures to try to combine the effects of
individual additives. Thus anti-oxidants may
back up the effects of desiccants; oil may
improve the handling properties, nutrient
value and other beneficial effects of starch,
sodium alginate or glycerol. The purity of oil
used as a carrier, and its properties/potential
for degradation (rancidity), are important and
may influence choice of oil; again it can be
speculated that free-radical formation in
impure oils results in microbial inactivation
(section 4.6.5).
10.7 APPLICATION AND AFTER-CARE OF
ORGANISMS
Some of the technical problems encountered
at application have been described in relation
to the special features of organisms, i.e. being
live (section 10.2) and particulate (section
10.3). These and other problems are most
apparent at the time of application. Users
reacted against the early products. Since
 10.7 Application and after-care of organisms
then, attempts to make products more user-
friendly have gained momentum and will
continue as a strong trend in the future. Here
application will be discussed as seen through
the user's eyes, because the user must be
attracted if the future potential of organisms
is to be realized. Formulation is the principal
means of improving performance and accept-
ability.
Supplier research and technology are
increasingly being directed towards improv-
ing user acceptance. For example, two wetters
favoured in experimental work on microbials,
Triton X-lOO and Tween 80, are unpopular in
practice because they are difficult to mix into
water, so they are replaced by more miscible
materials (sections 3.4.1, 4.3.6; AppendiX
Table 1.5). Much work has been done with
the conventional spray machines available
on site. However, user education about these
laboratory studies needs improvement by bet-
ter communication. Organisms are most
acceptable if they can be applied together
with other cultural practices, and information
on compatibilities of organisms with chem-
icals should be made more widely available
(e.g. section 6.5; Table 9.8 in section 9.5.3).
Organisms can be partially partitioned in
sprays (section 7.7.3a) and seed treatments
(section 8.2) by formulation. For example,
two bacteria and a fungicide have been incor-
porated in pre-treated seed.
The relatively slow speed of action and
initially invisible effects of organisms after
application do not impress users and are
seen as major disadvantages - although
some chemicals, such as insect growth regu-
lators, have the same problems but have still
won a market. Improvement in speed of
action has been a research topic with micro-
bials only in recent years and will need much
future work, largely involving formulation
(section 4.8.1b). Both constitutive, endogenous
dormancy (deep) and quiescent exogenous
dormancy (shallow) are valuable features in
extending shelf-life, but must be broken to
synchronize and activate organisms at appli-
323
cation, usually by wetting and ensuring the
presence of appropriate trigger materials and
nutrients (sections 4.3.2, 4.8.lb, 5.3.5, 7.4,
7.5.lc, 7.6.8). Spore-forming bacteria have
powerful endogenous dormancy. This is not
a problem with B. thuringiensis, because it
appears to be terminated promptly in the
insect gut. No one has investigated whether
premature germination and consequent early
death occurs at application on the leaf. It
would probably not be important because
only a few spores are needed to cause septi-
caemia of insects poisoned by the crystal toxin
(section 3.7.2b). However, the breaking of dor-
mancy of other spore formers must be
ensured. Many fungal resting spores have
deep endogenous dormancy, and careful
investigation is needed to prevent this delay-
ing activity after application. It makes some
insect pathogens unsuitable to use for predict-
able insect control. Organisms in exogenous
dormancy become emaciated in store, then
have to recoup energy supplies and switch
physiology to an active state after application,
and possibly flush out accumulated waste
materials. This can be speeded by formulating
some organisms with a wetter to facilitate
moisture uptake, and others with nutrients
(section 4.8.lb), e.g. sugar for rhizobia. Stimu-
lants can be added to formulations to increase
and speed germination, e.g. the insecticide
imidacloprid (section 4.3.7). Some organisms
benefit from pre-soaking in water before
application (section 4.3.2). Again, ideas can
be taken from seed treatment technology.
With other organisms, gentler treatment is
best, e.g. to avoid structural damage dry
Metarhizium flavoviride conidia need a gentle
rehydration in moist air to absorb moisture
before wetting, possibly mimicking a typical
series of events in nature (section 4.6.7). Moist
air treatment is unnecessary if dry conidia are
mixed into oil and applied as a ULV spray.
These ideas should be tried with other organ-
isms. It should be established whether drying
nematode juveniles for storage delays their
response at application and whether gentle
324 Trends in formulation of microorganisms and future research requirements
rehydration would be beneficial. Speeding the
recovery of organisms at application is a sub-
ject needing extensive further study.
A modern improvement to unreliable foliar
treatment has been formulation of organisms
in oil. Although oil is an expensive carrier,
ULV sprays give good cover in foliage cano-
pies under suitable conditions (sections 3.3.3a,
4.3.1). Oil effectively wets hydrophobic struc-
tures such as spores, insect cuticle and plant
epidermis. It carries spores into less accessible
places, e.g. between intersegmental folds of
insects (section 4.3.1), a feature that should
be studied more with pathogens applied to
plants for weed control and with antagonists,
etc. for plant disease control. The low specific
gravity of oil also causes spores to settle
rapidly in store and spray tank, which can
be overcome by formulation as oil-in-water
emulsion (sections 2.3.2b, 3.3.3b). Oil also
helps to combat unreliability by acting as a
useful nutrient for some organisms in the pre-
sence of water after application. It may also
have nutrient value for organisms when used
as a binder in seed treatment (section 8.3.3).
For many reasons, there has been a trend to
turn to oil and emulsions, which may increase
in future, despite the cost. This mimics trends
in agrochemical formulation.
Unreliability due to harsh conditions on
foliage has been reduced by a variety of addit-
ives, such as wetters, stickers, humectants,
sunscreens, nutrients, masks against plant
allelochemicals, synergists and phagostimu-
lants (Appendix I). By lengthening the survi-
val period of organisms on foliage, these
additives have widened the window for
application, thus easing timing restrictions,
which users often find difficult. This modern
trend has developed rapidly with insect
pathogens and formed a large part of the
research on formulation with these organisms
(sections 3.3, 3.4, 4.3). It should accelerate, and
the information is of great value for use with
other types of organism. Manufacturers have
often not fully explained to users the improve-
ments and advantages of new products, for
fear of revealing new additives when confi-
dentiality is the only trade protection. This is
another area for improvement of user educa-
tion. Food-grade additives are desirable for
application to food plants to back up the safe
nature of the organisms and to minimize the
addition of contaminants to products in
which the load of foreign organisms is often
a sensitive issue.
Many organisms require free water or per-
iods of high water activity / relative humidity
in order to take effect (sections 4.3.2, 5.3.5,
6.3). Their activities are often arrested and
they may be killed by daytime desiccation.
Formulation with oil or humectant delays
evaporation; humectants reabsorb moisture
from air overnight. This is a critical research
area, the a~m being to achieve adequate effect
from organisms in all weathers; it involves a
need to study the target leaf or insect/patho-
gen interface and effects on penetration of the
target. The potential of invert emulsions is
being examined to protect weed pathogens
(section 6.6), an idea that could also be
extended to antagonists of plant diseases and
to fungal insect pathogens (although some
experimental oils were phytotoxic on weeds).
Nutrients are also added to improve growth
of insect mycopathogens on plants in the most
favourable weather and in greenhouse condi-
tions, aiming at devastating insect kill in the
most favourable conditions (section 4.3.2),
and tolerating acceptable kills in average- to-
poor conditions. Analytical research is needed
to prove the value of each additive alone and
also in the presence of other tank-mix ingre-
dients. As knowledge increases it may be pos-
sible to express desirable, or suppress
undesirable, traits of organisms by manipulat-
ing the nutrient composition of formulations.
A recent trend has been application of
organisms to soil instead of to foliage, in
order to gain extra protection from desicca-
tion and sunlight, and in order to avoid expo-
sure to leaf allelochemicals. This has been
successful with some plant pathogens (section
6.8) and plant-disease antagonists (section
 10.7 Application and after-care of organisms
5.3.3), which tend to establish poorly on foli-
age because moisture is not available for long
enough on the leaf surface after application. It
could also be tried with pathogens of more
insects that live or hide in the soil (sections
4.4.1 to 4.4.3), particularly insects that feed on
seeds soon after planting (e.g. Invade, section
10.4). Treated seeds thus act as lethal bait, and
insects that succumb to disease would spread
pathogens in the soil. With particular organ-
isms, more research is desirable to decide
whether application to the soil surface gives
adequate protection. Better protection is
obviously obtained by subsurface application,
but at the expense of extra work.
Surface treatment of soil is relatively unreli-
able because penetration of organisms is lim-
ited by the excellent filtration power of the
soil (section 4.4.1). Filtration from liquid sus-
pensions might be reduced by research with
wetters, particularly organosilicones. Free-
flow agents might improve dusts. Another
approach might be to use oil-in-water
emulsions. A fuller understanding of the
cation-exchange capacity of soils in relation
to retention of different microorganisms
might be beneficial.
Subsurface inoculation of soil avoids wast-
ing organisms that lodge on the upper faces of
surface soil particles, where they are exposed
to desiccation and sunlight. Inoculation can be
made by drilling, which is most convenient at
seed planting, or by surface placement then
incorporation by rotovation or ploughing.
Sprays, slurries and granules can be used (sec-
tion 5.3.2, 5.3.3, 7.5), also treatment of seed
(section 5.3.1; Chapter 8). Application with
seed places organisms where they are most
needed, whether the objective is to treat the
plant or to start soil populations, as with rhi-
zobia. Pre-treated seeds are user friendly,
because they need no formulation by the
user. With many organisms there is a critical
need to compare these different methods.
Subsurface inoculation can be integrated
with many routine agricultural practices,
including application of fertilizers and pesti-
325
cides or ploughing. For example Pasteuria
penetrans, used experimentally to control
plant nematodes, is incorporated in the soil
in infected plant roots (Daudi et al., 1990),
probably best achieved by 'ploughing in'.
Information on compatibilities of organisms
with materials such as fertilizers and chemical
pesticides is accumulating (section 4.3.7).
However, in all subject areas the same flaw
has appeared in the experimental strategy.
Materials have been applied to organisms
during their growth in broth and on agar,
but these data have consistently shown little
correlation with limited field results and can
be regarded as a waste of time (section 6.5).
Laboratory tests using sprays with soil are a
little better but only field tests are conclusive,
so the best strategy is to start initially with
field tests, despite their greater expense. For
example, some chemical herbicides synergize
and others inhibit weed pathogens. A variety
of effects have been demonstrated in the
laboratory, including synergism and inhibi-
tion of both spore germination and appres-
soria development, but prediction of field
results from these data is tenuous because
the most important factors in the field are
unclear. The effect of mixing chemical insecti-
cides with microbials can vary with insecti-
cide dose and target stage of the insect
(sections 2.2.4, 3.4.6, 6.5).
The interpretation of field data in relation to
laboratory results may be a good subject for
modelling.
A great disadvantage of soil inoculation is
competition from soil biota. Nutrients to give
the inoculants a good start (section 4.4.2, 5.3.4,
6.8, 7.6.9) and protective additives can be
applied by formulation. This is most effective
in granules in which a centre of intense inocu-
lant growth itself gives the best protection,
arising from the inoculant's own chemical
defences. Nutrients should have the greatest
possible specificity for the inoculant organ-
isms. Oil used to regulate water availability
in formulation, or as a binder on seed, can also
serve a secondary function as a nutrient, for
326 Trends in formulation of microorganisms and future research requirements
which it could be optimized for the chosen
inoculant. This modem trend towards protec-
tion and support for inoculants could gain
momentum in the future.
Users have reacted against complicated
instructions and an element of unreliability.
This tends j to be a vicious circle, because
when reasons for certain unreliabilities are
discovered more remedies are incorporated
in packet instructions. Greater effort should
be made in future to deal with complexities
by formulation rather than by actions
required of the user; when formulation is
improved, the improvement should be
advertised in sales literature.
10.8 LIMITS OF IMPROVEMENT BY
FORMULAnON
Formulation has its limits. These may be real,
when no more progress can be made, or effec-
tive, when the little improvement left to be
made is not worth the cost of development
and materials. However, targets for improve-
ment are often ill-defined so that formulators
are uncertain when practical requirements
have been achieved. A more systematic
approach is required, where organism mode
of action and target behaviour are under-
stood, while acceptable windows for effective
use are well established. Sudden progress in
one direction can change the goal in another
at any stage, from organism production,
through storage, to care of the organism after
application. A new trend is modification of
yield-optimized production to improve har-
vest and formulation (sections 3.2.1, 4.6.2,
4.7.2), e.g. by media modification to make a
more friable product at harvest, and by use of
nutrients with wetting properties. This pays
when the benefit outweighs both extra costs
and any loss of yield. Shelf-life may be influ-
enced by production factors. For instance,
decreasing water potential in the culture med-
ium results in conidia of Trichoderma with
increased desiccation tolerance (Whipps and
McQuilken, 1993). Culturing M. favoviride at
30C rather than 26 C results in improved
high-temperature storage (section 4.7.3). The
addition of humectants during fermentation
may influence conidial survival. A large
enough effect on shelf-life may have a pro-
found influence on the economy of a product.
Less than 6 months' shelf-life requires direct
order service, 0.5-2 years is good enough for
conventional off-shelf sales, longer is helpful
but the further development work required to
achieve over 4 years is not worthwhile.
What are the limits of shelf-life? With the
help of dormancy, some insect eggs and bac-
terial spores survive over 10 years in an inert
condition, often at periodically high tempera-
tures. Once a constitutive dormancy is bro-
ken, the prerequisite for staying dormant is
that they remain dry enough. However,
water is necessary for life and some organ-
isms die if they become too dry. Water short-
age need not limit shelf-life, because the
moisture content of all materials fluctuates to
some extent by equilibrating with ambient
relative humidity; some organisms, e.g. fun-
gal conidia, can supplement their water
reserves by absorption from moist air. Moist-
ure is also partially replenished by water pro-
duced during respiration, albeit only slightly.
In contrast, by absorbing moisture from
humid air, an organism can exceed the low
moisture content needed to prolong shelf-life,
even to a level high enough for physiological
spoilage or damage due to growth of fungi.
Present thinking is to prolong shelf-life of cer-
tain organisms by keeping them very dry.
More knowledge might show that drying
could have a safe low limit, and there may
be an optimum low moisture content. In the
economy of life, energy is limited by finite
food reserves. If the physiology of an organ-
ism is known well enough, this energy limit
might be calculated from cumulative respira-
tion (section 10.6). Auto-intoxication may be
limiting, depending on how well the organ-
ism can excrete wastes or store them
harmlessly. Fundamental studies on the
mechanisms of inactivation, mentioned

above, might lead to novel areas of formula-
tion research to combat these mechanisms. An
example from early formulation research is
the use of /oxygen sinks' to mop up free radi-
cals that may be formed by exposure to ultra-
violet light (section 3.4.4c and e; part III of
Table 3.16 in section 3.4.4; Appendix Table
1.11). Would these sinks generally improve
storage characteristics? Information from the
food industry on storage and stability of
some foods (e.g. vegetable oils), as well as
on the anti-oxidants used, might prove useful
here.
Each species/strain as we know it today
presumably has shelf-life limits controlled by
its genetic make-up. These limits might be
improved as more strains are studied. Spec-
ulating/ could each species be converted to a
supersurvivor by transferring genes by
genetic engineering? Would this order of
research investment be worthwhile? How
would supersurvivors respond to formula-
tion? Would the development of a supersur-
vivor be ecologically desirable?
The limits of application efficiency depend
on spray machines, which in tum depend on
formulation, physical characteristics of the
spray, spray volume, terrain, crop and target
(Table 2.2 in section 2.2.2). The limit for ULV
spray in oil is demonstrated by ready-to-use
formulations, with proprietary diluent for fine
adjustment of viscosity, logistically developed
for direct delivery in bulk to North American
forests (section 3.3.3a). Formulations in water
for other machine/pest/crop applications
need individual adjustments. With these,
economy of large-scale manufacture is
achieved by marketing basic on-shelf formu-
lations tailored for the minimum tank-mix
requirements at many different volumes,
with the intention that tank mixes should be
supplemented, where necessary, for each
individual situation. For example, more wet-
ter can be added for waxy foliage. The limit
for user-friendly efficiency at HV may be
granular or encapsulated formulations, but
even these can waste organisms and additives
10.8 Limits of improvement by formulation 327
(section 10.4). They hold protective additives
in close juxtaposition to the organisms and
require only a decision as to the spray volume
to be used, independent of dosage and cali-
bration for individual machines. Beyond this,
efficiency is limited by information transfer to
the user and user education. Achievable
efficiency of application and efficiency of
organism survival afterwards depend on the
performance and variety of available addit-
ives (Appendix I). Limits are then determined
by a cost/benefit exercise: is it most econom-
ical to use more additives, or to supply a
larger quantity of organisms, or to make
more than one application? An example is
given in section 10.4.
Practical factors, such as new plant growth,
the time taken to complete a pest generation,
and the degree of asynchrony of generations,
exert fresh limits. Thus if important new
growth must be treated every week, extension
of deposit life by formulation beyond a week
is usually of limited use, because the organ-
isms have to be renewed each week anyway.
In addition to the practical limit of cost/
each type of additive has its own unique lim-
its. Wetters not only make a water spray stay
on leaves but, like oil carriers, they also enable
organisms to reach otherwise inaccessible
places between leaf hairs, into depressions,
even into stomata and between intersegmen-
tal membranes on the insect body. This
improves the chances of establishment of
organisms for weed, plant-disease and insect
control. In this respect there is no upper limit
to desired efficiency, provided activity is not
extended to non-target biota. The new super-
wetters (organosilicones) may be followed
with advantage by even better ones. How-
ever/ wetters also reduce spray retention if
the volume applied results in run off, acting
as detergents during rainfall and increasing
wash-off of organisms, so amounts used
should be limited to those needed to give
efficient wetting and organism dispersal.
Stickers need to be as rainfast as possible
without impairing the activity of organisms.
328 Trends in formulation of microorganisms and future research requirements
One possibility that has not been investigated
is that they may fix conidia of fungal insect
pathogens to the leaf firmly enough to reduce
pick-up of conidia by passing insects. Also, in
the tropics and sub-tropics the sticker may be
baked hard so that the insect just walks over it
uncontaminated (G. A. Matthews, personal
communication). It may become insoluble in
the insect gut. The need to protect against
physical removal by the action of wind and
sand needs to be studied further - is the best
strategy, at least with stomach-poison insect-
icides, to aid the microorganism to flow to
protected areas of the target surface, e.g.
stomata and the lower surface of the leaf
where the physical loss of organisms is least
(Jones and McKinley, 1987)?
Many insect phagostimulants are versatile
in that they stimulate many species of insect
(section 3.4.3), a useful feature with a broad-
spectrum pathogen such as B. thuringiensis.
However, if the stimulants also increase feed-
ing by non-susceptible insects in the pest
spectrum, plant damage may be increased.
This effect is most important with species-
specific pathogens such as baculoviruses.
When applied to the soil surface in bait they
may be attractive food for birds and rodents.
Limits on formulation have to be measured
against improved control of target insects,
which may well predominate, particularly
with B. thuringiensis in pest/crop situations
where insect feeding is inhibited - whether
by the bacterial toxin or by plant allelochem-
icals.
Sunscreens cannot protect organisms too
much (sections 3.4.4b, e, 4.3.3). Any curbing
of plant photosynthesis would be transient.
Some fungal weed pathogens may require a
light stimulus of unmeasured intensity for
germination. The limit for sunscreens is that
of high concentration in terms of added bulk
and cost, as well as disfiguring colouring of
the plants. They function only when in close
juxtaposition to organisms, which puts physi-
cal limits on formulation. Their value is best
measured in terms of organism half-life,
which may be only 12 h for unprotected
organisms on exposed foliage. A typical
objective could be to extend this to at least
72 h in exposed positions, which would auto-
matically be much longer on leaves protected
within the foliage canopy, an important deci-
sion largely resting on the relative importance
of pests at different positions. Larger aggre-
gates, as in dusts and baits, are probably the
most effective. Little work has been done on
the interaction of mixtures of UV protectants.
There is no upper limit on the effectiveness
of synergists, provided they do not extend the
effect of the organisms to non-target biota
(sections 3.4.6, 4.3.2, 4.3.7, 6.5). Some are
specific, others have a general action, e.g. by
phagostimulant, anti-allelochemical, or even
humectant. Specific synergists, other than
low dosages of chemical pesticides, do not
appear to have been studied in detail for
plant disease or weed control.
Some additives are multifunctional. While
each function imposes limits of its own, the
summed practical value of the functions eases
the limitation imposed by cost.
The limits of a new organism and asso-
ciated formulation must be understood when
deciding whether to abandon, improve or
commercialize it. To some extent, the poten-
tial of a new organism can be predicted from
the results of others, and the likelihood of
progress can be gauged somewhat from pro-
gress in other subject areas.
10.9 BASIC RESEARCH
Most of the formulation research discussed
above is aimed at short-to mid-term problem
solving. In the longer term, we need to delve
deeper, to basic effects. Basic research is
needed to open up new directions and set
new goals, as well as to seek explanation of
the science involved. Crucial to this is a
detailed examination of the ecology of the
organisms in relation to their targets. For
example, pathogens of insects, weeds and
plant-disease organisms have evolved to

exploit host species without making them
extinct, to allow host populations to establish
(ensuring adequate substrate for the patho-
gens) and, in some cases, to exploit host
populations only in certain environmental
conditions. Thus natural safeguards prevent
complete natural control of hosts. Typically,
profligate nature distributes huge numbers of
the pathogens, or their survival stages, to
passively exploit chance encounters with
their targets, usually when target popula-
tions are well established. Mass natural pro-
duction of pathogen ensues on the target
populations, and the cycle starts again.
Antagonists and symbionts follow a similar
biological cycle.
We try to improve on nature's performance
by artificial but economic production of
pathogens. These are stored in good condition
until needed, then applied directionally,
inundatively and effectively - ideally before
target populations reach damaging propor-
tions. On balance, we appear to improve on
nature by intensive production, by eliminat-
ing deaths in storage, and by employing
formulation to improve application and pro-
tect pathogens afterwards, albeit often incur-
ring a severe disadvantage by applying
pathogens at times that are relatively unfa-
vourable to them. However, do organisms,
growing in nature and on nature's own
substrates, perform differently from our
artificial, formulated products? If so, why?
Both better and poorer performances are
informative. It is intriguing to note that
short exposures of baculoviruses to UV
appear to increase activity, and some UV
wavelengths appear to negate the harmful
effects of others (Jones et al., 1993a). Also,
exposure to some temperatures (40-50 0c)
shows initial inactivation followed by reacti-
vation (McKinley, 1985; Jones, 1988). Can this
be exploited?
Although production yield can be optim-
ized and organisms can be grown in apparent
peak condition (sections 3.2.2, 3.2.1, 4.8.3,
9.4.1), we need to establish what exactly is
10.9 Basic research 329
this peak condition, what is really needed to
achieve it, and how it can best be preserved.
Quicker action of products used inundatively,
and an unexpected constitutive dormancy
that might lead to better storage, are two feat-
ures particularly to be sought. Experimental
application still in the natural medium, nat-
ure's carrier, could be compared with formu-
lated products. In nature, many organisms
grow with a cover of mucilage, a natural
encapsulation matrix (Potts, 1994). At least
with conidial heads of Verticillium lecanii,
there is some evidence that the presence of
mucilage improves survival of desiccation.
This may not be found in artificial liquid
media, or may be washed away if organisms
on solid media are harvested in water.
Possibly mucilage could be collected and
experimentally replaced, even in greater con-
centration than produced naturally (section
6.2). If this performs better than artificial for-
mulation additives, chemical analysis might
be inspirational and possibly lead to new
materials such as artificial mucilage and thix-
otropic gel. It is already well established that
protein and insect debris in unpurified sus-
pensions of baculovirus are good sunscreens
(section 3.4.4e; part I of Table 3.16 in section
3.4.4; Appendix Tables 1.11 and 1.12), which
are removed by purification. Probably these
screens are closely stuck around occlusion
bodies. Can the virus be purified to remove
or kill unwanted contaminants, but retain the
protective substances? And is this desirable in
the light of indications that unpurified virus
stores less well than purified virus (section
3.2.2)?
We can choose the most suitable strains and
improve them, if necessary, with the aid of
genetic engineering. Specific factors can be
added. For example, melanins which may be
nature's supersunscreens (Table 3.16 in sec-
tion 3.4.4; this work needs confirmation - see
above and section 3.4.4e), also other natural
pigments in spores, could be tried as sunsc-
reens (section 4.3.3a). Melanins, which are
usually cell wall-bound, have other desirable
330 Trends in formulation of microorganisms and future research requirements
features. There is some evidence of improved
desiccation tolerance, antibiotic activity, sur-
vival in soil, also system-dependent virulence
and protective action (e.g. against the mam-
malian immune system) and hydrolytic
enzymes, such as chitinase produced by
plant pathogen antagonists (Bell and Wheeler,
1986; Perpetua et al., 1996). A melanin-pos-
itive, UV-resistant B. thuringiensis strain was
x4.5 as active as its parent strain, although it
was not demonstrated whether this was due
to the melanin or some accompanying differ-
ence in toxin content (section 3.4.4.e). The mel-
anin would protect from sunlight only the
spore, which is less important than the crystal
as a potency factor (section 3.7.2b). Strains of
insect pathogens would probably be easiest to
improve by transfer of known genes from
melanin-positive strains (e.g. section 4.3.3a)
into normally hyaline strains with superior
insect-control properties. This is because syn-
thetic pathways for melanin are complex and
varied, as shown by the many enzymatic
changes responsible for converting pigmen-
ted organisms to hyaline mutants (e.g. Bell
and Wheeler, 1986; Perpetua et al., 1996). In
the short term, sunscreen protection by mela-
nin can be obtained by formulation with
extracted melanin (section 3.4.4e; Table 3.16
in section 3.4.4) or with melanin-rich natural
materials, which can protect both spore and
crystal of B. thuringiensis. A search is needed
for other suitable genes, e.g. those expressing
factors that may counter certain plant allelo-
chemicals (section 3.4.5). An alternative
approach is to introduce genes into extremely
photoresistant organisms, e.g. the B. thurin-
giensis ssp. israelensis 8-endotoxin genes into
the aquatic bacterium Deinococcus radiodurans,
which has an exceedingly efficient DNA
repair mechanism and a red pigment thought
to scavenge free radicals (Manasherob et al.,
1997).
Mugnier and Jung (1985) obtained large
differences in survival of bacteria held in
dried biopolymer gels due to the type of nutri-
ents present during growth of the bacteria.
This phenomenon may apply also to the
fungi and may be a lucrative area of research
into survival during storage (section 10.8).
We can apply organisms earlier and more
directionally than in the natural cycle.
Accumulating information on population
dynamics of pests and survival of their patho-
gens enables predictive modelling to play an
increasing role in research design. Applica-
tion must be made as directionally as possible,
and the organisms need protection by formu-
lation for reasons of economy. When feasible,
ecological conditions (e.g. humid) should be
chosen in which the organism's natural
powers of reproduction can be supplemented
by formulation, for example by applying
nutrients (section 4.3.2). At every stage,
investigation should continue to optimize the
operation and modelling should show up lim-
itations (section 10.4). Within this system, for-
mulation is the servant of function.
A feature which is advantageous in the use
of an organism when seen from one aspect,
may be problematic from another. For exam-
ple, spores tailored to promote rapid action
after application (section 4.6.7) may not be
physiologically suited for long storage and
so require protection by formulation. Metarhi-
zium conidia grown with trehalose stored bet-
ter than those with sucrose, but yield was less
(Potts, 1994). Ways round such problems
must be sought, compromises reached, or
priorities chosen in the context of the practical
use of an organism.
Basic research produces ideas that must be
cherished and developed (research sections in
all chapters); several examples are listed
below.
The moisture content of M. flavoviride conidia
responds with remarkable speed to chang-
ing ambient relative humidity. Is this differ-
ent in other fungi? Would it reveal
differences in the storage capabilities and
the need for formulation?
Research has greatly improved survival of
infective juveniles of insect-pathogenic

nematodes (sections 9.3.1, 9.4.1). This
implies that advanced research on nema-
tode physiology and biochemistry could
improve still inadequate shelf-life and lead
to the development of granules and pellets
that could be applied directly to soil, avoid-
ing the need to release infective juveniles
before application.
Some baculovirus occlusion bodies are
remarkably adherent, even to waxy, hydro-
phobic plant surfaces. Many bacterial
spores adhere well to leaves, essentially
two hydrophobic surfaces attracting each
other. Some initial research on the forces
involved with baculoviruses has been
done (section 3.4.2), but further research
might show whether the effect can be repro-
duced by formulation. Surface charges are
complex, but may be instrumental in these
effects. The complex charge systems on
materials like clays might be intermedi-
aries, or effective in their own right.
Some termites reject particular pathogens,
possibly as a result of smell (Begum and
Jackson, 1994). This might be masked by
termite phagostimulants or by extracts of
fungi that they use as food.
Some aphid species move more than others
and so pick up pathogens more readily.
B. thuringiensis makes caterpillars more
active, uncoordinated and prone to fall off
foliage, becoming vulnerable prey to
ground predators. Formulation with irrit-
ants may sometimes be useful, but not if it
reduces feeding on peroral pathogens.
A basic search for new additives is desirable.
Tinopal LPW, the sunscreen with strong
synergism for many baculoviruses (section
3.4.4e; part I of Table 3.16 in section 3.4.4), is
inspirational for the future. It would be useful
to see whether this synergist, which is most
effective with moderately active viruses, will
synergize partially inactivated viruses and
extend their persistence. Studies should
include expensive, sophisticated materials as
well as cheap, practical ones, in order to
References 331
search for novelty which may lead to new
groups of affordable additives.
REFERENCES
Anon. (1997) Cautious optimism for biopesticides
in the transgenic era. AGROW 274 (February 14),
18-9.
Bateman, R. P., Godonou, 1., Kpindu, D. et al. (1992)
Development of a novel technique for assessing
mycoinsecticide ULV formulations, in Biological
Control of Locusts and Grasshoppers (eds C. J.
Lomer and C. Prior), CAB International, Wall-
ingford, pp. 255--62.
Begum, H. A. and Jackson, C. W. (1994) Quantitat-
ive measurement of avoidance of entomopatho-
genic fungi by Reticulitermes flavipes Kollar, in
Abstracts of the VIth International Colloquium on
Invertebrate Pathology and Microbial Control, Mont-
pellier, August-September 1994, Society for
Invertebrate Pathology, p. 325.
Bell, A. A. and Wheeler, M. H. (1986) Biosynthesis
and functions of fungal melanins. Ann. Rev. Phy-
topathol. 24,411-51.
Bohmont, B. L. (1990) The Standard Pesticides User's
Guide. Prentice Hall, New Jersey.
Chapple, A. C. and Bateman, R. P. (1997) Applica-
tion systems for microbial pesticides: necessity
not novelty, in Microbial Insecticides: Novelty or
Necessity?, BCPC Symposium Proceedings
No. 68 (ed. H. F. Evans), British Crop Protection
Council, Farnham, pp. 181-90.
Daudi, A. T., Channer, A. G., Ahmed, R. and
Gowen, S. R. (1990) Pasteuria penetrans as a bio-
control agent of Meloidogyne javanica in the field
in Malawi and in microplots in Pakistan, in Pro-
ceedings of the Brighton Crop Protection Conference,
Pests and Diseases, Vol. 1, British Crop Protection
Council, Farnham, pp. 253-7.
Dent, D. R. (1997) Integrated pest management and
microbial insecticides, in Microbial Insecticides:
Novelty or Necessity?, BCPC Symposium Proceed-
ings No. 68 (ed. H. F. Evans), British Crop Pro-
tection Council, Farnham, pp. 127-38.
Ellis, R. H. (1988) The viability equation, seed via-
bility monographs, and practical advice on seed
storage. Seed Sci. Techno/. 16, 29-50.
Gazzoni, D. L. (1993) Programa de Manejo de Pragas
de Soja no Brasil: Uma Abordagem Historica, Ver-
sao, Empresa Brasileira de Pesquisa Agrope-
cuaria (EMBRAPA), Centro Nacional de
Pesquisa de Soja (CNPSO), Londrino, October
1993.
332 Trends in formulation of microorganisms and future research requirements
Jones, K. A. (1988) Studies on the Persistence of Spo-
doptera littoralis Virus on Cotton in Egypt, PhD
thesis, University of Reading.
Jones, K. A (1994) Registration and use of
microbial insecticides in developing countries,
in Proceedings of the VIth International Colloquium
on Invertebrate Pathology and Microbial Control,
Montpellier, August-September 1994, Society
for Invertebrate Pathology, pp. 82-8.
Jones, K. A., Cherry, A J. and Grzywacz, D. (1996)
Use of microbial pesticides in IPM strategies in
developing countries, in Abstracts of the 29th
Annual Meeting of the Society for Invertebrate
Pathology, University of Cordoba, Cordoba,
Spain, p. 4l.
Jones, K. A and McKinley, D. L. (1987) Persistence
of Spodoptera littoralis nuclear polyhedrosis virus
on cotton in Egypt. Asp. Appl. Bioi. 14, 323-34.
Jones, K. A., Moawad, G., McKinley, D. L. and
Grzywacz. D. (1993a) The effect of natural
sunlight on Spodoptera littoralis nuclear poly-
hedrosis virus. Bioi. Sci. Technol. 3, 189-97.
Jones, K. A, Westby, A, Reilly., P. J. A and Jeger,
M. J. (1993b) The exploitation of microorganisms
in the developing countries of the tropics, in
Exploitation of Microorganisms (ed. D. G. Jones),
Chapman & Hall, London, pp. 343-70.
Lisansky, S. G., Quinlan, R. J. and Tassoni, G. (1993)
The Bacillus thuringiensis Production Handbook,
CPL Scientific, Newbury.
Manasherob, R., Myasnik, M., Ben-Dov, E. et al.
(1997) Introduction of Bacillus thuringiensis sub
sp. israelensis (-endotoxin genes to the extremely
photoresistant bacterium Deinococcus radiodurans
R1, in Abstracts of the 30th Annual Meeting of the
Society for Invertebrate Pathology, Banff, Alberta,
p.45.
McKinley, D. L. (1985) Nuclear polyhedrosis virus
of Spodoptera littoralis Boisd. (Lepidoptera, Noc-
tuidae) as an infective agent in its host and
related insects. PhD thesis, University of London.
Mugnier, J and Jung, G. (1985) Survival of bacteria
and fungi in relation to water activity and the
solvent properties of water in biopolymer gels.
Appl. Environ. Microbiol 50, 108-14.
Perpetua, N. S., Kubo, Y., Yasuda, N. et al. (1996)
Cloning and characterization of a melanin bio-
synthetic THRI reductase gene essential for
appressorial penetration of Colletotrichum lagen-
arium. Molec. Plant-Microbe Interact. 9, 323-9.
Potts, M. (1994) Desiccation tolerance of prokar-
yotes. Microbial Rev. December 1994, 755-805.
Roberts, E. H. and Ellis, R. H. (1989) Water and
seed survival. Ann. Bot. 63,39-52.
Whipps, J. M. and McQuilken, M. P. (1993) Aspects
of biocontrol of fungal pathogens, in Exploitation
of Microorganisms (ed. D. G. Jones), Chapman &
Hall, London, pp. 45-79.
APPENDIX I: A CATALOGUE OF
FORMULATION ADDITIVES: FUNCTION,
NOMENCLATURE, PROPERTIES
AND SUPPLIERS

Konrad Bernhard, Peter J. Holloway and H. Denis Burges

KEY TO CONTENTS
Introduction 334
Adjuvant 1.7 350
Amino acid 1.11 360
Anti-oxidant
stabilizer 1.8 353
sunscreen 1.11 360
Binder 1.4 341
B-vitamin 1.11 360
Carrier
liquid 1.1 336
mineral 1.2 337
miscellaneous 1.3 338
Desiccant 1.4 341
Dispersant 1.4 341
Dye
sunscreen 1.9 355
tracer 1.9 355
Emulsifier 1.4 341
Floatation agent 1.4 341
Free-flow agent 1.4 341
Gellant 1.4 341
Humectant 1.4 341
Lubricant 1.4 341
Nitrogenous compound 1.11 360
Nutrient 1.8 353
Oil 1.1 336
Optical brightener 1.10 359
Phagostimulant 1.7 350
Preservative 1.8 353
Protein 1.11 360
334 Appendix I
Reflector 1.10
Spreader 1.6
Stabilizer 1.8
Sticker 1.6
Sunscreen
cosmetic 1.9
general 1.9-12
miscellaneous 1.12
Surfactant 1.5
Suspender 1.4
Synergist 1.7
Thickener 1.4
Wetter 1.5
INTRODUCTION
This appendix has been compiled as an easy-
to-use overview of additives which have been
tried in formulations of microorganisms.
These additives are often dubbed by formu-
lators as 'inerts'; usually they are anything but
inert.
There are five objectives.
To list alphabetically all additives tried with
microbials, whether successful or not.
To collect together key information about
their side-effects on microorganisms, often
not published elsewhere and probably the
most important function of this Appendix,
together with other useful information.
To classify the context in which materials
were used, either functionally or with
regard to organisms. This is critical infor-
mation when starting a research project.
To list alternative names.
To help trace suppliers.
In-depth information about work on each
additive with different organisms in the var-
ious chapters can be obtained by using the
comprehensive index at the end of the book.
Additives described in the various chapters
of this book were entered into a database with
additional information contributed by the
359
347
353
347
355
355-362
362
345
341
350
341
345
authors of all chapters and extracted from
suppliers' catalogues. It currently contains
706 materials. These comprise commonly
used laboratory chemicals, speciality chem-
icals, names of commercial trade products
(given in capitals), generic product names
such as 'clay' or 'skimmed milk', as well as
rather poorly defined materials such as 'aqu-
eous clover extract' or 'blowfly faeces'.
Tables 1.1 to 1.12 are extracts from this data-
base classified by functions listed in the head-
ings of each table. Some additives have been
used with different functions in mind. For
15% of them at least two functions, and for
3% even three different functions could be
identified. These additives therefore appear
41 several tables. For easier reading, some
major additive categories are subdivided by
their nature between several tables, and the
same format is maintained for all tables.
Names of the additives are listed in the first
column, and of organisms in the second. To
save space, the following abbreviations are
used:
B, Beauveria
Bm, beneficial microorganism
Bt, Bacillus thuringiensis
Bti, Bt ssp. israelensis
F, fungi

H, Hirsutella
M, Metarhizium
Mh, microbial herbicides
NPV, nuclear polyhedrosis virus
R, Rhizobium
T, Trichoderma
V, insect virus
V, Verticillium.
With some of the materials, names of specific
forms of the organisms, e.g. blastospores, are
given. If, for example, 'V' or 'F' is given, this
indicates either usage with a wide range of
organisms, or that more specific information
is unavailable. Side effects and other informa-
tion, including alternative functions, appear
in the third column.
Table 1.13 lists suppliers of additives, cross-
referenced by numbers to Tables 1.1-1.12.
These have been confined to suppliers of
trade products and speciality chemicals. For
some no supplier or address could, alas, be
Appendix I 335
found in the literature. Common laboratory
chemicals and materials for which only gen-
eric names are given can be obtained from
many suppliers, so no cross-reference number
is given. For most international companies,
only addresses of corporate headquarters are
given. In attempting to identify local suppli-
ers, it should be kept in mind that some trade
products were intended for local markets only
and thus are not available in every country, or
have been discontinued. With speciality che-
micals, mention of one supplier does not
imply that this is the only one. Industrial rear-
rangements by licensing, acquisitions and
mergers cause changes in suppliers; often
Table 1.13 is only a starting point in the search
for suppliers.
We intend to update the database regularly
and would therefore be grateful for further
information on additives listed, as well as on
new ones.
336 Appendix I

Table I.1 Liquid carriers

Name Organisms* Supplier t Useful information

ACTIPRON V, V. lecanii 16 Spray additive; carrier; sticker;
emulsifiable mineral oil (97%)
Almond oil M. anisopliae Vegetable oil
ASSIST Mh Mineral oil
BIO-VEG Mh Vegetable oil
BIVERT V Emulsifiable crop oil
BP ARACHIS OIL NPV 16 Vegetable oil
BP LIGHT PARAFFINIC NPV 16 Mineral oil
MINERAL OIL
BP MINERAL SEAL OIL NPV 16 Mineral oil
BP 150 SOLVENT NEUTRAL NPV 16 Mineral oil
BP 50 SPINDLE OIL NPV 16 Mineral oil
Canola oil Vegetable oil from rapeseed
CARRIER 038 NPV 74,119 Proprietary ULV carrier for NPV for
gypsy moth control; contains sunscreen
and anti-evaporant; proprietary
ingredients 95% v lv, water 5% v /v
Cedar wood oil F Essential oil
Chainsaw oil Mh Mineral oil
Coconut oil Conidia Vegetable oil
Cod liver oil Conidia 84 Fish oil
Corn oil F, Mh 26 Unrefined vegetable oil from maize;
refined, e.g. Mazola
Cotton seed oil NPV, conidia 86 Phagostimulant; vegetable oil; for
prolonged storage, dry by adding
anhydrous silica gel
Deodorised kerosene M. jlavoviride 13 Light mineral oil
EDELEX EL M. jlavoviride 85 Carrier for oil-based products; refined
medium viscosity index naphthenic oil,
low aromatic content
ENERGOL WT-l Bt 16 Highly refined white paraffinic oil
Grape seed oil Mh Vegetable oil
Groundnut oil F,Mh 86 Vegetable oil
ISOPARM NPV 39 Refined mineral oil; also code V
Kerosene Conidia 85 Odourless paraffinic oil; low viscosity
Lactic acid Conidia
Linoleic acid Conidia Fatty acid
Linseed oil F,Mh Vegetable oil
Liquid paraffin Conidia, Stabilizer; refined mineral oil for
blastospores, medicinal use, mixture of alkanes with
Mh > 12 C atoms/molecule
Milk, skimmed V, Bt, F, Bm Carrier; sunscreen
Motor oil Mh Mineral oil
Naphthol, heavy aromatic M. anisopliac 93 Petroleum-based oil; long-chain saturated,
aliphatic compound (unidentified
naphthol)
Neem oil Conidia Spray additive; vegetable oil
No.2 fuel oil NPV Mineral oil
NORPAR 12 NPV 39 Refined mineral oil, also codes 12 and 15
 Appendix I 337
ODINA OIL EL M. flavoviride 85 White mineral oil, a purified form of
RISELLA OIL used for ULV sprays
Olive oil Conidia, Mh 86 Vegetable oil
Palm oil Bt, conidia Vegetable oil
Rapeseed oil Conidia, Mh 65 See canola oil
RISELLA OIL F 85 Light mineral oil
SEATON cottonseed oil NVP 83 Vegetable oil, refined and deodorized
grade; crude oil may contain toxic
gossypol
SEATON rapeseed oil NVP 83 Vegetable oil, refined and deodorized
grade
SEATON soya oil NVP 83 Vegetable oil, refined and deodorized
grade
Sesame oil Mh Vegetable oil
SHELL RISELLA OIL (L) NVP 85 Mineral oil, also code (EL)
SHELLSOL K M. flavoviride 85 Light mineral oil; also Shellsol T
Soya oil Conidia, Mh 86 Vegetable oil
Sunflower oil Conidia, Mh Vegetable oil
T-400-100 M. anisopliae Mineral oil
TOP OIL 79 Emulsifiable oil
Vegetable oil Bt, Conidia Carrier, binder
Walnut oil Mh Vegetable oil
Wheatgerm oil Conidia Vegetable oil
Abbreviations, see text.
t Suppliers as given in Table LB.

Table 1.2 Mineral carriers (= fillers)

Name Organisms' Suppliert Useful information

ATTACLAY 24/48 M. anisopliae Attapulgite; also code X-250
Attapulgite clay Conidia
BARNET CLAY M. anisopliae Kaolinite
Bentonite clay Rhizobium, Mh
BENTONITE GO-II M. anisopliae Montmorillonite
Calcium carbonate Rhizobium spp., Synergist
Bt
Calcium montmorillonite R. japonicum
CELATOM F 112 Free-flow agent; diatomaceous earth;
prevented conidia in dust sticking to
applicator
CELITE 209 M. anisopliae Diatomaceous earth
Clay F, Bm 120, 121 Dispersant; sunscreen, carrier; numerous
suppliers, e.g. Englehard Corp., George
C. Brand Inc.
CONTINENTAL CLAY M. anisopliae Kaolinite
Diatomaceous earth T. harzianum, Free-flow agent; see CELATOM
Mh
JORDAN CLAY M. anisopliae Kaolinite
Kaolinite Pseudomonas
spp., Mh
338 Appendix I
Table 1.2 Mineral carriers (Cont.)

Name Organisms* Supplier t Useful information

Limestone, ground R. japonicum
Montmorillonite Pseudomonas
spp.
PYRAX Bm 78 Kaolin
PYRAXABB Conidia, Bm 78 Pyrophyllite, pH 7.0
Pyrophyllite Bm,Mh Nutrient
REX CLAY M. anisopliae Kaolinite
Sand Bt, Nematodes
SPESWHITE china clay NPV 56 Dispersant; kaolin; fine-grade cheap filler
Talc Bm
TENNKCLAY M. anisopliae Kaolinite
Vermiculite F,Bm 123 Illite; e.g. VERMICULITE codes 35/60 and
8/35
VERTAL-5 M. anisopliae Talc, also code 15
Zeolite Pseudomonas 127 Various grades available, selective
spp., Mh binding capacity for ammonium ion
Abbreviations, see text.
t Suppliers as given in Table 1.13.
Table 1.3 Miscellaneous carriers and polymerizers (= fillers)

Name Organisms* Supplier t Useful information

AATARA 430 R. japonicum 43 Vinyl pyrrolidone-styrene
AC-DI-SOL Bm 42 Dispersant, cross-linked sodium
carboxymethylcellulose
AGRO-LIG Bm Lignaceous shale, pH 4.1
ALCOSORB AB3 Serratia entomophila 125 Thickener; water-absorbing
polyacrylamide; high liquid
absorption capacity (> 50 times its
own weight)
Alder bark Talaromyces flavus
Alginate F, Bm,Mh
Alginate prill Bm,Mh Calcium alginate granules
ALPHACEL 116 Cellulose carrier; caused no reaction
from larvae
AVICEL Bm 42 Dispersant; microcrystalline cellulose
Azotobacter vinelandii alginate Plant disease
control agents
Bituminous coal Bm
Bran Nematodes, F Phagostimulant, bait
Calcium alginate Nematodes
Calcium sulphate, granular Bm
CARRAGEENAN Bm 86 Binder in pellets; carrier in capsules;
polyanionic
Cereal flour M. anisopliae Nutrient
Charcoal, activated Bm
Compost Bm
Corn cob grits Bt, F, Bm, Mh Nutrient; lignin content harms some
organisms
 Appendix I 339
Corn meal Bt, B. bassiana Phagostimulant; carrier; nutrient
Corn starch V, B. bassiana 26 Carrier; sunscreen; see e.g. MIRAGEL
463
Croscarmellose Bm 42 Cross-linked sodium
carboxymethylcellulose
DACAGIN NPV 33 Hydroxycellulose
Dextran Gliocladium virens Polysaccharide
DICAPERL 920 Bt 49 powdered perlite; flotation compound
EMCOCEL Bm 38 Dispersant; microcrystalline cellulose
EX-CEL Bm 87 Dispersant; microcrystalline cellulose
EXPLOTAB Bm 38 Sodium starch glycolate
FLOUR 961 B. bassiana 53 Cornflour, pregelatinized
/,-Butyrolactone M. anisopliae Dihydro-2(3-H)-furanone
GASILGM2 Conidia 29 Desiccant; silica zerogel powder
Gluconate Bm Polymerizer
Glycerol F,Mh Humectant; nutrient, osmotic
protectant
Gum carob Rhizobium spp. Vegetable gum
KARIONF F 63 Sorbitol;
KELGIN Plant disease 57
control agents
Lactose Bt, blastospores, Stabilizer; sugar
Pseudomonas spp.
LAPONITE Gliocladium virens
Leonardite shale Enterobacter cloacae
Lignite Bm
MIRAGEL starch Bt, B. bassiana 107 Fully pregelatinised pearl corn starch
used in food; contains 25% amylose
(ca same content as regular corn
starch)
MIRAGEL463 Bt, B. bassiana 89 Corn starch
Molasses V, Bt, Bm 88 Nutrient; sunscreen; carrier; e.g. MO-
MIX; dark brown, viscous residues
from sugar production; rich in
sugars, particularly sucrose
Muck soil T. harzianum
NEOSYLGP NPV Synthetic silica powder; free-flow
agent
Oat grains Bm Nutrient
Oat meal Fusarium solani
Paraffin wax Mh Thickener; long-chain paraffins
Pearl com starch 26
Peat Bm,Mh Many grades, e.g. granular
Perlite Fusarium spp., Bm
Polyacrylamide gel Bm,Mh Humectant
Polyamide Streptomyces spp.
POLYSULF Gliocladium virens
POLYTRAN Gliocladium virens
Poplar bark compost Fusarium spp.
Propylene glycol M. anisopliae
Rice flour Mh
Sawdust Bm
Shale Bm
340 Appendix I
Table 1.3 (Contd.)

Name Organisms* Supplier t Useful information

Silica gel F,Mh Desiccant; non-indicating harmless to
conidia, indicator grade lethal
Silica powder conidia, Mh 32 Desiccant; see WESSALON S;
hydrophilic
SMA-2625A 7 Polymer of styrene maleic anhydride
half-ester
SODIUM ALGINATE IG-350 Bm,Mh
Sodium caseinate R. japonicum
SodiumCMC Bacillus sphaericus S6 Medium viscosity polymer with 0.7
substitution degree; 1% solution
used with 0.05 M AI 2 (S04)3 solution
pH 3.3 to form extended release
microcapsules; did not harm spores
and improved stability at 50C
Sorghum powder Pandora de/phacis Nutrient
Sorghum straw Mh
Soya flour NPV, Bm, Mh .Phagostimulant; nutrient
SPAN-SO V 9 Sorbitan monooleate
Sphagnum moss Bm
STA-RX 1500 Bm 122 Pregelatinized starch

STAR-TAB Bm 54 Partially pregelatinized starch

Starch Bt, Bm, Mh
Starch, gelatinized Bt, V, Bm Starch treated with urea, alkali
solutions or heat
Succinate Bm Polymeriser
THIXCIN R M. anisopliae Modified, dried castor oil derivative
Wheat bran Bt, F, Bm Phagostimulant; carrier; nutrient
Wheat flour Mh,Bm Nutrient
Wheat straw Bm Nutrient
Wood chips M. anisopliae
Abbreviations, see text.
t Suppliers as given in Table LB.
 Appendix I 341
Table 1.4 Compounds affecting physical properties of formulations (binders, desiccants, dispersants,
emulsifiers, flotation and free-flow agents, gellants, humectants, lubricants, suspenders, thickeners)

Name Organisms* Supplier t Useful information

ACCUREL POWDER 99 Flotation agent; polypropylene; 106-
400 Itm particles; 70% void; flotation
agent in granules
AC-DI-SOL Bm 42 Dispersant, cross-linked sodium
carboxymethylcellulose
Adipic acid Bm Lubricant
ALCOSORB AB3 Serratia entomophila 125 Thickener; water-absorbing
polyacrylamide; high liquid
absorption capacity (> 50 times its
own weight)
Alginic acid Mh,Bm Dispersant, sticker
Aluminium silicate Bm Dispersant
AMBERLITE Bm 80 Dispersant; range of granular resins for
chromatographic separations
AMEA Mh Humectant; acetaminde-8-
mercaptoethylamine
Amylose Bm Dispersant; polysaccharide
AVICEL Bm 42 Dispersant; microcrystalline cellulose
BENTONE 38 F 91 Suspender in oil-based products;
organic derivatives of smectite clay
BEVALOID 116 M. anisopliae 14 Dispersant; sodium polyacrylate;
deflocculant; neutralized low M r
anionic polymer powder
BEVALOID 211 14 Dispersant; 40% solution of low M r
sodium salts of polycarboxylic acid
BIVERT Bt, V Thickener
Boric acid Bt, Bm Lubricant; synergist
CAB-O-SIL, see silica, fumed Bm 113 Lubricant; gellant; fumed silicon
dioxide; small, very light feathery
flakes, insoluble in water
Calcium chloride M. anisopliae, Desiccant; gellant
Calcium gluconate Plant disease Gellant
control agents
Calcium stearate Bm Lubricant
Carboxymethylcellulose Bt, F 21 Thickener; sticker; binder; stabilizer;
CMC-R295F sodium carboxymethylcellulose;
water-soluble; food additive;
compatible with Bt
CARGILL INSECTICIDE Bt 19 Thickener; stabilized molasses
BASE CONCENTRATE
CARRAGEENAN Bm 86 Binder in pellets; carrier in capsules;
polyanionic
CELATOM F 112 Free-flow agent; diatomaceous earth;
prevented conidia in dust sticking to
applicator
Cellulose acetate Bm Binder
CLARCEL FLO M. anisopliae Dispersant; silica powder
Clay F,Bm 120 Dispersant; sunscreen; carrier;
numerous suppliers e.g. Englehard
Corp., George C. Brand Inc.
342 Appendix I
Table 1.4 (Contd.)

Name Organisms' Supplier t Useful information

COMPRITOL Bm 44 Lubricant; glyceryl behenate
Corn syrup R. japonicum Binder
DARVAN NO. 4 78 Dispersant; lignosulphate; inactivates
or flocculates spores if used with
excessive amounts of soaps or
sodium and calcium salts
Dextrin Bm Binder; polysaccharide
DICAPERL HP 920 Bt 49 Floatation agent; perlite powder
DOWANOLTPM Thickener
DRIERITE 118 Desiccant; anhydrous calcium sulphate
powder
EMCOSOY Bm 38 Dispersant; soya polysaccharide
EMCOCEL Bm 38 Dispersant; microcrystalline cellulose
Ethyl cellulose Bm Binder
EUDRAGIT Bm Binder; methacrylate polymer
EX-CEL Bm 87 Dispersant; microcrystalline cellulose
GASILGM2 Conidia 29 Desiccant; silica zerogel powder (water
removed, pores in gell full of air);
synthetic amorphous silicon dioxide
Gelatin V,F Nutrient; protein; partly degraded
collagen; widely used in
pharmaceutical and food industry;
slow-release binder for Bti powder
on rice hulls
GELLAN Mh Humectant
Glucose NPV, blastospores, Binder; stabilizer; phagostimulant;
Bm sugar
Glycerol F,Mh Humectant; nutrient; osmotic
protectant; plasticizer
Gum arabic 57 Binder; vegetable gum; stabilizer
during spray drying; see gum acacia
Gum guar V,Mh, Bm 86 Binder, vegetable gum
Gum tragacanth NVP,Bm 86 Binder; vegetable gum; complex
mixture of polysaccharides
Gum xanthan NVP, Bm,Mh 86 Suspender; bacterial polysaccharide
produced by fermentation of
dextrose with Xanthomonas
campestris; see KELZAN
HIPURE LIQUID FISH Bm 73
GELATIN
Honey Blastospores Binder; stabilizer; rich in sugars
Hydroxypropyl- V,Bm Binder
methylcellulose
INDULIN AT 97 Dispersant; lignin purified from Kraft
pine; free of hemicelluloses
KELGINMV Mh 57 Humectant; alginate; also code LV
Keltose NPV 57 Humectant; alginate
KELZAN Bt 57 Thickener; stabilizer; food grade
xanthan gum; high M r
polysaccharide; extreme
pseudoplasticity
 Appendix I 343
KLX 106 Binder; lipid binder in pellets, solid
phase at 20C; mixture of 47%
partially hydrogenated cottonseed
and soybean oils
LAMEA Mh Humectant; lactamide-,B-
mercaptoethylaminel AMEA blend
Lecithin Mh 86 Emulsifier; e.g. LECITHIN II-S
Leucine, 0, L- Bm Lubricant, amino acid
Lignite flyash Conidia Dispersant
LUBRITAB Bm 38 Lubricant; hydrogenated vegetable oil
METAMUCIL Mh Humectant; methyl cellulose?
Methyl cellulose Bt, NPV, Bm 35 Thickener
Milk, evaporated R. japonicum Binder
MYVEROL 18-D6 Mh Emulsifier; saturated monoacyl
glycerol
MYVEROL 18-99 Mh Emulsifier; unsaturated monoacyl
glycerol
N-GEL Mh Humectant
NALCONTROL Bt, V Thickener
NITRACOAT R. japonicum 72 Binder
NITRIGUM R. japonicum 72 Binder
Paraffin wax Mh Thickener; long-chain paraffins
PEG 6000 Bm Lubricant; polyethylene glycol
Polyacrylamide gel Bm,Mh Humectant
Polyethylene glycol 8000 T. harzianum, F Humectant; high M r polyethylene
glycol
Polyethylene monostearates Bm Lubricant
POLYPLASOONE Bm 43 Dispersant; cross-linked
polyvinylpyrrolidone
Polypropylene carbonate F 76 Activator for BENTONE suspender
Polyvinyl alcohol NPV, Gliocladium 7,130 Humectant; sunscreen; see SMA-2625A
virens
Polyvinylpyrrolidone PVP- Bt, R. japonicum 21 Binder; humectant;
K30 polyvinylpyrrolidone; water-soluble
polymer; protective colloid;
humectant; reduces activity of some
toxins and microorganisms by
forming complexes but not Bt;
hygroscopic
PRECIROL Bm Lubricant; glyceryl palmitostearate
PRIMOJEL Bm 45 Dispersant; sodium starch glycolate
PROPYLTEX Bt Floatation agent; polypropylene
powder
PVP IV A-5-630 R. japonicum 43 Binder; vinylpyrrolidonelvinyl acetate
copolymer
REAX 907 97 Dispersant; low sulphonated Kraft
pine lignin; low free electrolyte
content; low conductivity; neutral
pH
Silica, fumed, TS-720, see F 113 Free-flow agent; hydrophobic dust
CAB-O-SIL sticker and free-flow agent;
improved adhesion of conidia dust
to ants and dispersion of dust forced
into nests under pressure
344 Appendix I
Table 1.4 (Contd.)

Name Organisms* Suppliert Useful information

Silica gel F Desiccant; non-indicating harmless to

conidia, indicator grade lethal
Silica powder F 32 Desiccant; see WESSALON S,
hydrophilic
SILOID Bm Lubricant; silica hydrogel
SIPERNAT Bt Free-flow agent, desiccant
SODIUM ALGINATE S-211 108 Binder
Sodium benzoate St, Bm Lubricant; synergist; preservative
Sodium lauryl sulphate Bm Lubricant
Sorbitol St, NVP, F, Bm, Mh Humectant; stabilizer, sugar
SOY-DEX Mh Humectant
SPESWHITE CHINA CLAY NPV 56 Dispersant; kaolin; fine-grade cheap
filler
Starch Bm Binder; corn, potato, pregelatinized,
rice and wheat types
Stearic acid Bm Lubricant, fatty acid
STEROTEX Bm 18 Lubricant; hydrogenated vegetable oil
Sucrose NPV, nematodes, Binder; phagostimulant; stabilizer;
conidia sugar
SUPERSORB St 105 Polymer; absorbent; slow-release agent
in Sti briquettes
Synthetic glues Bm Binder
TRITON X-IOO St, NPV, F 80 Emulsifier; octylphenol ethoxylate
with ca 10 mol of ethylene oxide;
non-ionic; water-soluble; insoluble
in oil without a coupling agent e.g.
oleic acid; low mammalian toxicity
VEEGUM (regular grade) Dispersant; suspender for water-based
products, complex colloidal
magnesium aluminum silicate for
smectite clays
Vegetable oil St, conidia Carrier, binder
Wallpaper glue R. japonicum Binder
WESSALONS M. anisopliae 32 Dispersant; silica powder
* Abbreviations, see text.
t Suppliers as given in Table I.13.
 Appendix I 345
Table 1.5 Surfactants and wetters

Name Organisms* Supplier t Useful information

AGRAL90 Mh 52 Nonylphenoxy polyethoxy ethanol;
non-ionic; stopped conidia
production
AI-1246 Bt Also codes 1280, 1364 and 1403
Alkyl phenols Bt, V,F
ARLACEL "C" Bt, V, F 9
ATLOX 3404/849 Bt Blend of anionic and non-ionic
surfactants; emulsifier; also codes
848,849 and 949
ATPLUS 300 Bt Also code 448
BIOFILM Bt
Butyldodecylamine NPV Cationic surfactant
hydrochloride
CARGILL INSECTICIDE Bt 19 Thickener; stabilized molasses
BASE CONCENTRATE
Cetyltrimethylammonium NPV, Bt Cationic surfactant; synergist;
bromide disinfectant
CHEVRON X-77 H. thompsonii 79
COLLOIDAL X 77 Bt, V, F 25
CRODOFOS N3N V Emulsifier and gelling agent
Dodecylamine hydrochloride NPV Cationic surfactant
ENHANCE Mh Tallow amine ethoxylate +
nonylphenoxy polyethoxy ethanol
ETALFIX Mh 61 25% CITOWETT in 20% methanol
ETOCAS 30 V 27 Castor oil ethoxylate; liquid; miscible
with acetone; harmless to NPV;
palatable to insects
FOLICOTE 351 Bt 92 Antitranspirant wax emulsion
Hexylamine NPV 37 NPV synergist
HI-SPREAD-CASEIN Bt 90
IGEPAL CO-630 Bt, V, F Nonylphenol ethoxylate; non-ionic
LATER'S SURFACTANT Bt, V, F 58
LOVO 192 Bt 40 Amine stearate
MANOXOLOT 62 Sodium dicotyl sulphosuccinate 20%
solution in a 60% water alcohol
solution, anionic
MAYWOOD SURFACTANT Bt Emulsified animal-derived protein
MILLER NUFILM H. thompsonii 68
MULTIFILM BUFFER X NPV
NONOXYNOL Mh Nonylphenol ethoxylate; non-ionic
NOVEMOL Bt, V, F
Octyldodecylamine NPV Cationic surfactant
hydrochloride
Oxysorbic Sorbitan monolaurate ethoxylate
PETROL AG Bt, V, F
PETRO MORWET EFW Bacillus sphaericus Wetter used in spray-drying
PINOLENE 1882 Bt 68
Polymer-forming terpene PITSULIN V
and emulsifier
PLURAFAC A-24 Bt Alcohol ethoxylate
346 Appendix I
Table 1.5 (Contd.)

Name Organisms* Supplier t UsefuL information

PLYAC Bt, H. thompsonii, 4 Octylphenol ethoxylate + emulsifiable
NPV A-C polyethylene; non-ionic;
compatible with Bt product FORAY
48 B
SANDOVIT Bt, V, F
SAS90 F Spreader
Silicobenzone GV Inactivated 70% of GV on glass,
probably due to pH 2.5; no effect
seen on apple trees
SILWET L77 Mh Organosilicone containing ca 8 mol
ethylene oxide; non-ionic. Can be
toxic to Mh
SOVIX 95 Nonylphenol ethoxylate; non-ionic
SPAN-85 Bt 9 Sorbitan trioleate; emulsifier
SPRAY OIL 435 H. thompsonii
SPREADITE 70 Nonylphenol ethoxylate; non-ionic
SURFINO TG-E 2 Acetylenic diol; non-ionic
SURFYNOL 104 S M. anisopliae 2 46% acetylenic diol on HI-SIL silica
TEEPOL L V 85 Blend of secondary alkyl sulphates and
alkylbenzene sulphonates
TRITON AG-98 Collego 80 Compatible with Colletotrichum
gloeosporioides; non-ionic
TRITON B-1946 NPV 80
TRITON B-1956 Bt 80 Modified phthalic glyceryl alkyd resin
TRITON CS-7 NPV 80 Polyvinyl alcohol
TRITON GR-5 80 Sodium dioctyl sulphosuccinate; 60%
solution in 2-propanol-water (1:1);
anionic
TRITONGR7M Bt 80 Sodium dioctyl sulphosuccinate;
anionic
TRITON X-35 Bt 80 Octylphenol ethoxylate containing ca 3
mol ethylene oxide; non-ionic
TRITON X-45 Bt, V, F, Mh 80 Octylphenol ethoxylate containing ca S
mol ethylene oxide; non-ionic
TRITON N60 Bt 80 Nonylphenol ethoxylate containing ca
6 mol ethylene oxide; non-ionic
TRITON X-lOO Bt, NPV, F 80 Emulsifier; octylphenol ethoxylate
with ca 10 mol ethylene oxide; non-
ionic; water-soluble; insoluble in oil
without a coupling agent e.g. oleic
acid; low mammalian toxicity
TRITON X-1l4 Bt, V, F 80 Octylphenol ethoxylate containing 7-8
mol ethylene oxide; non-ionic
TRITON X-152 NPV 80
TRITON X-ISS Bt, V, F 94 Methylene bisdiamyl phenol ethoxylate
TRITON X-I72 NPV 80
TRITON X-363M Bt 80 Octylphenol ethoxylate; non-ionic
TWEEN 20 Bt, V, F, Mh 86 Sorbitan monolaurate ethoxylate; >
0.1% inhibited thermophilic
actinomycete Thermospora curvata;
can be toxic to Mh
 Appendix I 347
TWEEN 40 Bt,Mh 86 Synergist; sorbitan monopalmitate
ethoxylate; >0.1% inhibited
thermophilic actinomycete
T. curvata; can be toxic to Mh
TWEEN 60 Bt,Mh 86 Synergist; sorbitan monostearate
ethoxylate; > 0.1% inhibited
thermophilic actinomycete
T. curvata; can be toxic to Mh
TWEEN 70 Aschersonia placenta Nutrient
TWEEN 80 Bt, F, V, Mh 86 Synergist; stabilizer; sorbitan
monooleate ethoxylate; < 1.0% no
lasting effect on thermophilic
actinomycete T. curvata; can
stimulate Mh
TWEEN 85 Mh 86 Sorbitan trioleate ethoxylate; non-ionic;
can be toxic to Mh
VATSOLOT Bt, V, F
WITCONOL H-31A Bt Polyethylene glycol oleate containing
ca 8 mol ethylene oxide
Abbreviations, see text.
t Suppliers as given in Table 1.13.
Table 1.6 Stickers and spreaders

Name Organisms Supplier Useful Information

Acrylic polymer Bt, V
ACRYLOCOAT Bt, NPV Acrylics
ACTIPRON V, V. lecanii 16 Spray additive; carrier; sticker;
emulsifiable mineral oil (97%)
Alginic acid Mh,Bm Dispersant, sticker
BIOFILM Bt
BOND Bt, NPV 60 Synthetic latex and alkylphenol
ethoxylate; at 2% no effect on insect
feeding; incompatible with Bt
product FORAY 48 B
CARBOSET Bt Depressed activity of Bt
Carboxymethylcellulose Bt, Plant disease 21 Thickener; sticker; binder; stabilizer;
CMC-R295F control agents sodium carboxymethylcellulose;
water-soluble; food additive
CHEVRON SPRAY Bt 22 Mixture of olefinic and aromatic
STICKER polymers; compatible with Bt
product FORAY 48 B; did not impair
germination of Bt in broth
Citric acid by-product Wetter-sticker; sunscreen; Institute of
Microbiology, Latvia
CUTINOL V7 F 126 Spray additive; emulsifiable rapeseed
oil
DUPONT SPREAD STICKER Bt 36
EMULTEX V.lecanii Spray additive; sticker gum
ENERGOL WT-1 Bt 16 Highly refined light paraffinic oil
ESTEROL 123 V.lecanii 126 Ethyl oleate
Gelatin Bt 73 Sticker for Bti on granules
348 Appendix I

Table 1.6 (Colltd.)

Name Orgallisms' Supplier t Useful illformatioll

GEON LATEX 652 Bt, NPV 11 Colloidal vinyl chloride polymer;

harmless to NPV; insoluble in water;
palatable to larvae
Gum acacia NPV, Bm, Mh 86 Vegetable gum from acacia tree; see
gum arabic
Gum arabic 57 Binder; vegetable gum; stabilizer
during spray-drying; see gum acacia
Gum ghatti Mh 86 Vegetable gum
Gum guar V, Bm, Mh 86 Binder; vegetable gum
Gum karaya NPV, Bm, Mh 86 Vegetable gum from sterculia tree
Gum, locust bean NPV, Mh 86 Vegetable gum from seeds of Ceratonia
siliqua
Gum tragacanth NPV, Bm 86 Binder; vegetable gum; complex
mixture of polysaccharides
Gum xanthan NPV, Bm, Mh 86 Suspender; bacterial polysaccharide
produced by fermentation of
dextrose with Xanthomonas
campestris; see KELZAN
HIGH TACK FISH GLUE Bt
HUGTITE Potato dextrin; polysaccharide
KELGIN HV F 57 Alginate material
KELZAN Bt 57 Thickener; stabilizer; food grade
xanthan gum; high M r linear
polysaccharide; extreme
pseudoplasticity
Larval extract NPV
LATRON CS-7 + or - Bt 80 Alkylphenol ethoxylate + sodium salt
of alkylsulphonated aIkylate;
nonionic; attracts oviposition of
Plutella xylostel/a; spreader-sticker
LYSINE KKL Wetter-sticker; lysine factory, Latvia
MANUCOL DM Mh 57 Sodium alginate; polysaccharide
MANUCOL DMF Mh 57 Alginate; polysaccharide
MANUGELGHB Mh 57 Alginate; polysaccharide; also code
GMB
MANUTEX KPR Mh 57 Alginate; polysaccharide; also code RH
Methyl cellulose Bt, NPV, Bm 35 Thickener; water soluble
Milk, skimmed V, Bt, F, Bm Carrier; sunscreen
Milk, whole NPV
Molasses of peat Sunscreen; wetter-sticker;
experimental product; Institute of
Wood Chemistry; Latvia
MOWIOL V. lecanii Polyvinyl alcohol
NACRYLIC X 4260 Bt Also code X4445
NUFILM 17 Bt, V. lecanii 68 Polymer-forming terpene + surfactant;
compatible with Bt product FORAY
48 B
ORTHO X-77 109 Alkylphenol ethoxylate + glycols, free
fatty acids and isopropanol; non-
ionic; spreader-sticker; attracts
oviposition of Plutella xylostel/a
 Appendix I 349
PELGEL Bm 59 Sticker for seed coating
PLYAC Bt, H. thompson ii, 4 Octylphenol ethoxylate + emulsifiable
NPV A-C polyethylene; non-ionic;
compatible with Bt-product FORAY
48 B
POLYNOX-WSR-N-lO T. harzianum 94
POLYSURF-C GEL Laetisaria arvalis 50 Modified hydroxyethyl cellulose
Polyvinyl alcohol NPV, Gliocladium 7 Humectant; sunscreen; see SMA-2625A
virens
Polyvinylpyrrolidone PVP- Bt, R. japonicum 21 Binder; humectant; water-soluble
K30 polymer; protective colloid; reduces
activity of some toxins and
microorganisms by forming
complexes; hygroscopic
Polyvinyl sticker Bt Adheres semipermanently to car
finishes
RHOPLEX AC 33 Np Bt 80 Acrylic polymer; also code B60A;
blocked flow meter and spinning
cage with Bt
Silica, fumed, TS-720 F 113 Free-flow agent; hydrophobic dust
sticker; improved adhesion of
conidia dust to ants and dispersion
of dust forced into nests under
pressure
SODIUM ALGINATE IG-350 Bm,Mh
Sorbitol Bt, NPV, F, Bm, Mh Humectant; stabilizer; sugar
Sorghum silage Bm Nutrient; sticker
SUTRO Bt
TENAC 85 Spreader-sticker
TOPWET SPREADER Bt 82
STICKER
VICCHEM EOP V.lecanii Emulsifiable ethyl oleate
X-LINK 2873 Bt
Abbreviations, see text.
t Suppliers as given in Table 1.13.
350 Appendix I
Table 1.7 Phagostimulants, synergists and proprietary spray adjuvants

Name Organisms* Supplier! Useful information

Acetamide Bt Reduces feeding; synergizes Bt
ACTIPRON V, V. lecanii 16 Adjuvant; carrier; sticker; emulsifiable
mineral oil (97%)
Alanine, -0, L Bt Synergist; amino acid
p-Aminosalicylic acid Bt Synergist
Almond hulls Bt Phagostimulant
Ammonia salts Bt Synergists; benzoate, hydrogen
phosphate and thiosulphate
Apple pomace Bt Phagostimulant
Aqueous plant extracts NPV Phagostimulant; clover, com, com silk,
cotton
Aqueous plant extracts Mh Germination, hemp sesbania,
Jimsonweed
Ascorbic acid Bt, V, blastospores Synergist; antioxidant; = vitamin C
Asparagine, L- Bt Synergist; amino acid
Aspartic acid, OL- Bt Synergist; amino acid
Borax Bt Synergist
Boric acid Bt, Bm Synergist; lubricant
Bran Nematodes Phagostimulant
Caffeine Bt Feeding inhibitor; 3,7-
trimethylxanthine
Calcium carbonate Rhizobium spp., Bt Synergist
Calcium salts Bt Synergists; acetate, hydroxide, nitrate,
oxide, sulphate
Canavanamine, L- Bt Synergist
Cetyl trimethylammonium NPV, Bt Synergist; disinfectant; cationic
bromide surfactant
Chitinase Bt Synergist
Citrus pulp Bt Phagostimulant
COAX Bt, NPV 20 Phagostimulant adjuvant; sunscreen;
cottonseed flour + sucrose +
vegetable oil + ethoxylated ester
CODACIDE F 65 Adjuvant; emulsifiable rapeseed oil
(95%) used at 5% in water; non-
irritant; non-phytotoxic to most
plants
Copper compounds Bt Synergists; oxide, phosphate, sulphate
and CuC03 x Cu(OHh
Cornmeal Bt, B. bassiana Phagostimulant; carrier; nutrient
Cottonseed flour NPV Phagostimulant
Cottonseed oil NPV, conidia 86 Phagostimulant; vegetable oil, for
prolonged storage dry by adding
anhydrous silica gel
CUTINOL V7 F 126 Adjuvant; emulsifiable rapeseed oil
Cycloserine, 0- Bt Synergist
Cyfluthrin Aspergillus JIavus Insecticide
Cypermethrin M. JIavoviride Synergist; insect growth retardant
advanced locust mortality 48 h; not
toxic to conidia
Cystine, L- Bt Synergist; amino acid
Deltamethrin NPV, Bt 23 Synergist, pyrethroid insecticide
 Appendix I 351
Dichloran Aspergillus flavus
Dimethyl sulphoxide Conidia, Bm Synergist, stabilizer
Dipicolinic acid Bt Synergist
Dipotassium Bt Synergist
hydrogencarbonate
Disodium-,B- Bt Synergist
glycerophosphate
Dodecylamine Bt, NPV 3 Synergist of NPV
EDTA Bt Synergist; ethylenediaminetetra-acetic
acid; chelating agent
ENTICE 30 Phagostimulant adjuvant; wettable
powder contains vegetable flour, oil,
sugar
Fructose NPV Phagostimulant
Fruit pectin Mh Phagostimulant
Fumaric acid Bt Synergist
Gallic acid Bt 86 3,4,5-trihydroxybenzoic acid; common
in cotton leaves; reduces feeding;
low concentration enhances Bt, high
inhibits; denatures proteins;
Glucose NPV, blastospores, Phagostimulant; binder; stabiliser;
Bm sugar
Glutamic acid, L- V Synergist; amino acid
Glutamine, 0, L- Bt Synergist; amino acid
Grape pomace Bt Phagostimulant
Grass meal Bt Phagostimulant
Guanosine Bt Synergist
GUSTOL Bt, V 10 Phagostimulant adjuvant; liquid
flowable contains vegetable flour, oil
and sugar
Histidine, 0, L- V Synergist; amino acid
Isoleucine, L- Bt Synergist; amino acid
Lauric acid Bt Synergist; fatty acid
Leucine, L- Bt Synergist; amino acid
Lysine, 0, L- Bt Synergist; amino acid
Magnesium chloride Bt Synergist
Magnesium sulphate Bt, Bm Stabilizer; synergist
Malic acid Bt Synergist
Methionine, 0, L- Bt Synergist; amino acid
Methyl-p-hydroxybenzoate Bt Synergist; preservative in insect diet;
highly soluble; high concentrations
inhibit Bt, low concentrations
synergize
Milk powder NPV Phagostimulant
MO-MIX NPV 88 Phagostimulant; anti-evaporant;
molasses
NEEMAZAL-T Synergist; neem extract-based
insecticide
Neem oil Conidia Spray additive; vegetable oil
Ornithine, 0, L- Bt Synergist; amino acid
OUTPUT F Adjuvant; mineral oil
PHEAST 1 Phagostimulant adjuvant; wettable
powder contains vegetable flour, oil,
sugar and inactive yeast
Phenylacetic acid Bt Synergist
352 Appendix I
Table 1.7 (Contd.)

Name Organisms' Supplier t Useful information

Phenylalanine, D, L- V Synergist; amino acid

Piperonyl butoxide NPV Synergist; inhibitor of mixed function
oxidase in insects
Potassium salts Bt Synergists; carbonate, hydrogen
carbonate, tartrate
Potato dextrose agar V.lecanii Nutrient adjuvant; nutrient agar
formulation
Proline, L- V Synergist; amino acid
Resorcinol Bt 86 Synergist; plant phenolic compound;
1,3-dihydroxy benzene; synergises
Bt crystal
Sabouraud dextrose agar V.lecanii Nutrient adjuvant. Nutrient agar
formulation
Salicylic acid Bt Synergist
SAN-285-WPG 6 Bt 81 Nutrient adjuvant for microbial
insecticides
Serine, D- and D, L- Bt Synergist; amino acid
Sodium benzoate Bt,8m Lubricant; synergist; preservative
Sodium dodecyl sulphate Bt Synergist; sodium lauryl sulphate
Sodium salts Bt Synergists; acetate, carbonate, formate,
nitrate, nitrite, salicylate
Sodium thioglycollate Bt Synergist; reducing agent; solubilizes
proteins
Sorbic acid Bt Synergist; preservative
Soya flour NPV, F, 8m Phagostimulant; nutrient
Sucrose NPV, nematodes, Phagostimulant; binder; stabilizer;
conidia sugar
Tannic acid Bt Synergist
Thidiazuron Mh Synergist; herbicide
Threonine, D, L- Bt Synergist; amino acid
Tryptophane, D, L- Bt, V Synergist; amino acid
TWEEN 40 Bt, Mh 86 Synergist; sorbitan monopalmitate
ethoxylate; > 0.1% inhibited
thermophilic actinomycete
Thermospora curvata
TWEEN 60 Bt,Mh 86 Synergist; sorbitan monostearate
ethoxylate; > 0.1% inhibited
thermophilic actinomycete T. curvata
TWEEN 80 Bt, F, NPV, V, Mh 86 Synergist; stabiliser; sorbitan
monooleate ethoxylate; < 1.0% no
lasting effect on thermophilic
actinomycete T. curvata
Tyrosine, L- Bt, NPV, V Synergist; amino acid
Urea NPV Synergist; nitrogenous compound
Valine, D, L- Bt Synergist; amino acid
WHEAST NPV Phagostimulant adjuvant
Wheat bran Bt, F, 8m, Mh Phagostimulant; carrier; nutrient
Wheat germ Bt Phagostimulant; nutrient
Zinc salts Bt Synergists; sulphate, sulphite
Abbreviations, see text.
t Suppliers as given in Table 1.13.
 Appendix I 353
Table 1.8 Compounds which stabilize biologicals in formulations (anti-oxidants, nutrients, preservatives,
stabilizers)

Name Organisms* Supplier t Useful information

AGELESS, TYPE Z 117 Iron oxide oxygen-absorber
a-Tocopherol Blastospores 63 Antioxidant; stabilizer in water; harmless
to blastospores; hydrophobic
Arginine Bm,Bt Nutrient; amino acid
Ascorbic acid Bt, V, blastospores Synergist; anti-oxidant; vitamin C
BAYSILlCONE E M. anisopliae Antifoam agent
Benzalkonium chloride Bt Preservati ve
Cereal flour M. anisopliae Nutrient, carrier
Chloramphenicol Bm 86 Antibacterial antibiotic
Corn cob grits Bt, F, Bm, Mh Nutrient, carrier
Corn gluten Bm Nutrient, carrier
Corn meal Bt, B. bassiana Phagostimulant; carrier; nutrient
Crystal Violet Bm Preservative
Cycloheximide Bm 86 Fungicidal antibiotic from Streptomyces
spp.
Dimethyl sulphoxide Conidia, Bm Synergist, stabilizer
Gelatin V,F, Bm Nutrient; protein; partly degraded
collagen; widely used in
pharmaceutical and food industries;
slow release binder for Bti powder on
rice hulls
Gentomycin F 114 Antibacterial antibiotic, 0.05% used in
production of conidia
Glucose NPV, blastospores, Binder; stabilizer; phagostimulant;
Bm nutrient, sugar
Glycerol F,Mh Humectant; nutrient, osmotic protectant
Hank's salt solution Blastospores Stabilizer
Honey Blastospores Binder; stabilizer; nutrient; rich in sugars
Horse serum Blastospores Stabilizer
Hydroxyethyl starch Blastospores 86 Stabilizer in water; harmless to
blastospores
Inositol NPV, blastospores Stabilizer; B-vitamin; sugar; harmless to
insect and virus
KELZAN Bt 57 Thickener; stabilizer; food grade xanthan
gum; high M r linear polysaccharide;
extreme pseudoplasticity
LABRAFIL M. anisopliae Anti-oxidant; glyceryl monooleate
ethoxylate
Lactose Bt, blastospores, Stabilizer; sugar
Pseudomonas spp.
LECITHIN II-S Blastospores 86 Stabilizer; from soybean; 10-20%
phosphatidykholine; stabilizer in
water; hydrophobic; harmless to
blastospores
LECITHIN IV-S Blastospores 86 Stabilizer; purified from LECITIN II-S
Liquid paraffin F Stabilizer; refined mineral oil for
medicinal use, mixture of alkanes with
> 12 C atoms/molecule
Magnesium sulphate Bt, Bm Stabilizer; synergist
Malt broth Mh Nutrient
Maltose F, Bm Stabilizer; nutrient; sugar
MANUCOLDH Blastospores Stabilizer; sodium alginate
Methyl-p-hydroxybenzoate Bt Synergist; preservative in insect diet;
highly soluble; high concentrations
inhibit Bt, low synergize
354 Appendix I
Table 1.8 (Contd.)

Name Organisms' Supplier! Useful information

Molasses v, Bt, Bm 88 Nutrient; sunscreen; carrier; e.g. MO-
MIX; dark brown, viscous residues
from sugar production; rich in sugars,
particularly sucrose
Oat grains Bm Nutrient
PEG 200 Stabilizer; low-Mr polyethylene glycol
Peptone Blastospores Stabilizer
Potassium chloride Conidia Stabilizer
Potassium dihydrogen Blastospores Stabilizer
phosphate
Potassium sorbate Bm 76 Food grade antifugal preservative
Proprionates Bt Preservative
Pyrophyllite Bm,Mh Nutrient
Ringer's solution Blastospores Stabilizer
Rose Bengal Bm Preservative
Sabouraud broth Blastospores 34 Stabilizer; nutrient; e.g. by Difco
Laborarories
Sodium azide Bm Preservative
Sodium benzoate Bt, Bm Lubricant; synergist; preservative
Sodium chloride Conidia Stabilizer
Sodium glutamate F Osmotic protectant
Sodium silicate Conidia Stabilizer
SORBA SPRAY ZIP Stabilizer
Sorbic acid Bt Synergist; preservative
Sorbitol Bt, NVP, F, Bm, Mh Humectant; stabilizer; sugar
Sorghum powder Pandora delphacis Nutrient
Sorghum silage Bm Nutrient; sticker
Soya flour NVP,F Phagostimulant; nutrient
Streptomycin sulphate F Antibacterial antibiotic; harmless to
blastospores at 25 fLg/ml
Sucrose NPV, nematodes, Binder; phagostimulant; stabilizer; sugar
conidia
Trehalose Bm Stabilizer; sugar
Tributyl citrate Conidia Stabilizer
TWEEN 80 Bt, F, V, Mh 86 Synergist; stabilizer; sorbitan monooleate
ethoxylate; < 1.0% no lasting effect on
thermophilic actinomycete Thermospora
curvata
Wheat bran Bt, F, Bm Phagostimulant; carrier; nutrient; bait
Wheat flour Mh,Bm Nutrient
Wheat germ Bt Phagostimulant; nutrient
Wheat straw Bm Nutrient
Xylene Bt Bacteriostatic preservative; stabilizer in
aqueous emulsions; application rate in
spray (1.7 p.p.b.) is 59000 times below
occupational health hazard rate (100
p.p.m.)
Abbreviations, see text.
t Suppliers as given in Table 1.13.
 Appendix I 355
Table 1.9 Sunscreens (cosmetic sunscreens, dyes, tracer dyes)
Name Organisms* Supplier t Useful information
Acid Black 48 V 86 Dye
Acid Orange 8 V 86 Dye
Acridine Yellow V 86 Dye
Acriflavin Bt 41 Dye; 3,6-diamino-l0-methyl acridinium;
cationic; forms photoprotective complex
with Bt crystal
Alcian Blue 8GX NPV 86 Dye
Alizarin Yellow R V 86 Dye; Mordant Yellow 3R or Mordant Orange
1
Alkali Blue V 86 Dyes; codes Alkali Blue 4B = Acid Blue 110
and Alkali Blue 6B = Acid Blue 119
Azocarmine V 86 Dyes; codes B = Acid Red 103, and G = Acid
Red 101
Benzilidine NPV Cosmetic sunscreen; water-soluble; harmless
sulphonic acid to plants and virus; absorptance high in
UVB
Benzopurpurin 4B V 86 Dye; Direct Red 3
Benzyl cinnamate Conidia 3 Cosmetic sunscreen; absorptance 220-300 nm
and 310-360 nm in cottonseed oil; 270-350
nm in groundnut oil + Shellsol K
Berberine sulphate Bt Cationic dye; alkaloid; high mammalian
toxicity
Bismark Brown V 86 Dyes; codes R = Basic Brown 4 and Y = Basic
Brown 1, Basic Brown G, GX, GXP,
Excelsior Brown, Leather Brown,
Manchester Brown, Phenylene Brown,
Vesuvin
Brilliant Blue G V 86 Blue dye, selectively stains proteins; =
COOMASSIE BRILLIANT BLUE G;
Brilliant Blue R V 86 Blue dye, selectively stains proteins; =
COOMASSIE BRILLIANT BLUE R
BRILLIANT Bt 75 Tracer dye; no harm to spores caused by 21-
SULFOFLAVINE day exposure to 1% aqueous solution
Brilliant Yellow V 86 Dye; Direct Yellow 4
Buffalo Black dye V 86 Dye; presumably Buffalo Black NBR =
Naphthol Blue Black or Amido Black lOB
CHIMMASORB 81 Conidia 24 Cosmetic sunscreen; = CYASORB UV 531
Chrome Axurol S V 86 Dye; Mordant Blue 29
Chrysophenine V 86 Dye; Direct Yellow 12
CIBACRON BLUE V 86 Dye; presumably CIBACRON BLUE 3GA =
Reactive Blue 2
CIBACRON V 24 Dye
YELLOW
Congo Red Conidia, Bt, V 3 Dye; Direct Red C, R, or Y; Congo Red 4B;
peaks 220, 340 and 500 nm; harmless to
insects and virus in diet; decreased virus
activity after storage with the virus
Curcumin V 86 Dye from Curcuma longa (turmeric); =
Natural Yellow 3; 1, 7-bis (4-hydroxy-3-
methoxyphenyl)-1,6-heptodiene-3,5-dione
356 Appendix I
Table 1.9 (Contd.)

Name Organisms* Suppliert Useful information

CYASORB UV 531 Conidia 24 Cosmetic sunscreen; = CIMMASORB 81; 2-

hydroxy-4-(octyloxy-phenyl)-
phenylmethanone; absorption peaks at
300-390 nm in cottonseed oil; 250-400 nm
in groundnut oil + Shellsol K
Direct Red 28 NPV 3 Dye
Direct Red 81 NPV 3 Dye
Disperse Blue 14 NPV 86 Dye
Disperse Orange 11 V 86 Dye
EAR XB 400 Bt 24 Tracer dye; presumably ERIO ACID RED XB
400
ERIO ACID RED XB Bt 24 Tracer dye; disodium formyl-m-
100 benzenedisulphonic acid; high
absorptance above 280 nm; no harm to
spores by 21-day exposure to 1% aqueous
solution; also not toxic to spruce budworm
la~vae; not bacteriostatic at 300 mg/l; not
biodegradable
Erio Yellow Bt 24 Tracer dye; no harm to spores by 21-day
exposure to 1% aqueous solution
2-Ethoxyethyl-p- NPV Cosmetic sunscreen
methoxy-
cinnamate
2-Ethylhexyl NPV Cosmetic sunscreen
salicylate
Ethyl cinnamate Conidia 3 Cosmetic sunscreen; absorption peaks 310-
370 nm in cottonseed oil; 270-340 nm in
groundnut oil + Shellsol K
Ethyl trans- Conidia 86 Cosmetic sunscreen; oil compatible; harmless
cinnamate to conidia
EUSOLEX 232 Bt, V 63 Cosmetic sunscreen; 2-phenylbenzimidazole-
5-sulphonic acid; water-soluble
EUSOLEX 4360 Bt, V, conidia 63 Cosmetic sunscreen; 2-hydroxy-4-
methoxybenzophenone; oil-soluble;
absorption peaks at 300-390 nm in
cottonseed oil; 260-400 nm in groundnut
oil + Shellsol K
EUSOLEX 6300 Conidia, Bt, V 63 Cosmetic sunscreen; 3-(4-
methylbenzylidene)-camphor; oil-soluble;
absorption peaks at 280-380 nm in
cottonseed oil; 250-390 nm in groundnut
oil + Shellsol K
EUSOLEX 8020 Conidia 63 Cosmetic sunscreen; 4-isopropyl-dibenzoyl
methane; absorption peak 270-410nm in
groundnut oil + Shellsol K
EUSOLEX 8021 Conidia, V 63 Cosmetic sunscreen; 61 % Eusolex 8020 +
39% Eusolex 6300; absorption peaks 270-
390 nm in groundnut oil + Shellsol K
Fast Blue V 86 Fluorescent neuronal tracer
Fluorescein Bt Dye
 Appendix I 357

GRAESSORB D Conidia 71 Cosmetic sunscreen; diethanolamine-4-

methoxycinnamate; water-compatible;
harmless to conidia; peaks at 320-350 run
in cottonseed oil; 250-400 run in groundnut
oil + Shellsol K
GRAESSORB S Conidia 71 Cosmetic sunscreen octyl-salicylate; oil-
compatible; harmless to conidia;
absorption peaks at 320-350 nm in
cottonseed oil; 280-370 nm in groundnut
oil + Shellsol K
Haematoxylin V 128 Dye for various cytological stains; Natural
Black 1
Homomenthyl NPV Cosmetic sunscreen; low UVB absorption;
salicylate water-soluble
Indigo Carmine V 86 Dye; Acid Blue 64; 5'5-indigosuphonic acid
Lissamine Green B V 86 Dye; Acid Green 50
Maximillion Brilliant Bt 75 Tracer dye; no detectable effect on Bt activity
Flavine BSF or physical characteristics of spray; no
harm to spores by 21-day exposure to 1%
aqueous solution
Maximillion Brilliant Bt 75 Tracer dye; no harm to spores by 21-day
Pink exposure to 1% aqueous solution
Mercurochrome V 86 Dye; Merbromin; 2',7'-dibromo-5'-
(hydroxymercuri)-fluorescein
Methyl Green Bt 63 Cationic dye, absorption peak in water 630-
635 run
Methyl Orange NPV 63 Indicator dye; Acid Orange 52; absorption
peak (pH 3.1) 501-504 run, absorption peak
(pH 4.4) 467-471 run
Methyl Red V 63 Indicator dye; absorption peak (pH 4.5) at
523-526 run, absorption peak (pH 6.2) at
427-437 run
Mexenone Conidia 71 Cosmetic sunscreen; 2-hydroxy-4-methoxy-
4-methyl benzophenone; absorption peaks
at 310-390 run in cottonseed oil; 270-
400 nm in groundnut oil + Shellsol K
Mordant Blue 1 NPV 86 Dye = Chrome Azurol B
Mordant Brown 33 NPV 86 Dye
Neutral Red V 63 Indicator dye, dark red at pH 6.8 and yellow
orange at pH 8.0 in aqueous solution
Nigrosin Bt Tracer dye; no harm to spores by 21-day
exposure to 1% aqueous solution
NIPASORB D Conidia 71 Cosmetic sunscreen; water-compatible;
harmless to conidia
Orange IV V 86 Dye; = Acid Orange 5 or Tropaeolin 00
Orcin V 86 Dye; Orcinol; 5-methylresorcinol; glycosides
and supholipids
Oxybenzone NPV; conidia 86 Cosmetic sunscreen; 2-hydroxy-4-
methoxybenzophenone; see Eusolex 4360;
harmless to conidia; oil-compatible;
absorption peaks 270-390 run in
groundnut oil + Shellsol K
358 Appendix I
Table 1.9 (Contd.)

Name Organisms* Suppliert Useful information

PARSOL 1789 Conidia 47 Cosmetic sunscreen; butyl-methoxy-
dibenzoyl methane; oil-compatible;
harmless to conidia; absorption peak 270-
350 nm in groundnut oil + Shellsol K
PARSOLM<!:X Conidia 47 Cosmetic sunscreen; octyl-p-
methoxycinnamate; oil-compatible;
harmless to conidia; absorptance peaks
340-370 nm in cottonseed oil; 270-360 nm
in groundnut oil + Shellsol K
Reactive Blue 4 V 86 Dye
Rhodamine B Bt, R. japonicum 63 Cationic dye; tracer dye; no harm to spores
by 21-day exposure to 1% aqueous
solution; mutagenic to Salmonella
Saturn Yellow Tracer dye; not a true solution, some droplets
1 j.m in diameter would not contain a grain
of dye
URANINE Bt Tracer dye; no harm to spores by 21-day
exposure to 1% aqueous solution
UVINAL DS-49 Bt 12 Tracer dye; 2,2'-dihydroxy-4,4'-dimethoxy-
5,5' disulphobenzophenone; no harm to
spores by 21-day exposure to 1% aqueous
solution
UVINUL D49 Bt, conidia 12 Cosmetic sunscreen; sodium 2,2'-dihydroxy-
4,4'-dimethoxy-5-sulphobenzophenone;
absorption peaks 250-380 nm in
groundnut oil + Shellsol K; compatible
with Bt
UVITEX ERN-P Bt 21 Cosmetic sunscreen; bis-benzoxalate
ethylene; non-ionic; high absorptance
above 280 nm; no harm to spores by 21-day
exposure to 1% aqueous solution
* Abbreviations, see text.
t Suppliers as given in Table 1.13.
 Appendix I 359
Table 1.10 Sunscreens (optical brighteners, reflectors)

Name Organisms* Supplier t Useful information

ACLARAT 8678 V 81 Optical brightener
Aluminium powder V Reflector
BLANKOPHOR Conidia 24 Optical brighteners; codes DML, HRS, LPG and
RKH; water-compatible; marginally water-
soluble; harmless to conidia
BLANKOPHOR BBH Conidia, V, Bt 17 Optical brightener; = Tinopal LPW; water-
compatible; marginally water-soluble; harmless
to conidia; strongly synergizes NPV
BLANKOPHOR BSU Conidia 17 Optical brightener
CALCOFLUOR V 6 Optical brighteners; codes LD and RWP
CALCOFLUOR WHITE NPV, conidia 86 = TlNOPAL LPW; di-sodium 2,2'-(1,2-ethenediyl)
M2 bis (5(4-(4-morpholinyl)-6-(phenylamino)-1,3,5-
triazin-2y-yl)amino-benzenesulphonic acid
Columbia Blue V 31 Optical brightener
Fluorescent brightner 28 V See TlNOPAL LPW
INTRAWITE V 28 Optical brighteners; codes ABL, EBF, ERN and
WGS
INTRAWITE CF V 28 Optical brightener; synergises NVP
INTRAWITE CF V 28 Optical brighteners; codes BS and BSB
LEUCOPHOR
LEUCOPHOR V 81 Optical brighteners; codes EFR, EHB, KNR, PAB,
PAL, PAT and WGS
LEUCOPHOR BS V 81 Optical brighteners; stilbenes, pH 7.6; synergize
NVP; also code BSB
P167 Conidia Optical brightener
PHORWITE AR V 69 Optical brightener; 2,2'-(1,2-ethenediyl)bis (5(4-
methylaminol-/3-(phenylamino)-1,3,5-triazin-2 yl
arninobenzene sulphonic acid; synergizes NPV;
pH 8.6
PHORWITE BKL V 69 Optical brightener; pH 4.2
PHORWITE BRU V 69 Optical brightener; pH 5.5
PHORWITECL V 69 Optical brightener; stilbene
SYNACRIL WHITE V 51 Optical brightener; pH 1.7
TINOPAL CBS-X V 24 Optical brightener; a naphthotriazole stilbene;
whitening agent; harmless to NPV; absorptance
good <380 nm; water-soluble; neutral pH
TINOPAL DCS V 24 Optical brightener; a stilbene fluorescent whitening
agent; harmless to NPV; absorptance good
< 380 nm; water-soluble; neutral pH
TlNOPAL LPW Conidia, NPV 86 = BLANKOPHOR BBH, CALCOFLUOR WHITE,
fluorescent brightener 28; a stilbene optical
brightener; high absorptance >280nm; pH 8.6;
synergizes NPV
TlNUVIN 328 Conidia 24 Optical brightener; 2-(2-hydroxy-3,5-di-tert-amyl-
phenyl)-2H-benzotriazole; absorptance peaks
260-400 om in cottonseed oil; 280-420 om in
groundnut oil + Shellsol K
TINUVINP Conidia 24 Optical brightener; 2-(2-H-benzotriasol-2-yl)-4-
methylphenone; absorptance peaks at 310-
390 nm in cottonseed oil; 270-380 nm in
groundnut oil + Shellsol K
Titanium dioxide NPV Reflector

* Abbreviations, see text.

t Suppliers as given in Table 1.13.
360 Appendix I
Table 1.11 Bioorganic sunscreens (B vitamins, amino acids, anti-oxidants, nitrogenous compounds,
proteins)

Name Organisms' Supplier t Useful information

Adenine v Amino acid
Albumins V Class of blood proteins; many e.g. bovine
serum albumin (BSA) available in pure
form at relatively low price
Allantoin V Nitrogenous compound
p-Aminobenzoic acid Bt, NPV, Steinemema B vitamin; harmless to insect and virus,
feltiae absorptance between 220 and 320 nm, max
at 280 nm
Amyl-dimethyl-p- V B vitamin
aminobenzoic acid
Apterin NPV Nitrogenous compound
Ascorbic acid Bt, V, blastospores Synergist; antioxidant; vitamin C
Brewer's yeast V Rich in protein, amino acids and nitrogenous
compounds
Casein V, Bt Inexpensive protein; frequently used in
culture media and buffers
Catalase Bt, V Protein with enzymatic activity; antioxidant
because it degrades peroxides
Choline chloride NPV B vitamin; harmless to insect and virus
Egg albumen A 5253 V 86 Protein; see albumins
Ethoxylated p- V B-vitamin
aminobenzoic acid
Folic acid Bt, NPV B-vitamin; harmless to insect and virus;
naturally occurring zoochrome,
absorptance at 22D-400nm, peaks 280 and
350nm
Gelatin V,F Nutrient, protein; partly degraded collagen;
widely used in pharmaceutical and food
industry; slow release binder for Bti
powder on rice hulls
Glutamic acid, L- V Synergist; amino acid
Guanine V Nitrogenous compound
Haemolymph V Rich in proteins and nitrogenous compounds
Histidine, D,L- V Synergist; amino acid
Hypoxanthine NPV Nitrogenous compound
Inositol NPV, blastospores Stabilizer; B vitamin; harmless to insect and
virus
iso-Octyl-p- V B vitamin
aminobenzoic acid
iso-Octyl-p-dimethyl- NPV Cosmetic sunscreen; B vitamin
aminobenzoic acid
Milk, peptonized V 34 Protein; complex mixture of water-soluble
peptides generated by partial degradation
of casein, a major constituent of milk
Nicotinic acid NPV B vitamin; harmless to insect and virus
Pantothenic acid NPV B vitamin; harmless to insect and virus
Peroxidase V Protein; enzyme; acts as antioxidant because
it degrades peroxides
Phenylalanine,D,L- V Synergist; amino acid
 Appendix I 361
Phenylthio- V 86 Anti"oxidant
carbamide
Proline, L- V Synergist; amino acid
Propyl gallate V Anti-oxidant
Protein hydrolysates Bt See gelatin, peptonized milk, soya
hydrolysate
Pyridoxine NPV B vitamin; harmless to insect and virus
Riboflavin Bt, NPV B vitamin; a fluorochrome, naturally
occurring zoochrome in insects, harmless
to insect and virus
Sodium ascorbate V Anti-oxidant; see ascorbic acid
Soya hydrolysate V See protein hydrolysates
Superoxide V Protein; enzyme; acts as anti-oxidant because
dismutase it degrades peroxides
Thiamine NPV B vitamin; harmless to insect and virus
Tryptophane, D,L- Bt, V Synergist; amino acid
Tyrosine, L- Bt, V Synergist; amino acid
Urea V Synergist; nitrogenous compound
Uric acid Protozoa, NPV, Nitrogenous compound
Steinernema feltiae
Wheat gluten Bt,Mh 66 Rich in proteins
Xanthine NPV Nitrogenous compound
Xantopterin NPV B vitamin; harmless to insect and virus
Abbreviations, see text.
t Suppliers as given in Table I.13.
362 Appendix I
Table 1.12 Miscellaneous sunscreens

Name Organisms* Supplier t Useful information

Blowfly faeces Protozoa
Carbon black Bt, V Insoluble; absorbent; very strong absorption
270-350 nm
CARBON TYPE RB V 77 Carbon black; high porosity activated carbon
Charcoal V Carbon black
Citric acid by-product Wetter-sticker; Institute of Microbiology, Latvia
COAX Bt, NPV 20 Phagostimulant; cottonseed flour + sucrose +
vegetable oil + ethoxylated ester
Corn starch V, B. bassiana 26 Carrier; see e.g. MlRAGEL 463
DAR-MOL, DE-MOL, NPV See molasses
DRI-MOL MOLASSES
2,2-Dihydroxy-4- Conidia 111 Oil-compatible; harmless to conidia
methoxy-
benzophenone
Dodecylamine Bt, V 3 Synergist of NPV
FLORISIL 60-100 mesh Conidia 108 Magnesium silicate; oil-compatible; low
solubility in oil; harmless to conidia
2,2-Hydroxy-4- Conidia 111 Oil-compatible; harmless to conidia
octobenzophenone
IMC 90-001 UV screen V SHADE
INDIA INK V Carbon black
Keltose NPV 57 Humectant; alginate
Lignin suphonic acid Bt, V 15 Sodium lignosulphonate; absorptance poor, low
peak at 280 nm; see RAYMIX POWDER
LIGNOSITE AN 46 Lignosulphonate
Melanin from Bt Dark brown pigments produced by S. lividans in
Streptomyces lividans presence of tyrosine gene; insoluble in water,
acid and common organic solvents
Melanins V Dark brown pigments in e.g. bacteria, insects
and vertebrates; natural sunscreens; see
melanin from S lividans
3-(4-Methylbenzylidene)- NPV
camphor
Molasses V, Bt, Bm 88 Nutrient; carrier; e.g. MO-MIX; dark brown,
viscous residues from sugar production; rich
in sugar, particularly sucrose
Molasses of peat Wetter-sticker; experimental product; Institute
of Wood Chemistry; Latvia
ORZAN LS-50 V 55 Lignosulphonate
Polyvinyl alcohol NPV, Gliocladium 7 Humectant; see SMA-2625A
virens
PROTEC-lOO NPV
RAYMIX L-3 V 55 Wood derivative; lignosulphonates; water-
soluble; low surface tension, acidic pH
RAYMIX POWDER V 55 Wood and bark derivative; lignosulphonates;
water-soluble; low surface tension; neutral
pH
SHADE Bt, V 81 =!MC 90-001 = I+T-193; natural polyflavonoid;
water-soluble buffer; absorbs peroxide
radicals; harmless to NPV; low absorptance
peak at 280 nm
Sulisobenzone GV 2-Hydroxy-4-methyl-5-sulphobenzophenone;
1% aqueous pH 2.5 dried on glass inactivated
(70%) GV; less inactivation in field
UVAL V 67
Abbreviations, see text.
t Suppliers as given in Table 1.13.
 Appendix I 363
Table 1.13 Suppliers' names

Reference Company Location

No.
1 AgriSense Fresno, California, USA
2 Airproducts pIc Walton-on-Thames, Surrey, UK
3 Aldrich Chemical Company, Inc. Milwaukee, Wisconsin, USA
4 Allied Chemicals
5 American Casein Company Burlington, New Jersey, USA
6 American Cyanamid Wayne, New Jersey, USA
7 Arco Chemical Company
8 Atlas Chemical
9 Atlas Powder Company
10 Atochem North America, Inc. Philadelphia, Pennsylvania, USA
11 BF Goodrich Chemical Co. Kitchener, Ontario, Canada
12 BASFAG Ludwigshafen, Germany
13 Local retailers Benin, West Africa
14 Bevaloid Ltd Flemingate, Beverley, UK
15 Borregaard Industries Sarpsborg, Norway
16 BP Sunbury on Thames, UK
17 Burlington Chemical Burlington, North Carolina, USA
18 Capital City Products Co. Columbus, Ohio, USA
19 Cargill
20 CCT Corporation Lichfield Park, Arizona, USA
21 Chemical Development of Canada Ltd Montreal, Quebec, Canada
22 Chevron Chemicals
23 Ciba Geigy See Novartis (98)
24 Ciba Geigy Speciality Chemicals Basel, Switzerland
25 Colloidal Products
26 CPC International Englewood Cliffs, New Jersey, USA
27 Croda Chemical
28 Crompton & Knowles Charlotte, North Carolina, USA
29 Crossfield Chemicals
30 Custom Chemicides Fresno, California, USA
31 Day-Glo Colour Corporation Cleveland, Ohio, USA
32 Degussa AG Frankfurt-am-Main, Germany
33 Diamond Shamrock
34 Difco Laboratories Detroit, Michigan, USA
35 Dow Chemical
36 Du Pont Wilmington, Delaware, USA
37 Eastman Kodak Rochester, New York, USA
38 Edward Mendell Co. Patterson, New Jersey, USA
39 Exxon
40 Fisons Ltd See AgrEvo
41 Fluka Buchs, Switzerland
42 FMC Corporation Philadelphia, Pennsylvania, USA
43 GAF Corporation Wayne, New Jersey, USA
44 Gattefosse Corporation Montvale, New Jersey, USA
45 Generichem Corporation Little Falls, New Jersey, USA
46 Georgia-Pacific Bellingham, Washington, USA
47 Givaudan and Co. Ltd Surrey, UK
48 K. B. K. Greef Chemicals Ltd Croydon, UK
49 Grefco Address not given
364 Appendix I
50 Hercules Inc. Wilmington, Delaware, USA
51 ICI America Inc Wilmington, Delaware, USA
52 ICI See Zeneca (129)
53 Illinois Cereal Mills Paris, Illinois, USA
54 Ingradient Technology Pennsauken, New Jersey, USA
55 m Rayonier Stamford, Connecticut, USA
56 ECC St Austel, Cornwall, UK
57 Kelco International Ltd London, UK
58 Later Chemical
59 Liphatech Inc. Milwaukee, Wisconsin, USA
60 Loveland Industries Inc. Greeley, Colorado, USA
61 Maag See Novartis (98)
62 Manchem Ltd
63 E. Merck Darmstadt, Germany
64 Microbio Rhizogen Co.
65 Microcide Ltd Bury St Edmund, Suffok, UK
66 Midwest Grain Production Inc. Pekin, Illinois, USA
67 Miles
68 Miller Chemical and Fertilizer Corp. Hanover, Pennsylvania, USA
69 Mobay Chemical Corp. Union, New Jersey, USA
70 Murphy No longer exists.
71 Nipa Laboratories Clwyd, UK
72 Nitragin Co. Address not given (USA)
73 Norland Products Inc. Address not given
74 Novo Nordisk Bioindustrials Danbury, Connecticut, USA
75 Organic Dyestuffs East Providence, Rhode Island, USA
76 Pfizer Ltd Sandwich, Kent, UK
77 Pittsburg Activated Carbon Co. Pittsbur& Pennsylvania, USA
78 R.T. Vanderbilt Company, Inc. Norwalk, Connecticut, USA
79 Ring-Around Products
80 Rohm & Haas Co. Philadelphia, Pennsylvania, USA
81 Sandoz See Novartis (98)
82 Schering See AgrEvo (110)
83 Seatons Hull, North Humberside, UK
84 Seven Seas Ltd
85 Shell Direct Ltd Southampton, UK
86 Sigma Chemical Co. St Louis, Missouri, USA
87 SITCO Bound Brook, New Jersey, USA
88 Southern States Corporation, Inc. Richmond, Virgina, USA
89 Stanley Manufacturing Decatur, Illinois, USA
90 Stauffer Chemical Co. No longer exists
91 Steetley Minerals Ltd Worksop, UK
92 Sun Oil Co.
93 Teneco Corporation
94 Union Carbide Danbury, Connecticut, USA
95 Vitax Address not given (UK)
96 W.R. Grace & Compariy Columbia, Maryland, USA
97 Westvaco Chemical Division Charleston Heights, South Carolina, USA
98 Novartis Basel, Switzerland
99 Armak Co. McCook, Illinois, USA
100 BDH Ltd Poole, UK
101 Hopkins Agricultural Products Division Madison, Wisconsin, USA
102 Matheson, Coleman and Bell Norwood, Ohio, USA
 Appendix I 365
Table 1.13 (Contd.)

Reference Company Location

No.

103 pfaltz and Bauer, Inc. Stamford, Connecticut, USA

104 Mallinckrodt, Inc. St Louis, Missouri, USA
105 Supersorbent Co. Lumberton, North Carolina, USA
106 Durkee International Foods Louisville, Kentucky, USA
107 Staley Inc. Decatur, Illinois, USA
108 Fisher Scientific Co. Springfield, New Jersey, USA
109 Valent Walnut Creek, California, USA
110 AgrEvo Frankfurt-am-Main; Germany: Saffron
Walden, Essex, UK
111 Mycotech Butte, Montana, USA
112 Eagle-Picher Minerals Reno, North Virginia, USA
113 Cabot Tuscola, Illinois, USA
114 TechAmerica Inc. Elwood, Kansas, USA
115 Ross and Rowe Inc. USA
116 ICN Costa Mesa, California, USA
117 Misubishi International Corp., Food Division New York, USA
B
118 W.A. Hammond Drierite Company Xenia, Ohio, USA
119 Abbott Laboratories Chicago, Illinois, USA
120 Englehard Corporation Edison, New Jersey, USA
121 George C. Brandt, Inc. Denver, Colorado, USA
122 Colorcon Inc. West Point, Pennsylvania, USA
123 W.R. Grace of Canada, Ltd. Ajax, Ontario, Canada
124 Activated Carbon Co. Pittsburg, Pennsylvania, USA
125 Allied Colloids Australia Proprietary Ltd Address not given (Australia)
126 Victorian Chemical Co. Pty. Ltd Richmond, Victoria, Australia
127 Trifolio-M GmbH Lahnau, Germany
128 Boehringer Bioproducts Heidelberg, Germany
129 Zeneca Agrochemicals Fernhurst, Surrey, UK
130 Matheson, Colman and Bell Norwood, Ohio, USA
APPENDIX II: SPRAY APPLICATION
CRITERIA
Keith A. Jones
CONTENTS
11.1 Spray application rates, droplet size and number of
droplets
11.2 Effect of formulation and other parameters on droplets
11.3 Interaction of droplet size and particulate active
ingredients
References
This AppendiX summarizes in tabular and
numerical form the physical criteria for appli-
cation of different types of spray, with part-
icular reference to the application of microbial
pesticides. The first section deals with physical
characteristics of droplets. The second section
describes some effects of suspension carriers
and formulation additives on droplet forma-
tion, size and behaviour. The final section
relates droplet size and placement to the action
of organisms, and illustrates the great impor-
tance of appropriate formulation and applica-
tion parameters on successful use of these
organisms in the field. For more information
about the general aspects of spray physics, the
reader is referred to Matthews (1992).
11.1 SPRAY APPLICATION RATES, DROPLET
SIZE AND NUMBER OF DROPLETS
Classification of volume application rates var-
ies with target (field crop, forest, etc.). A
broad guide is given in Table 11.1 and more
information in section 2.2.2b.
367
369
372
374
Droplet size is often measured in terms of
number mean diameter (nmd) or, more
usually, volume mean diameter (vmd).
These are, respectively, the diameter at
which half the number, or half of the spray
volume, is smaller. Bateman (1991) classifies
sizes as in Table 11.2.
Spray applicators will produce a range of
droplet sizes. By formulation and good
sprayer design, this range of droplet sizes
can be minimized. Fig. 11.1 gives an example
of a droplet spectrum produced by a commer-
cial sprayer.
Smaller droplets result in higher numbers
of droplets per unit area. Fig. II.2 shows the
number of droplets that would land on a flat
surface, assuming that there is no loss through
evaporation or drift. Usually a spray will not
be applied to a flat surface, but to a complex
surface such as a crop. Leaf area indices for a
crop (l.a.i. = ratio of leaf surface area of a
plant to area of ground occupied by the
plant) will give an idea of the maximum num-
ber of droplets that could impinge on the sur-
368 Appendix II
Table 11.1 Volume application rates (l/ha)

High volume HV 600-1000+

Medium volume MY 200-600
Low volume LV SO-200
Very low volume VLV 5-S0
Ultra low volume ULV <S

Table 11.2 Droplet classification (Bateman, 1991)

Drop size (/-lm) Description

1O-2S Fine aerosol (fine mist) for flying insects
2S-S0 Aerosols (mist) for drift and air-blast spraying
SO-I00 Standard mist or fine spray for oil-based ULV spraying
7S-lS0 Fine spray suitable for water-based controlled-droplet
applications (CDA) or higher volume applications
IS0-300 Fine/coarse spray suitable for herbicides
2S0-S00 Coarse spray suitable for soil treatment and avoidance of drift

face of leaves etc. (for example, for an l.a.i. of 4
the number of droplets from Fig. 11.2 should
be divided by 4). However, l.a.i. does not take
into account such factors as upper and lower
leaf surfaces (the latter can often be the target
site of action for a beneficial microorganism)
or losses resulting from lack of impaction or
retention of droplets (section 2.2.2b).
Under operational conditions, droplets
are subjected to evaporation, which is influ-
enced by temperature, relative humidity
and droplet size (surface area/volume ratio).
The surface/volume ratio (s/v) is calculated
as:
S/V = 4nr /(4/3)7r? = 3/r

CIl
CIl
ell
"0
.5
~ 0 +---~.......L""""',.J--'-r''-
~ 50 100
~ Droplet diameter (~m:log scale)
Droplet volume (pi)
Ii.. , 'Ii
Q)

Q. I, "'I i i" "Ii I i i i Ii i "'1 ' , i Ii "'I , I i 1111 i

1 10 100 1000 10,000 100,000
Expected number of particles per droplet
12
in a formulation containing 10 particles/I

Figure 11.1 Droplet size distribution of a Micron Ulva+, 7000r.p.m., blank carrier oil, Odina EL and
Shellsol T 1:1 (Bateman, in press).
 Appendix II 369

7
11.2 EFFECT OF FORMULAnON AND OTHER
10 PARAMETERS ON DROPLETS
,,
6
,,
10 ,,
,,
Both the size and size range of droplets pro-
5 "" ,, 21/ha
Q)
0;
10
"" '" 10l/ha duced by a sprayer are affected by the flow
"
,,"~x/ ""h, rate of the suspension through the sprayer
<.> 4
(J)
10
Cl
0 (Fig. 11.3).
-=- 10
3
'" E<.> " " ,,
Flow rate of a suspension through a sprayer
"" ,, depends on the size of inlet to the nozzle

*
2
10 ,,
0.. "" ,,
,, (Fig. 11.4), on whether the suspension is
e 10
1
"" ,,
"C ,, pumped into the atomizer, and on the viscos-
Z
.; ""
10 "" ity of the suspension.
10-1 " With spinning disc sprayers, disc speed
also influences droplet size (Fig. 11.5). Disc
10-2 speed is determined by sprayer design,
10 100 1000
Drop size 11m (log scale) power supply (number of batteries) and
spray liquid viscosity.
Figure 11.2 Theoretical relationship between Final size of droplets reaching the target
droplet size and number of droplets per cm2 on a also depends on the amount of evaporation.
flat surface at three application rates (2, 10 and Addition of viscous non-evaporative oils or
100l/ha). humectants to volatile formulations reduces
evaporation. Table 11.4 gives the viscosity of
some UL V carriers.
Therefore as the droplet size decreases the Many carriers are too viscous for use alone
ratio, and hence evaporation, increases. and need to be mixed with less-viscous mater-
Small droplets also take longer to fall in air ials. For example, it has been recommended
and are subject to being blown by wind (Table that the viscosity of products for ULV
11.3). application to cotton in West Africa should

Table 11.3 Effect of spray droplet size on spray drift (Ross and Lembi, 1985)
Droplet diameter Type of droplet Time to fall 3 m Distance (m) droplet travels in falling 3 m*
(p,m) in still air
Wind 1 mls Wind 3 mls Wind 5 mls
5 Fog 64 min 1953 5859 9765
57 s
10
1 16 min
44s
503 1509 2514

20 Mist 3 min 56 s 118 355 592

50 Fine spray 39.4 s 19.8 59.5 99
100 1 10.8 s 5.2 15.5 25.9
200 Coarse spray 3.9 s 2.0 5.9 9.9
400 2.0 s 1.0 3.0 5.0
600 1 1.7 s 0.8 2.5 4.2
1000 1.0 s 0.5 1.5 2.5
Bold figures indicate droplets likely to evaporate before reaching the target. Anti-evaporants/non-volatile oils (Table
11.4) are added to reduce evaporation. It is normally recommended that spraying should not be undertaken at wind
speeds greater than 3 m/s; this is particularly important with smaller droplets.
370 Appendix II
be around 7 cP, allowing standardization on 160.,-----------------,
spay nozzles (H. M. Dobson, Natural
Resources Institute, UK, personal communi-
cation). However, the viscosity of different 140

120
'[
~ 120
110 c..
Q)
N
'(jj
100
a; 100
e-
:::1. 90
Q.
e
Cl Cl
~
c..
Q)
80 80
N
'(jj 70
a;
Q. 60+---.---~--"T""""-__,...--.--_l
e
Cl
60
3000 4000 5000 6000 7000 8000 9000
Speed (rpm)
50

40 Figure 11.5' Effect of disc speed on droplet size for a

typical spinning disc sprayer (Shell, 1987).
30
o 2 3 4 5 6 7
Flow rate (ml/sec) carriers varies enormously (Table 11.4);
Shellsol, or other light oils, have been added
Figure 11.3 Effect of liquid flow rate on droplet size to the more viscous materials. Despite this,
for a range of UL V applicators (Shell, 1987). currently available commercial insecticides
have wide viscosity ranges - H. M. Dobson
(personal communication) measured viscos-
ities ranging from 3-30 cP for UL V products
..... _ _ 2.0mm available in Kenya for locust and armyworm
2 4 ...~
',,,- 1.7 mm control. However, it should be noted that visc-
4-4~ osity also depends on other factors, such as
. .'4
. '~ 1.4 mm temperature (Table 11.4) and presence of
, ...
,
~
'~
.
,
1.0 mm

",",
4'4,-','Y.
particulate materials (the latter being non-
Newtonian in behaviour). For example,
with the Bacillus thuringiensis formulation
/1;,. ..........

'* Dip-88-EE, the pseudoplastic behaviour of

bacterial cells and other formulation ingredi-
ents results in lowering of viscosity at high
shear rates (Sundaram and Retnakaran,
1987).
0+----.-----r-----.-----,--.----.----1 Impaction and retention of droplets on the
o 5 10 15 20 25 30 35 target surface are described in section 2.2.2b.
Flow rate (ml/sec)
Important factors include terminal velocity of
the droplet, which is related to droplet size.
Figure 11.4 Relationship between viscosity of spray Small droplets with low terminal velocity
liquid and droplet size for four nozzles: Tubair XJ
1.0, 1.4, 1.7 and 2.0 (numbers are sizes of orifice in remain airborne for long periods (Table 11.3)
rom) (Shell, 1987). and may be carried by the airstream around
 Appendix II 371
Table 11.4 Density and viscosity of some ULV formulations and formulation carriers (R. P. Bateman,
personal communication)

Carrier Density (at 15C) Apparent viscosity (cPY

(at 25C, other temperatures
in brackets)
Deodorised kerosene 0.783 1.6
Shellsol T 0.756 1.4
Shellsol K 0.79 1.55
Isophorone 0.918 2.15
Diesel oil 0.844 7.2 (20C)
Ethylene glycol 1.12 13.3
OdinaEL 0.869 31 (20C), 14.3 (40 0c)
Risella EL 0.875 14.8 (40C)
Isopar V 0.82 14.7 (25C), 8.24 (40C)
Isopar M 0.778 2.2 (20C)
Corn oil 0.941 64 (20C)
Actipron (Bayer) 0.879 75.3 (20C)
Groundnut oil (Benin) 80 (20C)
70% Shellsol K, 30% groundnut oil 0.875 8.4 (20C)
50% Shellsol T, 50% Odina EL 0.802 5.9 (20C)
Thiodan CO (25%, Hoechst) 1.03 11.8
Perfekthiom UL (40%, BASF) 1.05 2.75
Water 1.00 0.8
Viscosity of Newtonian solutions measured either as kinematic viscosity (cSt) or as dynamic viscosity (cP). cSt = cP
divided by density. However, particulate suspensions are non-Newtonian and should not strictly be measured in terms
of kinetic or dynamic viscosity; therefore the term 'apparent viscosity' is used (Bateman, 1996).

large surfaces such as cotton leaves. They may
settle, however, on small-diameter targets
such as pine needles (Maas, 1971). Formula-
tion additives and carrier are also important
factors acting on retention (section 2.2.2b).
Droplets on the target surface will spread
(and penetrate) the surface at different rates.
The degree of spread depends on forces of
attraction between the molecules in the dro-
plet, droplet size, degree of wetting of the
target surface (influenced by carrier and pres-
ence of surfactants), and viscosity (influenced
by carrier and formulation additives). Dro-
plets of viscous formulations and/or those

Formulation additives Microorganism

e.g sunscreen particles

Contact areas Target surface

Figure 11.6 Contact angle and contact area of spray droplet on target surface (modified from Smith, 1993).
372 Appendix II

that do not wet the target surface will stand
proud of the surface with a large contact angle
and small area of contact (Fig. 11.6). These are
more effective for pick-up of contact-active
materials by an insect from a target surface.
Droplets with low contact angles and large
contact area are preferable for materials that
need to be spread as evenly as possible over
ated number of different-sized organisms that
can be contained within a range of droplet
sizes at a concentration of 5%. This does not
include the amount (percentage) of particul-
ate formulation materials in a spray, which
6
10 .,..----------------,

the surface, e.g. direct application of an organ- 5 1001/ha

10
ism to a susceptible host insect and organisms
that need to be eaten by the target host.
Contact angle (degree of spread) also influ- ,/
,/
,;'

ences persistence of the droplet and the ,/

microorganisms inside it - e.g. high contact

angles mean that additives (e.g. sunscreens)
are kept concentrated around the microorgan-
isms (Fig. 11.6).

11.3 INTERACTION OF DROPLET SIZE AND

PARTICULATE ACTIVE INGREDIENTS
Beneficial organisms are particulate in nature 10 100 1000
and therefore for each application rate (e.g. Droplet size (11m)

number of organisms/ha; volume of suspen-

sion/ha) droplets will contain a certain num-
ber of organisms (Fig. 11.7).
With the less potent and the larger micro-
organisms, it may be physically impossible for
a droplet of a given size to contain an effective
dose (section 10.3). Table 11.5 gives the estim-
Figure 11.7 Theoretical number of microorganisms
per droplet for a rane of droplet sizes at an appli-
cation rate of 1 x 101 particles per ha. The shaded
area represents the optimum range of size and
number of particles based on Tables 11.2 and II.3
and potency ranges of most candidate beneficial
microorganisms.

Table 11.5 Number of organisms that can be accommodated in different drop sizes at an acceptable
viscosity (5% organism concentration)

Drop size (p,m) Drop volume (p,l) Organism size (p,m)

2 4 6 8 10

10 5.24 X 10- 7 6.25 0.78 0.23 0.1 0.05

20 4.19 x 10-6 50 6.25 1.85 0.78 0.4
40 3.35 x 10-5 400 50 15 6.25 3.2
60 1.13 x 10- 4 1350 169 50 21 10.8
80 2.68 x 10-4 3200 400 119 50 25.6
100 5.24 x 10-4 6250 781 231 97 50
150 1.77 x 10-3 21094 2636 781 330 168.75
250 8.18 x 10-3 97656 12207 3617 1526 781.25
500 6.55 x 10- 2 781250 97656 28935 12207 6250
1000 5.24 x 10- 1 6250000 781250 231481 97656 50000

may account for a further 5-15% volume, to
give a maximum of 20% particulate material.
Higher concentrations are likely to increase
viscosity of the spray to unacceptable levels.
With low concentrations of organisms and
small droplets the distribution of particles
amongst the droplets follows a Poisson
distribution. This means that a proportion of
the droplets may contain no particles at all
(Table 11.6). Products should be formulated
to give concentrations of particles high
enough to avoid large numbers of 'empty'
droplets.
Under field conditions, the majority of dro-
plets do not reach the target (section 2.2.2b).
Measurements of numbers of droplets on tar-
get sites in the field allow actual impact to be
predicted. For example, figures for the use of a
nuclear polyhedrosis virus (NPV) to control
noctuid larvae on cotton are presented in
Table 11.7. The assumed dose rate is 1 x 1012
polyhedral inclusion bodies (PIB)/ha. Spray
deposition data on cotton for two sprayers in
Egypt are used.
Barnett (1992) calculated the LD50 and
LD90 for neonate larva fed NPV to be 0.96
and 8.03 PIB, respectively. LDso values for
other noctuids, such as Spodoptera littoralis,
Appendix II 373
are in the range 1-10 PIB (e.g. Jones, 1988).
Thus a sufficient number of microorganisms
are reaching the target area for the product to
be effective in all positions except for the
lower canopy of the crop with the ULV
sprayer (Table 11.7). Similar calculations are
possible for varying dose rates, deposition
rates, droplet sizes and application volumes,
as well as for organisms of different potency.
For example, Jones (1988) calculated the mean
amount of infective S. littoralis NPV applied to
cotton leaves using a CP3 knapsack sprayer
fitted with upwardly directed nozzles. From
these figures, the mortality of first-instar lar-
vae was calculated and the degree of inactiva-
tion of virus over time (Table 11.8; Jones, 1988;
McKinley et al., 1989); from these data it is
possible to estimate the impact of virus
persistence on pest control. Notably, with the
higher volume spray the greatest mortality of
larvae would be obtained underneath leaves
in the lower canopy, where protection from
the sun is best (Table 11.8)/ but at ULV poor
droplet cover cancels this advantage
(Table 11.7). Data on degree of protection
from inactivation given by formulation addi-
tives could also be included in these estimates
Oones, 1988).

Table 11.6 Effect of application method (hydraulic flat fan, air blast sprayer and spinning disc) on number
of spores of the biofungicide Ampelomyces quisqualis per drop when applied at ca 1.25 x 1011 spores/ha
(Chapple and Bateman, 1987)

Sprayer* l!ha t percentage drops with percentage Amount Percentage Percentage

volume with with no drops> 1 drops
o spore 1 spore no sparest spores (I!ha) spore > 300p,m

2080-14 159 63.8 15.3 5.8 9.2 20.9 30.0

2080-16 242 66.2 14.6 5.4 13.1 19.2 50.3
2080-30 560 71.8 11.7 4.7 26.2 16.5 76.2
Airblast 250 95.6 3.9 57.1 142.7 0.5 0.0
Spinning disc 1.5 6.5 12.0 0.4 0.01 81.5 0.0
11.2 39.9 23.6 7.4 0.8 36.5 0.0
* Three hydraulic flat fan nozzles (Hardi International A/S, Denmark), under similar operating conditions, an orchard
air-blast sprayer, and a spinning disc sprayer (Ulva+, Micron Ltd, Bromyard, Hertfordshire, UK).
t Volume application rate under field operating conditions.
t Percentage of the applied drops containing no spores, by volume.
Volume application rate for spinning disc with oil was 1.51/ha and with water was 11.2 l/ha.
374 Appendix II

Table 11.7 Droplet cover of cotton by two different sprayers and estimated consumption of virus
polyhedral inclusion bodies by first instar larva of Heliothis spp. when applied at 1 x 1012 PIB/ha
(adapted from Topper, 1984; Barnett, 1992)

Application to Deposits and Upper canopy Lower canopy

cotton* amounts eaten
Upper leaf Lower leaf Upper leaf Lower leaf
surface surface surface surface

Upwardly Drops/cm t lea~ 165 88 199 73

directed LV Drops eaten/24h~ 12.5 6.6 15.1 5.5
sprayt Virus eatenl24h** 374 198 452 164
VLV fan-assisted Drops/cm t lea~ 67 50 7 0.6
sprayt Drops eaten/24h~ 5.1 3.8 0.5 0.04
Virus eatenl24 h** 172 87 11 1
* Cotton 42 cm high, windspeed 0.5 m/s.
t CP3 Knapsack sprayer (Cooper Pegler and Co. Ltd., Ashington, Northumberland, UK) and upwardly pointing TY3
nozzles (Spraying Systems Ltd., Godalming, Surrey, UK). Application rate, 120 I/ha. Flow rate (at 20 psi), 500 rnl/min.
VMD,190j.tm.
I Turbair Fox spinning disc, fan-assisted ULV sprayer (Turbair, Pan Britannica Industries Ltd, Waltham Cross, Herts,
UK). Application rate, 8.5 l/ha. Flow rate, 62 rnl/min. VMD, 72j.tm.
Mean values.
~ Barnett (1992) calculated that first instar noctuid larvae (Heliothis virescens) consumed 7.563 mm2 of leaf tissue in 24 h.
** Polyhedral inclusion bodies (rounded to nearest whole number).
Table 11.8 Estimated amount of infective Spodoptera littoralis NPV applied at low volume on different
regions of the cotton plant (McKinley et al., 1989)
Position on plant Infective virus (SE) 48 Estimated mortality of
h after application* neonate larvae (%)
Upper canopy Upper leaf surface 4.1 3.3 x 101 10.5
Lower leaf surface 5.7 3.2 x lQ2 61.0
Lower canopy Upper leaf surface 7.7 0.7 x 1Q2 69.0
Lower leaf surface 3.4 0.8 x 103 87.5
* Application rate,S x 1012 PIB/ha in 120 I water. Mean infective virus at application was 3.85 x 103 PIB/cm2 on the
upper canopy, lower leaf surface, where most neonate larvae feed, which would cause 92.5% mortality of neonate
larvae. Estimated inactivation is for an unformulated, purified virus suspension; the effect of growth dilution of the
plant is not included.

REFERENCES biological insecticides. OIBC/WPRS Bull. 19,

Barnett, A. L. (1992) An investigation into the
encounter of a baculovirus deposit on a cotton
leaf surface by Heliothis virescens (Lepidoptera:
Noctuidae) larvae, MSc thesis, University of
London.
Bateman, R. P. (1991) Controlled droplet applica-
tion of mycopesticides to locusts, in Biological
Control of Locusts and Grasshoppers (eds C. Lomer
and C. Prior), CAB International, Wallingford,
pp.249-54.
Bateman, R. (1996) Formulation strategies appro-
priate in the ultra-low volume application of
29-34.
Bateman, R. (in press) Delivery systems and
protocols for biopesticides, in Biopesticides: Use
and Delivery, Humana Press, Towota, New
Jersey.
Chapple, A. C. and Bateman, R. P. (1997) Applica-
tion systems for microbial pesticides: necessity
not novelty, in Microbial Insecticides: Novelty or
Necessity? Symposium Proceedings No. 68 (ed.
H. F. Evans), British Crop Protection Council,
Farnham, pp. 181-90.
Jones, K. A. (1988) Studies on the persistence of
Spodoptera littoralis nuclear polyhedrosis virus
 Appendix II 375
on cotton in Egypt, PhD thesis, University of Shell (1987) Hand-held ULV Insecticide Spraying: A
Reading. Shell Pocket Guide 1987, Shell, Sittingbourne.
Maas, W. (1971) ULV Application and Formulation Smith, A. (1993) Adjuvants in Crop Protection,
Techniques, N. V. Philips Gloeilampenfabrieken, Agrow Report DS86, PJB Publications, Rich-
Eindhoven. mond.
Matthews, G. A. (1992) Pesticide Application Meth- Sundaram, A. and Retnakaran, A. (1987) Influence
ods, Longman Scientific & Technical, Harlow. of formulation properties on droplet size spectra
McKinley, D. J., Moawad, G. M., Jones, K. A. et at. and ground deposits of aerially-applied pesti-
(1989) The development of nuclear polyhedrosis ..- -cides. Pestic. Sci. 20,241-57.
virus for the control of Spodoptera littoralis Topper, C. P. (1984) Report on the Research and Devel-
(Boisd.) in cotton, in Pest Management in Cotton opment of Nuclear Polyhedrosis Virus ofSpodoptera
(eds M. B. Green and D. J. de B. Lyon), Ellis littoralis (Boisd.) 1979-1983, Vol. 2, Department
Horwood, Chichester, pp. 93-100. for International Development, London.
Ross, M. A. and Lembi, C. A. (1985) Applied Weed
Science, Macmillan, New York.
APPENDIX III: GLOSSARY (including list
of product and additive types)
H. Denis Burges
The objective of this glossary is to list product
and additive types and to define specialist
meanings intended in this book, including
some terms and units in relation to organism
formulation. The terminology and codes for
product types are those recommended for
chemical pesticides by the Global Crop Pro-
tection Federation (GCPF, formerly GIFAP).
There is merit in using this global system to
standardize terms adopted for microbials
worldwide. To help foster this objective, all
potentially relevant GCPF terms have been
included, even though some of the product
types have not yet been used for microbials.
The stems of some words are indicated by
vertical lines, and '" denotes repetition of the
word or its stem. Some key information is
included with the definitions, also cross-
references are given to chapter sections in
which confused areas are discussed.
abaxial leaf surface = lower leaf surface Sur-
face facing away from the stem during early
development.
absorb Incorporate; '" ance log intensity of
incident or transmitted radiation (not used in
this book, see absorptance); ",ent having a
tendency to absorb, substance that absorbs;
absorptance measure of ability to absorb
radiation, ratio of ability to absorb incident
flux (formerly absorptivity).
acervuli us, '" i (pI.) Dense, cushion-like, fun-
gal mass of conidiophores and conidia. Inside
a host, it is covered by host tissue which rup-
tures at maturity.
adaxial leaf surface = upper leaf surface Sur-
face facing towards the stem during early
development.
additive = fonnulant Non-pesticidal ingredi-
ent added to a formulation for a specific pur-
pose to improve application, persistence, etc.
May be incorporated in a product or added to
a tank mix.
adhesive 1. See sticker. 2. Class of substances
used for sticking.
adjuvant Has many definitions. Regarded for
this book only as a proprietary additive,
marketed specially for microbial products or
commonly used with them to improve ap-
plication, persistence, etc. Has no pesticidal
activity.
adsorption Attraction of ions or compounds
to the surface of a solid.
agglomeration Size increase by particle colli-
sion and adhesion (section 7.7.3a).
anhydrobiotic organisms Able to survive
extreme desiccation.
appressori I urn. '" a (pI.) Swollen hyphal
structure produced by recently germinated
fungal spore to penetrate the host's surface
layers.
378 Appendix III
arthrospore Asexual spore. A type of coni-
dium resulting from hyphal fragmentation.
attapulgite A clay with pseudo-layer forma-
tion that prevents swelling in water.
bait Forms: AB grain ""'; BB block ""'; CB "'"
concentrate, for dilution; GB granular ""'; PB
plate bait; RB ready to use ""', 5B scrap "'.
bar = atmosphere Average air pressure at sea
level, 100 kPa, 1023 cm of water, 760 mm Hg,
14.7Ib/in2 .
binder 1. Liquid fixing organisms and addit-
ives to carrier or seed. 2. Holds formulated
product together into aggregates.
blastospore Thin-walled element budded
yeastwise off mycelium in haemocoel and
liquid culture. = hyphal body (quoted in this
book if used by a cited author). Typically lar-
ger, thinner-walled and probably less envir-
onmentally resistant than the conidium of the
same species. More variable in shape and size
and produced by a different morphological
process to a conidium.
briquette, BR Solid block designed for con-
trolled release of organisms into water.
bulking agent see carrier.
capsule 1. Solid or liquid surrounded by a
protective membrane, cf. granule (section
3.3.1). 2. See occlusion body of a virus.
carrier = bulking agent, diluent, filler, vehi-
cle Inert ingredient to hold or dilute organ-
isms to desired concentration and improve
coverage and distribution. If dry, has low
absorption but absorbs binder, free-flowing.
Often forms main bulk of a product. Hydro-
philic for aqueous sprays.
carry Distance downstream to which blackfly
control is nearly 100% effective (section 3.6,
3.7.3).
chelating agent Inorganic complex which
combines with unwanted ions. Used to treat
heavy metal poisoning, e.g. EDTA for lead
poisoning.
chlamydospore Asexual, single-celled, fungal
spore formed by contraction of the protoplast
of part of an existing cell and secretion of a
thick wall, often impregnated with hydropho-
bic material. Main function in nature is long-
term survival, not dissemination.
clay Fine-grained crystalline hydrous silic-
ates.
coacervation Reversible aggregation.
compaction Granule formation by applying
pressure to particles (section 7.7.3c).
conidi I urn "",a (pI.) = condiospore. Asexual,
non-motile fungal spore, produced by a vari-
ety of processes and structures, e.g. a distinct-
ive phialide. Typically formed aerially on
solid media; formed in liquids singly in a
few species; cf. blastospore, pycnidiospore.
contaminant Unintended microbe in a
product; "'" suppressant additive to suppress
its growth.
cosmetic sunscreen Sunscreen used in the
cosmetics industry.
critical micelle concentration Concentration
that provides a monomolecular surface cover
to a drop.
Dalton's law of partial pressures Partial pres-
sure of a gas in a mixture is the pressure it
would exert if it occupied the same volume
alone at the same temperature.
delta endotoxin = S '" Proteinous toxin in
crystal of Bacillus thuringiensis and B. sphaer-
icus.
desiccant Water absorber that dries a product
at harvest.
diluent see carrier.
dispers I ant 1. Neutralizes the attractive
interaction of like particles, maintaining
them in uniform suspension in liquids; can
act as wetter, deflocculant; "'ibility time that
particles remain without settling in water or
oil. 2. Additive that promotes release of
organisms from product after application.
dispersible concentrate DC Liquid, homo-
genous formulation applied as a dispersion of
solids (for the purposes of this book) in water.

dormancy State of extremely reduced activity;
constitutive '" endogenous constraints not
overcome simply by conditions suitable for
growth; exogenous environmentally
imposed by withholding conditions suitable
for growth or by the presence of inhibitors,
growth being resumed on return of good con-
ditions and/ or removal of inhibitors.
dust see powder.
emulsifier Surfactant used with oil, see wet-
ter.
end-use container Container for product on
sale; '" product product on sale.
epigeal Growing above ground.
feeding I attractant Volatile material that
attracts an animal to feed; '" stimulant mater-
ial that stimulates feeding; '" preference food
preferred to others given a choice, may be a
combined result of attractants and stimul-
ants, may be an indirect effect by acting as a
preservative of materials in a mixture, an arti-
ficial food mix may be preferred to foliage of
the natural host plants.
filler See carrier.
flowable product Suspension of particles in
complex liquid, emulsifiable for water sprays
but not (OF) for oil. Often synonymous with
SC suspension concentrate. SU ~ ready for
use through ULV equipment. FS ~ for seed
treatment.
formul I ate Mix ingredients (= "'ants) to aid
organism preservation, storage, application to
target, survival and activity. "'ation =
product.
free-flow agent Additive that improves the
flow of a powder.
free radical Radical capable of independent
existence, containing one or more unpaired
electrons, sometimes highly reactive.
fungistasis Inhibited growth of fungi.
gel I ant Additive that forms a gel; "'ation
gelling by lowering temperature or by a che-
mical reaction (section 7.7.3d).
Appendix III 379
granulating All types of size enlargement
(section 7.7.3).
granule, GR 1. Free-flowing solid product.
MG micro'" 0.1-Q.6mm diameter. FG fine ~
0.3-2. 5 mm. GG macro", 2-6 mm. WG water-
dispersible '" designed to disintegrate
rapidly in spray water. Fabricated ~ pro-
cessed into a hard aggregate, can have uni-
form shape. See also capsule. Sections 3.3.1,
7.5.3 and 7.5.3a. 2. Single grain of starch
(starch chemistry term).
growth retardant = ~ suppressant Additive
at concentrations that suppress further
growth of organisms while they are in a
product; its effects are later diluted out at
application to non-suppressing levels.
humectant Additive that increases the moist-
ure content of a product by absorbing water
from the air.
hydathode Specialized structure that actively
exudes water from plants.
hyphal body see blastospore.
Hyphomycetes Used in preference to the
alternative 'Fungi Imperfecti', 'Deuteromyco-
tina' or the new 'mitosporic fungi'.
induration Harden. Agglomeration with
heating to soften surface ingredients (section
7.7.3b).
inocull ant Formulated or unformulated bio-
logical product applied to a target; ",um for
the purpose of this book, initial 'seed' organ-
isms used to start a culture, often for mass
production.
inversion Where air temperature rises as alti-
tude increases, i.e. reversal of usual tempera-
ture decrease with altitude.
invert emulsion Water-in-oil emulsion.
marumerizer Machine for rounding irregu-
larly shaped particles into spheres (descrip-
tion, section 7.7.3c).
matrix Mass formed by a gelling process
entrapping active ingredient, leaving some
exposed on the surface; specialized meaning
for the purpose of this book.
380 Appendix III
microbead = microcapsule.
mil 0.001 inch
monomolecular layer = monolayer Sub-
stance spreading on the surface of water into
a layer only one molecule thick.
mycorrhiz Ia, "'ae (pI.) Symbiotic organisms
in plant roots. May assist uptake of nitrogen
and other nutrients by roots and may ramify
into the rhizosphere.
nictation Searching to-and-fro body move-
ment of infective nematode larva standing
on its tail.
occlusion body Survival stage of baculovirus
with virions embedded in protein matrix, =
polyhedral inclusion body (polyhedrosis), =
capsule (granulosis).
optical brightener Brightening agent (often
added to textiles) that increases the brightness
of a material by reflecting some incoming
ultraviolet radiation after conversion to vis-
ible light.
osmoconditioning Technique for increasing
the moisture content of seed.
partial pressure see Dalton's law of '"
pellet Solid product> ca 6 mm diameter for
rapid (e.g. fizzy) or slow release of organisms
in water; also called tablet, TB.
pesta Granule made by an extrusion process
similar to that for pasta.
phagostimulant = feeding stimulant.
phase inversion temperature Temperature at
which the hydrophilic and lipophilic tenden-
cies of emulsifiers balance causing a dramatic
change in viscosity.
plasticizer Maintains flexibility and adhesive
power.
polyhedral inclusion body see occlusion
body.
powder types: DP dustable '" free flowing;
OS '" to apply dry to seed; GP flo dust, very
fine for pneumatic dusting; WP wettable'" to
apply as suspension in water.
pre-gennination of fungus spores Germina-
tion of spores before application.
prill Granules solidified when drops of liquid
holding organisms are dripped into a reacting
liquid.
product Mixture formed by formulation for
packaging.
pseudoplasticity Low viscosity and excellent
flow under high shear, but elevated viscosity
at rest. After emission, droplets tend to coar-
sen, reducing spray mist, giving narrower,
more uniform spray pattern and good drift
control.
pycnidiospore Conidium formed in a pycni-
dium, a minute flask-shaped fungal fruiting
body, lined internally with conidiophores and
having an apical hole; cf. conidium.
relative humidity Ratio of vapour pressure in
air to the saturation vapour pressure of water
at the same temperature; see water activity.
rhizosphere Zone of soil in the immediate
vicinity of an active root, characterized by a
microbial flora different (more active, more
numerous and more diverse) than that in the
bulk soil.
sclerotium, micro'" Long-term survival stage
of fungus comprising compact, hyphal aggre-
gate, with a differentiated rind, usually
melanized.
seed I covering, PS 1. Filmcoating '" process
applying uniform dust-free, thin, water-
permeable membrane; 2. "'coating light
cover to improve handling, ca O. 1-2-fold
weight increase; 3. ",-pelleting encasing in
spherical mass for precision drilling, ca 2-
50-fold increase.
seedpiece Piece of potato tuber cut to a size
suitable for planting.
semiochemical Chemical produced by an ani-
mal and used in communication, e.g. phero-
mone, allomone.
siderophore Low molecular-weight, iron-
chelating molecule.

singulate Separate into units containing one
seed or other object.
sorption Absorptance and adsorption con-
sidered jointly.
spermosphere Zone of soil in the immediate
vicinity of a seed.
spreader Additive that improves the spread
of a spray over a plant surface by lowering
surface tension.
stabilizer 1. Additive that maintains the viab-
ility and activity of organisms in a product; 2.
= suspender.
sticker See also adhesive Additive used to
adhere organisms to foliage and seeds.
sunscreen used in wide sense, additive to
prevent/alleviate damage due to sunlight.
sunscreen protection factor Ratio of radiation
dosage required to cause a given degree of
damage on sunscreen-protected material to
that required on unprotected material.
surfactant See emulsifier, wetter.
suspender See also stabilizer; additive that
maintains in suspension solid particles in a
spray/powder in a spray tank/hopper.
suspension concentrate, SC See flowable.
suspo-emulsion, SE See flowable.
synergist Additive, which increases the activ-
ity of organisms but, when used alone, is
inactive or only slightly active. Often used in
this book in the broadest sense, even includ-
ing additive action.
tablet See pellet.
technical I material, TC, '" concentrate, TK
Active ingredient, associated impurities and
Appendix III 381
small amounts of necessary additives result-
ing from a manufacturing process.
thickener Additive that increases the viscos-
ity of a product.
thixotropic In a gel state when stationary,
rapidly converting to a flowable when agit-
ated.
tracer dye Dye added to a spray to reveal the
extent of the deposit left on foliage and
insects, often ultraviolet fluorescent.
vehicle See carrier.
vermiculite Spongy mineral silicate able to
hold up to 20% w /w spores without a binder.
water activity (a w ) = water vapour activity
Partial vapour pressure of product (p) divided
by that of water (Po)
aw = p/po
Germination of fungus spores and growth of
bacteria inhibited at aw < 0.7--0.8. Illustrates
the availability of water to organisms. Equ-
ivalent to soil water potential. Interchange-
able with relative humidity in that it gives
the equilibrium value of a material with a
particular relative humidity. See section 4.2
for discussion.
water availability Closely related to water
activity. See section 4.2.
water potential of soil = water activity Has
two components, soil matric potential + soil
osmotic potential. See water activity.
wetter surfactant used with water, d. emuls-
ifier. Improves mixing of a product with a
liquid. Reduces surface tension. Improves
spread of a spray over a plant surface.
INDEX
Page numbers appearing in bold
refer to figures; page numbers
appearing in italics refer to tables.
Aatara 430 269, 338
abamectin 107
abrasion 59,79, 81, 259, 274
absorbents 20,68-72,75,227
naturally occurring 76
see also individual compounds
absorptance 377
absorption of substrate 237
Abuti/on theophrasti, see velvetleaf
acacia, see gums
Acarina 108
accession numbers 318
Accurel Powder 93, 341
AC-Dl-SOL 249,338,341
acephate 65, 302
acervuli 225,377
acetamide 79, 82, 85, 350
acetamide-{3-mercaptoethylamine,
seeAMEA
acetone 345
acetylenic diol 346
acetylenic surfactant 52, 57
Acetylenic surfactant 5485 57
acids, synergistic 81-2
Aclarat 8678 71, 359
Acremonium diospyri 203
Acriflavin 69,74,355
acrylamide polymer 94
acrylic acid 94
acrylic polymer 60-1, 347, 349
acrylics 347
Acrylocoat 60-1,347
actinomycetes 193,272,347
Actipron 46, 141,336, 347, 350,
371
Activate 9-0 217
activators 49, 249-50
active ingredients
interaction with droplet size
372-4
nature of 312-13, 313-16
activity measures 320
acylamines 82
additives 18, 55--86
catalogue of 333-6
combining 211,216,259,315,322,
324
concentrations of 322
cost of 101,317-18
definition of 204, 377
food grade 324
functions of 333-362
harmful to biocontrol agents 103
hazard categories for 103
hygroscopic 55, 146
in insect control
formulations 102-4
interactions with pathogens 78-9,
213
for land use, trends in 102-4
multifunctional 97-8,105,328
neutralizing 55
new 109,176,331
nomenclature for 333-362
nutrient 140
see also nutrients
properties of 333-362
selective, to extend host
range 219
separation of 261
in soil formulations 247-50
suppliers of 363-5
synergistic 210-16
toxicity of 210-16
types of 377-381
units of quantification for 56
in water 162, 163, 164
see also individual additives
ade:1ine 68,360
adherence 136, 137, 259, 269, 315,
331
adhesives 92, 377
see also stickers
adipic acid 249, 341
adjuvants 62, 217, 319
commercial, compared 63
definition of 204, 377
spray 39
see also additives
adsorption 174,377
Aedes 87-9, 87, 91, 94, 96, 106
Aedes aegypti 88, 96
Aedes albopictus 93
Aedes vexans 95
aeration during fermentation 207
aerosols 18, 210, 368
generator 13
Aeschynomene virginica, see northern
jointvetch
aesculin 70
aftercare of organisms 322-6
agar 63,238-9,239,352
slanted 239
see also individual agars
Ageless, Type Z 257, 353
Agaricus spp. 190
agglomeration 93, 246, 251, 377
agitation 313,314, 319
Agra157, 211, 345
Agrobacterium 54,198
Agrobacterium radiobacter 190,193
Agrobacterium tumefaciens 190
Agro-Lig 194,264,276,338
Agropyron cristatum, see crested
wheatgrass
Agrotis spp., see cutworms
Agrotis ipsilon, see cutworms, black
Agrotis segetum, see turnip moth
aircraft 40, 155, 159, 169, 226, 274,
277
au-drying 40,155,159,169,226,
274,277
au-seeding 260
AL products 57, 345
alanine 80,350
albumin 76,360
see also egg albumen
Alcaligenes 54
Alcian Blue 8GX 69, 355
alcohol ethoxylate 345
alcohols 94
alkoxylated 147
alphatic 94
Alcosorb AB3 273,338,341
aldehyde, cross-linking 92
alder, red 203
alder bark 197,338
384 Index
alfalfa 137, 139, 189, 242, 260, 268,
270
see also lucerne
algae 86, 107
algal polysaccharide 262
alginate 44, 338
in humectants 342, 362
in microbial herbicides 215-16,
220
in mycoinsecticides 142, 151
in plant-disease
formulations 195-7
in seed formulations 267, 275
in soil applications 245, 247, 249
in stickers/spreaders 348
see also calcium alginate; sodium
alginate
alginate beads/granules/
pellets 208, 223, 224-5, 224,
227,245,249
alginate gelation 252
alginate prill 188,195-6,198,262
alginic acids 215, 215,216, 249,341,
347
alkalis 79
alkalinity, leaf surface 66
alkanes 353
alkylarylpolyoxyethylene
glycols 217
alkylarylsulphonates 24
alkylbenzene sulphonate 346
alkyl phenols 57,345
alkylphenol ethoxylate 347, 348
alkylpolyethoxyethanol 217
alkyl sulphates 346
alkyl sulphonated alkylate 348
alkyl sulphonates 24
allantoin 71, 360
allelochemicals 62, 78, 101, 103,
328
inleaves 55,66,84,324
almond 189
almond hulls 350
almond oil 161
Alnus rubra, see alder, red
Alphacel 337
alpha-tocopherol, see tocopherol
Alternaria 190, 214, 216, 242
Alternaria brassicicola 190,193,273
Alternaria cassiae 217,219,220,221,
225,226
Alternaria crassa 217,219,220,225
Alternaria macrospora 217,219,224
Alternaria raphani 273
Altox products 24, 58
aluminium carboxymethylcellulose
(CMC) 96
aluminium powder 72, 359
aluminium silicate 249, 341
aluminium sulphate 96, 340 Aphanomyces cochlioides 274
amberlite 249, 341 aphids 133,135,140,173,331
AMEA 214, 214, 341, 343 apple 61,62,75,189,193,265,346
amides 80 larvae on 41
amine ethoxylate, tallow 345 apple pomace 350
amine stearate 345 application methods 10-19,314-15
amino acids 54, 68, 75, 76, 80, 207, aerial 14,18,100,104,319
322,343,350-2,360-1 against mosquitoes 90, 91
balance of 79 of mycoinsecticides 139,172
p-aminobenzoic acid (PABA) 68, of nematode formulations 299,
360 306
p-aminosalicylic acid 82, 350 costs of 50
ammonia 24 effects on spore numbers 373
ammonium benzoate 83, 350 efficiency limits of 327
ammonium hydrogen equipment 11, 12-15
phosphate 81, 85, 350 on foliage 210
ammonium ion 338 integration with formulation
ammonium salts 350 205
ammonium secretion 86 for nematodes 299-300, 300-5,
ammonium sulphate 158 301
ammonium thiosulphate 81, 350 on-farm 271
Amoeba 151 problems of 8
Ampelomyces quisqualis (powdery for soilborne crop diseases
mildew) 188,373 189-92
amylase 83 trends/future research into
amyl-dimethyl-p-aminobenzoic 172-4,322-6
acid 68,360 see also sprays; sprayers
amylopectin 43 application parameters/rates
amylose 43,249,339,341 367-9,368,374
anaerobiosis 162, 322 see also sprays
annual bluegrass 218 appressoria
Anoda cristata, see spurred anoda adhesion of 207
Anomala schoenfeldti 304 chemical herbicides and 325
Anopheles 87-9, 87, 96, 106, 152 definition of 377
Anopheles albimanus 95 herbicide formulation effects
Anopheles gambiae 92 on 211-216, 214, 215, 217,
Anopheles stephensi 96 223
antagonism 21, 86, 237, 322 Apron 193
antagonists 195, 199,329 apterin 360
antibacterials 322, 353--4 AQlO Biofungicide 188, 189
antibiotics 10, 50, 248, 265, 290, 291, aquatic environments
322,330,353--4 problems of 87-9
in virus production 38 target insects in 87-9,100, 152
Anticarsia gemmatalis 40, 41 aquatic plants, submerged 225
anti-desiccants 152, 221, 224 Arachis oil 46, 51, 55, 57-8, 336
anti-evaporants 18, 45, 47, 100, 315, Archips pomoneUa 41, 75
336,351,369 arginine 79, 80, 84, 85, 197,353
anti-feedants 85, 173 Arlacel "C" 57, 345
antifoam agents 23,171,314,353 Armillaria 191
antifungal preservatives 354 armyworms 41,57, 103,370
Anti-Fungus, see Bio-Fungus aromatics 160, 347
antimicrobials 21,78,314 Arosurf 97, 106, 109
see also preservatives Artemia salina 247
antioxidants 9, 73, 74, 77, 157, 159, Arthrobacter 198
160,165,322,327,350,353--4, arthrospores 207,378
360-1 Aschersonia 133
anti-transpirant wax emulsion 345 Aschersonia aleyrodis 153, 156
ants 21,151-2,343,349 Aschersonia placenta 147,347
Aphanomyces 193 Ascochyta pteridis 214,215
 Index 385
ascorbic acid 55, 73, 75, 79, 82, 164, autoencapsulation 53-4 transgenic plants and 86, 270
165, 350, 353, 360 biology 35 UV-resistant mutant 76,330
ascospores 193,274 broth 37 vegetative cells 98, 100
ash 45,46 carriers 336-40 viscosity 370
asparagine 79, 80, 350 chemical insecticides and 312 in water bodies, see B.t. israelensis
asparagus 190 compatibility 56, 302 water-based flowable
aspartic acid 80, 350 concentration 100 concentrate 48
aspen 78 crystal toxin 18, 21, 35 wettable powders 51-3,91
Aspergillus 190 acid/ alkali effects on 77, 78 wetters and 56,57-8, 345-7
Aspergillus flavus 196, 350, 351 density of 91 Bacillus thuringiensis israelensis
Aspergillus niger cinnamomeus 143 enzymatic breakdown 98 87-97
Aspire 188, 189 as feeding inhibitor 17, 62, 109, gamma irradiated 106
Assist 211, 336 313 granules 91, 92, 94
aster, Chinese 194,275 inactivation of 67 photoresistant 330
Astrazone Orange/Yellow 69 mode of action 35, 323 potency 37
asulam 214 decontamination, high toxin gene transfer 107
Atlox products 57, 345 pressure 40 Bacillus thuringiensis kurstaki 49, 84,
atomization 251, 259 deterioration 54 107
atomizer, rotary 37,49 dispersible concentrate 48 flowable concentrate 49
Atplus products 57, 345 dormancy 323 potency 37
Attaclay products 154, 337 in dry baits 42 shelf-life 55
attapulgite clay 39, 142, 150, 153, in dusts 42, 42, 43 wettable powder 91
154,197,295,337,378 ecology 35 Bacillus thuringiensis tenebrionis 64
attractiveness of target 315 emulsions 90,96 bacteria
autoclaving, see sterilization encapsulation 26, 95, 103, 105 deterioration of 54
autoencapsulation 53, 67 evaporation rates 18 Gram negative/positive 248,273,
Autographa californica NPV 54 fermentation 36-8 318
autointoxication 321, 326 formulation and physical humidity /water requirements
autolysis 55 properties 341-4 of 20
aviation fuel 159 granules 51-3,91,92,94 long-term survival form of 198
Avicel 246,249,337,341 half-life 67 as pathogens 35
a w , see water activity as irritant 331 phylloplane 54
axillary buds 209, 213 microcapsules 101 as plant disease formulations 193
Azocarmine 69, 355 nematodes and 304 in seed applications 272-3,259,
Azospirillum 236, 237, 243 oils and 46, 51 262,265
Azotobacter 236,237 phagostimulants and 63-4,199, soil 240,241, 242, 248
Azotobacter vinelandii alginate 195 328,350-2 symbiotic 290, 291
azygospores 158 plant extracts and 21 vegetative 147
potency 37 wettable powders 95
Bacillus 236, 237, 242, 245, 261 preservation technology 322 see also Bacillus spp.
see also particles production of 36-9 bactericides 80
Bacillus megaterium 54 rain effects 59, 61 bacteriostatic action 354
Bacillus popilliae 42 sales, worldwide 35 preservative 354
Bacillus sphaericus 35, 50, 54, 106 shelf-life 52,55,99 Bactimos 93, 96
carriers for 340 sprays 48-51 baculovirus 35, 35, 46
in granules 92, 94 adjuvants 350-3 adherence 331
in pellets 93 in spruce budworm control alkaline damage to 77
pH in storage of 98 49-50,50 autointoxication by fats 321
prill 96 stability of toxin 35 bacterial invasion 38
sunlight and 89, 106 stabilizers 350-3 cell culture 38
wetters for 57-8, 345 standardization 321 contaminants in formulation 10
Bacillus subtilis 54,190,191,193,194 stickers 60-1,62,347-9 deterioration of 54
as seed treatment 264, 272-3, 278, sunlight and 67, 74, 89 dual infection 322
279 sunscreens 68-73,355-62 encapsulation 53
Bacillus thuringiensis 3,49,242 surfactants 345-7 inactivation by plant extracts 21
aerial application 18 synergists 79,80-3,85,350-2 in vivo production 38, 39
agitation 313 tank mixes 65-6 lethal dose 101
in agriculture/forestry, see B.t. technical material, costs of 317 microfiltration 40
kurstaki; B.t. tenebrionis toxin gene transfer 106-8 mode of action 35
386 Index
baculovirus (cont.) in baits 151 berberine, alkaloid 76
non-occluded 108 calcium and 173 berberine sulphate 355
photoinactivation of 67, 74 on leaf 139 Bevaloid products 37, 48, 52, 58,
production of 38-40 in oil 137-8, 159, 161 157,341
specificity 328 persistence 147 bicarbonates 20, 248
stability 52 rainfall and 146 Binab-T 188, 189
sunscreens and 331 stored in water 162, 166 binders 10,23,52,194,246,249,258,
surfactants 56 in submerged culture 170 261,324,341-4
suspension 25 sunlight on 142, 143, 144-5 definition of 378
temperature effects 53 young culture slow-release 360
UV exposure effects 329 germination 172 see also individual compounds
see also CPV; GV; NPV; occlusion dispersants 341 bioassays 279
bodies; viruses dusts 142, 152 Bio-Blast 135,155
bags endophytic growth in com 148 biochromes 76
autoclavable 168 flowables 141 bioencapsulation 105
fermentation 167, 168, 169, 175 fungicide effects on 148 Biofilm 57,60-1,345,347
growing 168-9 granules 150 Biofox C 188, 189
paper seed 248 growth in sterilized soil 150 Bio-Fungus 188, 189
plastic heat-sealable 239,243 hosts 133 BioMal 218, 242
water-soluble 218 nematodes and 304 biopolymers, polycationic 54
see also fermentation nutrients 353 BioSafe 246
Bairoisa-kamikiri 135 pellets 142 Bio-Save products 189,193
baits 21,63, 174, 303, 306, 325, 328, shelf-life 153, 155 Biotrol2 44
354 sunscreens 362 Bio-veg 211, 336
bacterial concentration in 95 survival 155, 156, 157, 165 Bipolaris sorghicola 219
dry 42, 64, 67 Beauveria brongnMrtii 135, 147, 150, bis-benzoxalate ethylene 358
fungal applications 151-2 158 bituminous coal 273,276,338
granules 99 Bedding technique 291 Bivert 46, 47, 336, 341
liquid 301 bee, honey 138 Blachere process 158
mosquito 95 bee-comb 41 black cherry 223
pellets 95, 300, 301 Beeconmist 65 blackfly (Simulium spp.) 19,35,87,
see also attractants; individual beeswax 56, 57 88,106,109
pathogens beet armyworm 300,304 mode of feeding 87
baking powder 93 beetles 35,138 oil effects on 95, 104
banana weevil 301, 303 see also scarabs particle size, ideal 89
bark 197,362 Bemesia tabaci, see whitefly virus and 105
barley 150, 189, 191 benazolin 212 black nightshade 219
Barnet clay 337 beneficial organisms 2 black scurf 276
barnyard grass 213 carriers 336-40 Blankophor products 66, 77, 82, 84,
barriers, plant physical/ dispersants 347 143,144,359
chemical 206 lubricants 342-4, 352 blastospores
basil 188,190 nutrients 353-4 antioxidants for 350, 353
batches soil applications 235-53 binders for 342, 351
basis 260 synergists 350, 351 carriers for 336, 339
machines 258 benefiting mechanisms 237, 237 definition of 378
size 259 Benlate 302 desiccation, tolerance of 171
Bathurst burr 218, 221 benomyl 43, 302 dimorphism 207
Baysilicone E 171,353 bentazone 212 formulations 170
beading 315 Bentone 38 49, 154, 341, 343 in oil 152
beans 189,191,194,264,272 bentonite 22,70,150,152,154,197, germination 132
BeauverM 135,149,236 225,262,262,268,337 hydrophilic 170
Beauveria bassiana Bentonite GO-ll 154,337 production 135
blastospores 133,151,155,171 benzalkonium chloride 50, 353 stability 164
carriers 339, 350 benzilidene sulphonic acid 68, 76, stabilizers 353-4
colony spread 150 355 sunscreens 360
conidia/spores 132,133,138 Benzopurpurin 4B 69,355 survival 155, 158, 163, 164, 171
annular aggregation of spray 2-(2-H-benzotriasol-2-yl)-4- virulence 164
deposit 145 methylphenone, see Tinuvin P yield 171
in aqueous spray 139, 149 benzyl cinnamate 68,144,355 see also individual species
 Index 387
blastospores (eont.) butyldodeclyamine carbofuran 148
Blatella spp., see cockroach hydrochloride 57, 345 carbon 70, 76,77, 150, 171, 197,207
blenders 92, 196, 220, 226 butyl-methoxydibenzoyl as UV blocker 146
ribbon 22 methane 358 high porosity activated 362
BlightBan A506 189, 193 -y-butyrolactone 161, 339 carbon black 70, 77, 362
Blissus leueopterus, see cinch bug carbon dioxide 158, 169, 175, 249,
blockages, see nozzles; screens cabbage 41,59,60,191,304 322
blood, dried 46,65 Chinese 141 carbon: nitrogen ratio 197,207
blotch 193 pests of 41, 42, 57 carbonates 20, 248
blowflies 108 cabbage looper 304 Carboset 60,62,347
blowfly faeces 362 Cab-O-Sil 97,249,341,343 carboxymethylcellulose 47,65,96,
Blue Circle 189 cadavers, insect 34,38,42, 133, 139, 194,269,274,276,341,347
bollworms 41, 65 147, 150, 155, 156, 168, 170, 321 cardboard bands 301
pink 63 caffeine 79,82,350 Cargill Insecticide Base
Bombyx mori, see silkworm caking 10,22,23, 36, 97, 167, 169, Concentrate 47, 57, 60, 341,
Bond 60, 62, 65, 66, 347 242,313,314 345
borax 81,84,85,350 calcium 20 carnation 188,189,190
boric acid 79,81,85,249,341, 350 calcium acetate 82 carrageenan 245,338,341
Botryosphaena 191 calcium alginate 21,154,195-6,292, K- 247,249,252
Botrytis spp. 188, 189, 190, 193 293, 294, 294, 295, 297, 298, Carrier 038 65, 336
Botrytis cinerea 189, 191 301,338 carriers 197-8,216,242-3,247,
bounce-off 49 calcium carbonate 79,81,85,167, 336-40
Boverin 135 168, 267, 270, 270, 337, 350 bulky 223,251
bovine serum albumin 155, 360 see also lime; limestone density of 371
BP products 46,336 calcium chloride 44, 53, 61, 64, 70, heavy core 92
bracken 214,215 153,154,158,173,195,195-6, hydrophilic 218
Bradyrhizobium japonicum 262, 268, 224,341 hydrophobic 152
269 calcium gluconate 195-6,196,262, inert 22
bran 21,36,63,67,151,227,275, 262,341 local 42
338,350 calcium hydroxide 81 seed application 266--8
prills 274 calcium ions 38,79, 173 shelf-life and 155
brassicas 41 calcium montmorillonite 151,269, soft 22
seed 257, 260 337 as sunscreens 143
Brightener 28 82 calcium nitrate 81 viscosity of 370, 371
Brilliant Sulfoflavine 355 calcium oxide 79,81,85 carrier-t!xtender 36
brine shrimp cysts 247 calcium salts 23, 342, 350 carrot seed 260,262,264,274
briquettes 11,19,22-3,93-5,96,105, calcium stearate 249,341 casein 57,60-1,62,72,360
252,315 calcium sulphate 79,81, 167, 168, Cassia obtusifolia, see sicklepod
definition of 378 244,245,247,338 Cassia oecidentalis, see coffee senna
bromoxyni1 212 anhydrous 158, 342 Cassia surratensis, see kolomona weed
broths, see individual types see also gypsum castor oil 152, 154, 340
brown plant hopper 141 Calcofluor products 71,82,144,359 castor oil ethoxylate 345
BSU 144,145 calibration 264, 313 catalase 73,75,360
buckwheat 167, 168 L-canavanarnine 350 cation exchange capacity, soil 325
budworms 41, 44, 58 Candida oleophila 188, 189 CDA, see droplet application,
Buffalo Black 69, 355 canola 189 controlled
buffers 315 canola oil 216, 221, 336 Cecaperl 158
electrical charge 174 capillarity 136 cedar shavings 292
pH 9,77, 78, 146, 163, 238, 243, capsules 23, 43-4, 100, 101, 102, 301 cedar wood oil 336
247,248-9, 314 buoyant lipid 88, 95-6 Celacol M2500 63
water-availability 140, 146 carrier 54 Celatom 337, 341
water-soluble 362 cellular 100 celery seed 257,260,262,265
bulking agents 224, 242, 247 definition of 378 Celite 209 154, 337
see also carriers see also autoencapsulation; cell paste 240
buoyancy 88,89,105 CellCap; encapsulation; cell solution leakage 222
Burkholderia eepacia 189, 193, 273 granules; microencapsulation CellCap 54, 100, 101, 104
n-butanol 217 captan 193 Cell-Tech 271
butylated hydroxyanisole 160 car paintwork damage 61, 62, 349 cellulose 24, 26, 95, 258, 268, 278,
butylated hydroxytoluene 160 carbamate 148 292, 294, 338
388 Index
cellulose (cont.)
microcrystalline 246, 338-9,
341-2
powdered 159
as stickers 59
cellulose acetate 249,341
cellulose acetate phthalate 250
cell-wall protectant 165
centrifugation 37,39, 169, 206, 251,
differential gradient 38
multi-orifice 26
Cephalosporium spp. 203, 220
Ceratonia siliqua, see locust bean
cereal grains 158
as granules 167, 169
small 257, 260
cereal grain flour 140,141, 157, 338,
353
cesspools 92
cetyl stearyl diethoxylate 97
cetyltrimethylammonium
bromide 57,80,85,345,350
Chaetomium globosum 273-4
chainsaw oil 216, 336
chalk 261
charcoal 70,245,268,362
activated 242, 292, 338
charge 85,172,174,331
charge-masking 59
cheesecloth 39
chelating agents 78,83,351
definition of 378
chemicals
compatibility with 323, 325
nematode 302
target substrate 20-1
see also fertilizers; fungicides;
herbicides; pesticides
Chenopodium album, see fathen
cherry 189
Chevron 46,65,345
Chevron spray sticker 50, 60-1, 65,
347
Chevron X-77 147
chickpea 78, 274, 275
chicory seed 260
Chimmasorb 81 145,355
chitin, colloidal 44, 173
chitin synthesis, interruption of 148
chitinase 60,79,330,350
chitosan 54
chlamydospores 204, 275
definition 378
chloramphenicol 248, 353
chlorides 314
chlorogenic acid 78, 79
choline chloride 68,360
Chondrosterium purpureum 191, 211,
216,223
Choristoneura fumiferana, see spruce coffee senna 226
budworm Coleoptera 79,108
Chrome Axurol 69, 355, 357 collaboration, research 228
chromophores 74 collagen 342,353,360
chrysanthemum 141,173,300 collection efficiency, target 16
chrysophenine 69,355 Collego 58, 204, 207, 216, 220, 346
chymoelastase synthesis 174 Colletotrichum 191, 224
Cibachron Blue/Yellow 69, 355 Colletotrichum coccodes 213, 217
Cimmasorb 81 356 Colletotrichum dematium 214,214,
cinch bug 138 215,215, 216
Citowett 218, 345 Colletotrichum gloeosporioides 200, 204,
citric acid 53 211, 212, 218, 220, 346
citric acid by-product 61, 70, 347, f.sp. aeschynomene 220
362 f.sp. malvae 212
citrus 188,189, 193,204 see also Collego
citrus cyst 190 Colletotrichum orbiculare 207, 210,
citrus pulp 63, 350 217,218,221
citrus root weevil 148 Colletotrichum truncatum 207, 218,
citrus seed 277 219,220,221, 223, 225, 226,
Clarcel products 157, 158, 341 226,227
clarsol 151, 158 Colloidal X-77 57, 345
Clavibacter xyli 87 colloids 51, 249, 343, 349
clays 51,90,95,105,135,139,150-2, see also clays
154,157, 159, 168, 174, 197, colonization patterns 278
227,242,244,245,246,261, colony-forming units 38
267,268,269,292,294,314, Colorado potato beetle 41,64
337,341 commensal microorganisms 107
coating 151 commercial products 188-93,204
colloidal 24 commercialization 311-12,316-20,
definition of 378 328
as dispersant 142 failure of 204,316-18
as sunscreen 144, 145, 146 of herbicides 207,
see also named clay types of nematode products 290,
clay minerals 22, 23 305-6
climate effects 137-42,172-4,205, of seed treatments 257,274, 278,
218,228 279
clopyralid 212 of soil formulations 236,238,241,
clover 151, 189, 270, 270 253
clumping 10,45,53, 174, 209, 323 compaction 252,378
during processing 23,38,51, 137, comparative studies 108,279
261 competitiveness 317
reversible 24 compost 150, 338
clustering 260 Compritol 249,342
coal 263, 264, 267, 268 concentrates 238, 239
see also bituminous coal aqueous 98, 240
coating, see seed coating frozen 239
Coax 44, 63-4, 66, 67, 70, 74, 76, 99, suspension 24-5, 37, 51, 89-90,
350,362 98, 100, 104, 381
Coccus viridis 147 dilution of, for spraying 164
see also scale insect dormant aqueous/oil 240
Cochliobolus lunatus 213 low-cost 49
cockchafers 147 research, fu ture 104
cockroach 301,303,306 viscosity of 24
cocoa weevil 138 technical 381
coconut oil 161,336 see also flowables, technical
Codacide 139, 141,161,350 concentrates
cod liver oil 161,336 Congo Red 20,69,76,77,144,145,
codling moth 39, 41, 301 199,355
GV 40,62,75 conidia 132, 378
 Index 389
conidia (con!.) sunscreens for 355-62 corn borer 41, 43, 44, 60, 148
additives in water, effect of 163 survival control of 43, 67, 99, 142, 169
aerial 156,170,175 during drying 155-6 corn cob 154, 157, 244, 247, 250
ageing 175 in storage 136, 154, 155, 171 corn cob flour 95, 250
aggregations 137 in sunlight 143, 144-5 corn cob granules 44
binders 344, 352 in water 162 corn cob grits 90, 91, 95, 105, 148,
carbon:nitrogen ratios unformulated dry 156 197,225,338,353
affecting 207 vegetation, applications to 139 corn flour 43,62, 63, 70, 227
carriers for 336-40 wettable powders 135, 218 nixtamalized 43
colour monitoring 171 see also macroconidium; pre-gelatinized 53, 339
concentration of 165, 175 microconidium; spore; corn gluten 250,353
dehydration of 166 individual organisms corn leaf/plant 63,64,67,148
delicate 133, 170 conidia beads 170,171 corn meal 67,142,198,208,339,350,
desiccants for 342 conidia chains 175 353
dimorphism in 207 conidiation 142,169,170,207 agar 275
dispersants for 343 Conidiobolus obscurus 158 corn oil 57,62,63--4,88,161,216,
dormancy of 155, 156, 158 conidiogenesis 132 217, 221, 336, 371
drying 155 conidiospores, see conidia corn, pests on 41
dusty 137, 156 conifer pests 41 corn rootworm 299
freeze-drying 155 Coniothyrium minitans 189 corn seed 63, 194
germination 139, 143, 148, 162, Conquer 189,193 corn silk, aqueous extract of 350
214,215,223,321 see also Victus corn starch 43-5, 53, 60, 64, 67,
accelerated 173,174,176 containers 70,142,227,249,339,344,362
in storage 152 autoclavable 168, 169 capsules 44
under sun lamp 224 airtight 159, 162 pearl 339
young/old 172 contaminant-proof 169 com syrup 269, 342
in granules 158 end-use 158, 169, 176 Cornitermes cumulans 152
half-lives of 153 heat-sealable 168, 239 Cosmopolites sordidus, see banana
harvesting 167, 168--9,320,321 moisture-proof 9,55 weevil
humidity and 156, 175 for nematodes 298 cost 43, 316-20
hydrophobic 137, 162, 175 sealed 175,322 of early microbials 98
immature 175 sterilized 239 as research priority 108--9
metabolism, survival by see also bags trends in 99-101
reducing 156 contaminants 324, 378 see also cost elements in individual
moisture content of 156, 174, 330 in fermenter 168,169 entries
in nature 166-7 in formulations 10, 98, 164 COST 816 programme 319
in oil 138, 216, 221, 222, 312, in storage 159-60,318,322 cost analysis 175,317-18
323--4,350 suppressants of 240, 248 cost/benefit analysis 102, 240, 253,
pelleted 224 Contans 189 300,327
physiological studies, future 173, Continental clay 154, 337 cost-efficiency 98, 206, 279
176 controlled droplet application Costelytra zealandica 273
powders 167 (CDA), see droplet application cotton 59, 189 190, 191
pre-drying, effects of 160 Convolvulus arvensis 221 aqueous extract of 350
production systems 135 cooling systems 168 dose rates for 373,374
on solid substrates 166-70 Coomassie Brilliant Blue 355 NPV distribution on 374
in submerged culture 207 co-polymers 269,271 pests on 41, 62, 63--4, 66
protein, role of 207 copper 160 refugia, planting 107
rehydration of 143, 166, 174, 323 copper carbonate 79,81,350 simulated rain and 60--1
rodlet layer on 137 copper hydroXide 81,302,350 spray viscosity for 369-70
seed 171 copper oxide 81, 350 transgenic 86
shelf-life of 156,172,176 copper phosphate 81, 350 treatment of 45, 47, 54, 66
size of 175 copper sulphate 81, 350 UV radiation and 67
stabilizers 351, 353--4 co-precipitation 37-8,40 cotton, aqueous extract of 350
sticky 170 cork 93 cotton bollworm 41
storage in oil 143, 159-62, 160, com 191, 262 cotton leaf 84,351
161, 166, 173,174, 175, 198 aqueous extract of 350 alkalinity 20, 77
storage in powder form 158, 165, endophyte 87, 148 cotton seed 257, 272-3
172,176 super-sweet 260, 264 cottonseed flour 62,63,350,362
submerged 170 transgenic 86 cottonseed meal 63, 67
390 Index
cottonseed oil 45,46,62,63,65, 161, cuticle Desmonium tortuosum, see Florida
162, 336, 337, 343, 350, 355, charge on 172 beggarweed
356,357,358,359 insect 21, 132-3, 137, 147, 174,315 detergents 23, 79, 80, 85, 175,327
tannin-rich 103 leaf/plant 213,315 see also surfactants
courts of action 237, 253 Cutinol 140, 141,347, 350 deterioration 54---5, 318
coverage 315 cutworms 41, 64, 79 see also individual organisms
cress seed 274,275 black 301,303,304,305 development costs 317
crested wheatgrass 137, 139 cyanobacteria 236, 237 DeVine 204,206, 207, 320
crickets 172! Cyasorb UV 531 145,355,356 dew 20,78,217,221,227
Crodofos N3N 57, 345 cyclamen 188, 189, 190 dew period 209-10,219-20,221
crop monoculture 62 cyclamines 57,58 dextran 156, 339
crop-protection chemicals 213 Cyclocephala hirta 304 dextrin 52,60,249,342,348
crop-spray residues 168 cyclohexane 96 dextrose 348
crops cycloheximide 248, 353 Diabrotrica spp., see rootworms
field 190, 191, 193 cyclones in extraction system 168 3,6-diamino-lO-methyl acridinium,
fruit 190 cycloserine 80, 350 see Acriflavin
nursery 190, 191 Cydia pomonella, see codling moth diamondback moth 41,63-4,348
ornamental 190, 193 cyfluthrin 196, 302, 350 diatomaceous earth 135, 152, 154,
vegetable 190,193 cynazine 212,213 155,159,197,207,224,227,
croscarmellose 249,339 cypermethrin 142,350 261, 295, 337, 341
crospovidone 249 cystine 80, 350 2! ,7'-dibromo-5'-(hydroxymercuri)-
Crotalaria spectabilis, see showy Cythion 302 fluorescein 357
crotalaria cytoplasmic polyhedrosis virus dicamba 212
crown gall disease 190 (CrV) 67 Dicaperl 92, 339, 342
crucifer flea beetle 299 dichloran 196,351
crucifer seeds 274 2,4-D herbicides 212 diclofop 212
crumbles 188 Dacagin 65,339 Dictyostelium 247
cryopreservation 298, 298 Dagger G 273 die-casting 252
cryoprotectants 175 Dalton's law 378 Diegall 193,190
Crystal Violet 248, 353 damping-off 188,193, 194,195,225, dielectric drying 251
crystals, toxin 34---5, 36 226,245,265,273,274,275, dienochlor 302
allelochemicallsynergist 276 diesel oil 159,371
reaction 103 Dar-mol Molasses 362 diethanolamine-4-
denatured in sunlight 89 Darvan No.4 342 methoxycinnamate 357
dissolved 88 Datura stramonium, see jimsonweed difenzoquat 212
encapsulated 96 decanoic acid 96 diflubenzuron 302
enzymatic breakdown of 98 de-dodecylamine hydrochloride 57 dihydro-2(3-H)-furanone 339
extraction technique 100 deep-freezing 153 1,3-dihydroxy benzene 352
genes for 108 deflocculants 341 2,2'-dihydroxy-4,4'-dimethoxy-5,5'
hydrophobic 44, 56 defoliation 85 disulphobenzophenone 358
partitioning into droplets 49 degradation of organisms 314-15 2,2-dihydroxy-4-
radiation effects on 106 see also individual organisms methoxybenzophenone 144,
rain and 59 dehydration 166,240,247,248,272 362
sunlight and 67, 74 Deinococcus radiodurans 330 2,2-dihydroxy-4-
temperature and 53 delicacy of organisms 312 octobenzophenone 146
cucumber 139, 191, 199 delivery systems 93, 95, 99, 278 diluents 16, 154, 247
cucumber mildew 148 deltamethrin 85, 350 see also carriers
cucumber seed 194,264,273,276 De-mol Molasses 362 dimethirimol 148
Cucurbita texana, see Texas gourd Dendrolimus, see tent caterpillar dimethylgliotoxin 198
cucurbits 189,193 density separation 365 dimethylpolysiloxane 217
Culex 87, 88, 92, 93, 94, 96, 97, 106 Deny 189 dimethyl sulphoxide 82, 163,248,
Culex pipiens 95 desiccants 22, 198, 321,322, 341-4, 351,353
Culex quinquefasciatus 88, 94 378 dimorphism 207
Culicinomyces clavosporus 133, 152, mycoinsectide 154, 156-7, 158, 159, dioctyl sulphosuccinate (DSS) 147
153 167,174 Diospyros virginiana, see persimmon
Culigel polymers 93, 94 desiccation 218,219,221,247,269, tree
Curcuma longa 355 278,324,325,329,330 Dip-88-EE 370
Curcumin 69,355 nematodes and 291, 292-3, 299, Dipel products 54,60-1, 65, 302
customization 320 300,305 dipicolinic acid 82, 351
 Index 391
dipotassium evaporation of 45, 65, 141, 210, skin staining from 8
hydrogencarbonate 351 368-9,369 sunscreen 69-70
Diptera 79,108 formulation effects on 369-72 tracer 47,60,137, 172,355-8
disodium formyl-m- number for impaction 17 see also named dyes
benzenedisulphonic acid 356 size 16, 44, 47, 48, 66, 75, 108, 109, Dylox 302
disodium-,6-glycerophosphate 351 313,316
dispersants 23, 24, 37, 52, 56, 93, 94, effect on liquid flow rate 370 EAR XB400 356
314,341-4,378 and efficacy 45 Echinochloa crus-galli, see barnyard
fungal 142,157 and evaporation 45 grass
for soil organisms 249 in fungal applications 137, ecology
disulphide bonds 79 139-40, 169, 172 host/target 312
DiTera 190 and interaction with microorganisms 312, 328, 330
DNA, see genetic engineering particles 372-4 ectomycorrhiza 236, 277
docks 218 in mycoherbicides 223, 251 Edelex oils 49
dodecylamine 82,85,351,362 relationship with number 369 EDTA 78,79,83,351
dodecylamine hydrochloride 85, size spectrum 16, 57, 146,367, education, user 253,319
345 368,372 effervescents 91, 249
dolomite 271 spore-loaded, high 223 egg albumen 61,62, 66, 72,360
Dormal 267 spread 17,75 egg lecithin 97
dormancy 156,198,238,239,240-1, surface/volume ratio 368-9 eggplant 197, 199
248,253,320-1,323,326,329, target impact/retention 370-2 Eichhornia crassipies, see water
379 see also crystals of toxin; sprays hyacinth
see also individual organisms dropping bottle 97 Elcar 39
dosage 56,206,218,220,238,313, drought 151 Eldana saccharina, see sugarcane stem
372,373 drums borer
DOT, see oxygen, dissolved coater 259 electrostatic forces 45, 59, 209, 299
Dowanol TPM 47, 342 priming process 263 Emcocel 249,339, 342
Dow Coming 550 Fluid 152 rotating 261, 271, 277 Emcosoy 249,342
dowels 188 side-vented 259 emulsifiable crop oil 336
downy mildew 313 dryers emulsifiers 25-6,141,147,162,219,
drag, frictional 209 fluid-bed granulator 170 220-2,220,314,341-4
drenches 149,174,276 forced-air convection 261 lipid 79
Dreschlera monoceras 213,214,224 ventilated cabinet 167 emulsions 25-6,96,314
Drierite 158, 342 dry-harvesting 168-9 cost of 173
drift, spray 26,45,99,208,315,368, drying 168,170,242,250-1,314, formulation from aqueous
369 321-2 concentrate 50-I
in water 105 atmospheric tray 251 fungal 137, 139, 147
see also wind costs of 241 invert 26,173,199,206,219,220,
drilling, fluid 264-5, 325 co/counter-current 251 220,222,222,227,313,324
Dri-mol Molasses 71, 362 dielectric 251 liquid, stability of 10
drip applications 238 microwave 251 oil 45,48, 139,216, 221, 223, 313
droplet application, controlled stabilization 36 oil-in-water 100, 139, 221, 324,
(CDA) 12, 13,45, 100, 101, of treated seed 258,259,261 325
137, 138, 156, 172, 173,313, see also air-drying; desiccants; preparation of 221
368 freeze-drying; pre-drying; water-in-oil 90
droplets spray-drying; tumble-drying; wetters as 56
active particle content 17 vacuum drying Emultex 141,347
behaviour of 16-18 Dupont Spread Sticker 60, 347 encapsulation 26, 61, 102, 148, 154,
classification by size 367-9, 368, durum wheat flour 226 225,227, 314, 315, 327, 329
369,369 dust applicators 13-15 alginate 154, 227
concentration of organisms dustiness 101,250,259 for aquatic products 95-6
in 313,316,372-3,372,372 dusts 22,42-3,74,142,151-2,156-9, buffering 78
see also dosage 174, 223, 242, 275 cost of 102, 103
contact area/angle 370-2,371 dyes electrostatic 26
coverage 315,373,374 all colour groups 19,20,69-70, mycelial 142
diameter of (NMD, VMD) 49,90, 242,269,355-8,359 seed 262-3,262,275
138, 223, 367 cationic 357,358 see also seed coating
distribution, fungal 137 in encapsulation 26 specialist companies 103
empty 316, 373 indicator 357 spray improvement with 53-4
392 Index
encapsulation (cont.)
sunscreen in water 89
see also autoencapsulation;
bioencapsulation; capsules;
microcapsules
encrusting, see seed coating
endive seed 260
endomycorrhizae 236
vesicular-arbuscular 236, 245,
277
endophytes 87, 106, 107, 148, 270
8-endotoxin 38,86,106, 107
see also Bacillus thuringiensis
crystals
end-use container 169,379
Energol WT-l 91,336,347
energy store 320, 326
energy use measured by
respiration 320
energy utilization strategies,
nematode 293
Enhance 211, 345
enhancin protein, GV 82
Enterobacter cloacae 194, 264, 273,
276,339
Entice 63, 351
Entomophthora 135
Entomophthorales 133,133, 170
entomopox virus 44, 64, 67
entrapment 93, 104, 105, 199, 226
environment
adaptation to 278
impact on 300, 306
soil 238
environmental friendliness 2,99,
205,312
environmental persistence 98
enzymes
antagonism and 197
germination stimulated by 24
inappropriate adhesion 207
oxidative 73, 75, 77
stability of 164
in sunscreens 360-1
toxin crystals attacked by 54,74,
98
Epic 190, 193,272
epiphytes 54, 100, 107
epithelium 79, 80, 81
epizootics 108, 173
equipment
calibration of 313
filmcoating 258
on-farm 258
pelleting 260-1
sowing 265
Erinnyis ello GV 53
Erio Acid Red 60, 65, 69, 356
Erio Yellow 356
Erwinia 54 autoxidation of 55
Erwinia amylovora 189,193 oxidized 160
Erynia 133 fathen 214
erythritol 171 fatty acids 24,96, 162, 173,217,247,
essential oils 336 292,302,336,344,348,351
Esterol123 141,347 feedback, researcher to user 311-12
Etalfix 218, 345 feeding attractants 379
Etcos 30 66 feeding avoidance 49
2,2'-(1,2-ethenediyl)bis(5(4- feeding behaviour
methylamino)-,B- aquatic 87,88,89,95,99-100
(phenylamino)I,3,5-triazin-2yl nematode 293-4,303,304-5
aminobenzene sulphonic acid, feeding depressants/inhibitors 79,
see Phorwhite AR 82,83,95,104,142,350,351
2,2'-(1,2-ethenediyl)bis(5(4-(4- feeding stimulants 79,91,95,199,
morpholinyl)-6- 378
(phenylamino)-1,3,5-triazin- see also phagostimulants
2y-yl)amine- fenoxaprop-ethyl 212
benzenesulphonic acid, see fermentation
Tinopal LPW Bacillus thuringiensis 36-8
ethoxyethanol 18, 45 bag 167, 168, 169, 175
2-ethoxyethyl-p- deep liquid 37,97, 135, 169,
methoxycinnamate 68,356 170-1,176,274
ethoxylated p-aminobenzoic Hyphomycete 171
acid 68,360 light regimes and 208
ethoxylated ester 350, 362 Metarhizium mycelium 170
ethoxylated isostearyl alcohol 97 moist powder 242-3
ethoxylated tridecyl alcohol 166 monoxenic liquid/semi-solid 291
ethyl acetate 47, 53 neutralization 244
ethyl cellulose 249, 250, 342 scale-up 170
ethyl cinnamate 144, 146, 356 semi-solid 36-7
ethyl hexanol 94 solid substrate 48, 167, 208, 241
ethyl oleate 141, 347, 349 in storage 243
ethyl trans cinnamate 356 submerged 206,207,241
ethylene bromide 239 wet 90
ethylenediaminetetra-acetic acid, see see also individual major organisms
EDTA fermenters 266, 291
ethylene glycol 371 air-lift 171
ethylene oxide 344, 345, 346 deterioration in 54
2-ethylhexyl salicylate 68,356 rotary 169
Etocas 30 51, 58, 345 fertilizers
Eudragit 250, 342 added to alginate 196
Euphorbia esula, see leafy spurge chemical, compatibility with 302,
Eusolex products 69, 76, 144, 356 314,325
evaporation 18, 65, 173, 209, 219, see also individual major
369 organisms
conidial 137 self-replicating 312
droplet 65, 141,210,368-9, 369 field activity 173, 174,204, 205
as a drying process 251 field bindweed 217, 221
from plant surfaces 210, 211 field crops, tank mixes for 65-6
reduction of 45, 146 field pansy 208,214,221-2,222
Ex-eel 249, 339, 342 field reliability 217, 228
fJ-exotoxin 38 field studies/trials 3,74-5,77,80-3,
Explotab 249, 339 99, 105, 108,325
extrusion 246, 249, 265 filamentous growth control 171
machines for 252 fillers 336-7
see also noodles cost-reducing 225
ratio to pathogen 51
Fast Blue 69, 356 for wettable powders 23
fats see also carriers
 Index 393
filmcoating, seed 276,380 foaming 23,303,314 spray-application
particle grade 278 fogging machines 13,18,45,46 intereactions 228
pelleting and 273,274 fogs 18,45, 140 sprays
techniques for 258-60 foliage dry climate 137-9
binder system 261, 274 applications to 210 wet climate 139-42
side-vented drum 259 deterioration of organisms on 55, sticky 209
spouted-bed system 258-9 324-5 sustained-release 224
filters, nylon mesh 23 lower surface of 374 tailored / on-shelf 327
filter-feeding larvae 95, 105 penetration of 45,90-1 trends/future 97-109,253,
filtration 98, 206 pH of 76 311-31
fire ants 151-2 protection of organisms on 77-8, ULV-eDA 172
fireblight 193 102 for use in water 104-9
fish, dried 108 rapid growth of 40, 106 water-based 51
fish glue 348 spore pick-up from 139 water-retaining 228
fishmeal 95 spray cover of 172,373 foxtail 227
fish oil 336 upper surface of 374 Frankia 236, 237
flamprop-methyl 212 waxy 327 free-flow agents 42, 91, 92, 92, 157,
flask culture 170, 171 water-repellent 62 174,314,325,341-4,379
Flavobacterium 54 wet/dry fluctuations on 146 free-flow material 52
flocculants 170 foliage ca!'opy 90, 100,300,316, see also flowabies
flocculation (reversible 324,374 free radicals 77, 247, 322, 327, 330,
clumping) 24, 317 folic acid 68, 76, 199,360 379
flooding 93 Folicote 351 57, 345 freeze-drying 38-9,51,98, 100,
Florida beggarweed 218, 219 Fames annosus 193 155-6,159,250-1,321
F10risil 144, 362 food-grade additives 10, 77, 103, freeze-thaw survival, spores 165
flotation 106 175,228,324 freezing
see also buoyancy; formulations, food sources for soil organisms damage from 247, 248
floating 250 deep- 153
flotation additives/agents 92,92, Foray 60-1,346,347,348,349 see also cryopreservation
93,341-4 formulations 1, 204, 378 friability 38
flotation coefficient 96 aquatic 106 friction 249
Flour 961 70, 339 aqueous 98 frost 189, 193
flour 62,64,88,95,140,142,146,301 buoyant 87-9 frozen cultures 239-40, 239
see also cereal flour; cornflour; costs 316-18 fructose 351
wheat flour nematode 294 fruit 189,190,195
flowability 23, 250, 252 deterioration in 54-5 fruitworms 41
flowable concentrates/products 48, dormant aqueous/oil 239 fuel oil 65
49,92, 141, 378 dry 22-4,42-4,97,98,99,154, Fulvia fulva 191
development of 51 204-6 fumaric acid 82, 351
oil-based 49 effects on droplets 369-72 funding, development 204
water-based 48 floating 88-9,91-5,104,152 international 319
flowables 89-90, 101, 104, 109, 142, frozen 239-40,239 fungal sprays 137-48
172,227 functions of 7, 9-21 fungi
flower seed 260, 263, 265 for germination acceleration activation of 343
flowers 189 173 additive effects on 210-16
fluid bed apparatus 168 for insect control 40-55 adjuvants for 350-2
granulator 170, 171 integration with application application to soil 149-52
separator 167 systems 205 application to water 152-4
fluid bed drying 170,251,261,263 for land use 99-104 carriers for 336-40
fluid bed spray-coating 26, 276 limits of 326-8 contact-acting 102
fluid drilling 194,264-5,276, 277 liqUid 16-19,24-6,48-51,98 desiccants for 343-4
fluid suspension, application to multipurpose 105,316 frozen 20
soil 237-8, 238-41, 239 nematode 287-306 herbicide interactions 213
see also sprays in oil 51, 169, 209, 241 humidity 20
Fluorescein 356 patents and 217 industrial flow-lines for 134
fluorescent brighteners 359 for plant disease control 193-9 insect pathogenic 2, 21, 208
see also optical brighteners principles of 7-27 life cycles 132-5
fluorescent neuronal tracer 356 ready-to-use 172 mitosporic 133
fluorochrome 76,361 for soil inoculants 237-47 nutrients for 342
394 Index
fungi (cont.)
osmotic protectants for 354
pathogens 133
plant disease pathogenic 188-93
plant pathogenic 218
plant pathogens and 2
seed applications and 273-6, 259
slow-growing 278
solid-substrate production
of 166-70
spore harvest 109
stabilizers and 344, 353-4
stickers for 347-9
storage of 9, 109
sunlight susceptibility of 142-6
sunscreens for 360
surfactants for 345-7
surfactant toxicity 214
suspenders for 341
synergists 352
temperature and 20
water relationships of 135-6
wetters for 57
see also conidia; hyphae; mycelia;
spores; individual genera
fungicides 99,162,174,265,269,272
added to alginate 196
compatibility with
organisms 148, 213, 241, 302
in granule formulations 295
resistance 317
fungistasis 48, 150, 174, 379
furnace oil 58
Fusaclean 188, 190
Fusarium 189,190,191,193,224,242,
272,339
Fusarium graminearum 276
Fusarium lateritium 217, 218, 225,
226
Fusarium moniliforme 188, 189
Fusarium oxysporum 188, 189, 190,
226,227
f.sp. dianthi 197
Fusarium solani 339
f.sp. cucurbitae 198, 224--5
Gaeumannomyces graminis 197
see also take-all
galactose agar 63
Galleria mellonella 41,50,57, 292
gallic acid 83, 351
Galltrol-A 190, 193
gamma irradiation 106,239,243,
252,267
gas exchange 168,169,264,266
gases in storage 10
Gasyl GM2 157, 159, 167, 170,339,
342
gel, thixotropic 329
gel beads 196,252 glyceryl palmitostearate 249, 343
gel encapsulation systems, glycosides 357
seed 262-3 glyoxal 92
gelatin 26,44,54,61,91,140,141, Gold-Coat 267
163,249,250,342,34~353,360 Golden Bear Oil 91
fish 92 gossypol 162, 337
Gelgrade 93 Graessorb products 144,146,357
Gellan 342 grain 168
gellan gum, see gums grain pests 104
gelation 195,252,378 gram, potted 64
gellants 341-4,378 granulation 251, 379
gelling 43-4, 249 granules 146,244--5,314,315,316,
gelling agents 97 325
gene, toxin 86, 106, 108 absorbent 149
genes, crystal protein 108 alginate 224, 245, 249
genetic engineering 2,74,86,98, applicators for 11,13-15
103,106-8,278,305,306,317, coated 227
327,329,330 controlled-release 105
see also transgenics cost of 91,99, 101
gentomycin 353 definition of 22-3, 43, 379
Geon Latex 60,348 dry 170, 171
Geotrichum candidum 189, 193 dual-purpose (land/water) 109
gerbera 190 fabricated 245-6
germination fast-release 91, 92
inhibition of 79,80, 82, 83, floating 91-3, 105
212-13,214,216 flow able 244
stimulation of 24 fungal 149-51, 158
see also conidia; spores grain 150
germling viability 216, 217 heavy sand 92
ghatti, see gums herbicidal 223-7
Gibbago trianthemae 217 for insect control 43-4
glasshouses 221 low-cost 167
see also greenhouses for LV /HV sprays 101
glidants, see lubricants mycelial 170
Gliocladium 236, 237, 241, 250 nutrient-soaked 168, 169, 225
Gliocladium roseum 195, 197 oil-coated 227
Gliocladium virens 188,191,195,197, for plant disease control 187,
199,245,274,339,343,346, 195-7
349,362 size of 225, 226
GlioGard 191, 245, 246 slow-release 91-3,94, 225
gliotoxin 198 for soil applications 237-8, 242,
gluconate 252, 339 243-6
glucose 63, 152, 155, 159, 163, 165, starch-based 99
170,171,249,342,351,353 sustained-release 92-3
glues trends for 100, 101
fish 60,348 for use in water 90-6, 104--5
synthetic 249, 344 water-dispersible (WDG) 24, 43,
wallpaper 269, 344 52-3,101,135,169,188,244,
glutamic acid 68,80,351,360 246
glutamine 79,80,351 nematode 292,294,294,295-8,
gluten 60,62, 72, 76, 197, 199, 225, 295, 297, 301, 306
226 see also capsules; pesta; prill
glycerin 65 granulosis virus (GV) 35, 35, 40, 42,
glycerol 92,140,141,146,161,164, 59,82,86
164,171,214,214,219,225, sunscreens 68-73
240,248,322,339,342,353 grape 66, 188, 189, 191, 193
p-glycerophosphate 83, 85 grape berry moth 66
glyceryl behenate 342 grape pomace 351
glyceryl monooleate ethoxylate 353 grape seed oil 216, 336

grass grub 273
grass meal 63, 351
grass seed 260, 263
grasses, weed 212
grasshoppers 21,35,44, 77, 301, 303,
318
fungal control of 135, 137-9, 146,
151,156,169,172,174,241
phagostimulants for 64, 67
greenhouses 18,53, 133, 135, 140,
143,191,223,225
foggers in 45
glass/sunlight factors 19
simulated rain in 60
see also glasshouses
Green Muscle 135,169
grinding 36,38, 101, 169,226,242,
314
equipment 39
heat during 55
Grip 269
groundbait 63
groundnut oil 159, 161, 162, 216,
336,355,356,357,358,359,371
growth promoters 188,194,236,
245, 272, 273, 277
growth regulators 213, 302
growth retardants/
suppressants 173, 209,237,
240, 248, 327, 350, 379
guanine 71, 360
guanosine 80, 351
guar, see gums
Guardcoat 270
gums 59,215,273,314
acacia 61, 215, 215, 216, 249, 348
arabic 37, 269, 270, 271, 342, 348
carob 267, 339
gellan 216
ghatti 215,215,216,348
guar 59,61,215,215,249,342,348
karaya 61,194,215,348
locust bean 61,194,215,348
plant 194,215
polysaccharide 24
sticker 141
tragacanth 61, 194, 342, 348
vegetable 59
xanthan 47,48,61,65,194,215,
215, 216, 249, 267, 273, 342,
348,353
Gusto 63
Gustol 63, 351
GV, see granulosis virus
Gypchek 39, 65, 77
gypsum 42,245,252
gypsy moth 41,60-1, 62, 65, 102-3
modelling 99
phagostimulants for 63-4, 66
sunscreens and 77
synergists and 84, 85
tannin and 78
gypsy moth NPV 77
gypsy moth polyhedrosis virus 39
haematoxylin 69, 357
haemocytes 82
haemolymph 34, 360
half-life 48, 328
handling 10, 65, 102, 317, 322
Hank's salt solution 163, 165,353
harvest 8, 167, 168, 169, 175, 176
semi-automated 291
see also individual major
organisms
hazard categories 103
hazards 99
heat, see temperature
heat, frictional 261
heat protectants 97-8
heat tolerance 314
heat transfer 168
helicopters 13
Helicoverpa armigera 56, 57-8, 59, 63
stunt virus 107
Heliothis 41, 46, 65-6, 107,374
Heliothis NPV 38, 45, 46, 57-8, 61,
63-4,74,75
Heliothis punctiger 61
Heliothis virescens 38, 63, 374
Heliothis zea 63, 78, 133
see also cotton bollworm
hemp sesbania 218,219,220,221,
223,226,226,227,350
herbicides, chemical 210, 212, 225,
302,325
herbicides, microbial 2, 203-28
adjuvants for 350-2
binders for 342
carriers for 336-40
commercial 204
dispersants for 341
efficacy of 208-10
foliar application of 210
fungicide reactions 213
humectants for 341-4
resistance to 209
selective 209
solid formulations of 223-7
specificity of 219,226
sprays 216-23,205,206
droplet size 368
stabilizers for 353-4
stickers for 347-9
surfactants for 345-7
Heterobasidium annosum 191,193
Heterorhabditis
biology 291-4, 292
Index 395
life cycle 289-90
see also nematodes
Heterorhabditis bacteriophora 293-4,
304,305
Heterorhabditis megidis 293, 293
hexylamine 57, 85, 345
High Tack Fish Glue 60, 348
Hipure Liquid Fish Gelatin 342
Hirsutella thompsonii 133, 141, 142,
147,170,345-6,349
HI-SIL silica 346
Hi-Spread-Casein 57, 345
histidine 68, 80, 351, 360
HLB, see hydrophile-lipophile
balance
homomenthyl salicylate 68, 357
honey 41,155,163,269,342,353
honey bee 138
hopper-boxes 193, 237, 244, 258,
271,272
homworm 44
horse serum 163,164,165,353
host/pathogen balance in
nature 329
host plant resistance 78
host plants, land 41
host ranges 133,219,220
hot spots in substrate bulks 168
hot water treatment (seeds) 265
house fly 301,303
HPPL HiStick 269
Hugtite 348
humectants 18, 20, 45, 75, 322, 324,
326,341-4, 379
for microbial herbicides 214,214,
218
for mycoinsecticides 141,146,
173
for plant disease control
formulations 198,200
humidification, seed 264, 273
humidity 20,55, 139--42, 156-8, 173,
175, 216, 221
effects on persistence 19
high 209,210,218-19,315
low 138,139
see also relative humidity; relevant
individual entries
humidity chamber 171
HV, see sprays, high volume
hyaline mutants 330
hydramethyluon 303
hydration 216, 264
hydrocellulose 65
hydrogen chloride 199
hydrogen peroxide 322
hydrophile/lipophile balance
(HLB) 24, 25, 25, 166
hydrophilic reaction 216
396 Index
hydrophobicity 44, 56, 59, 137, 152,
162, 170, 209, 324
hydroxycellulose 65, 339
hydroxyethyl cellulose 194,265,
276,295,349
hydroxyethyl starch 164,353
2-hydroxy-4-
methoxybenzophenone 356,
357
2-hydroxy-4-methoxy-4-methyl
benzophenone 357
2-hydroxy-4-methyl-5-
sulphobenzophenone 362
2,2-hydroxy-4-
octobenzophenone 144, 362
2-hydroxy-4-(octyloxy-phenyl)-
phenylmethanone 356
hydroxypropylmethyl cellulose 47,
249, 250, 342
hygroscopic additives 55,97, 146
hygroscopic powders 38
Hymenoptera 108
hymexazol 274
hyphal body fragments 240, 241,
245,277
see also blastospores
hyphal growth 197
Hyphomycetes 133, 133, 153, 155,
166, 170, 171, 379
hypoxanthine 71, 360
iatrogenic diseases 213
Igepal CO-630 57, 345
Illite 154
imazethapyr 212, 213
imbibition 174,242,250,264
IMC 90-001 UV screen 362
imidacloprid 148,323
immersion techniques, seed 263-5
immunoblotting method,
colony 271
impregnated sticks 188
impregnation, seed 270
inactivation 326-7
Incide 87
inclusion bodies, see polyhedral
inclusion bodies
incompatibility 314
India ink 70, 362
Indigo Carmine 69, 76, 357
5'5-indigosulphonic acid 357
indoles 227
indulin 70
Indulin AT 342
induration 251-2, 379
inert ingredients
and aquatic larvae 95
hazard categories for 103
infectivity, retention of 154
inhalation by operators 22 insecticide traps 303
injectors 15,262 insecticides, chemical 3, 85, 290,
inocula, solid 223 300,303
inoculants 205,206,379 compatibility with organisms 93,
application of 325 148,241,301,312,325
combination 241 synergistic 304
distribution on target 208, 209 see also pesticides
dry method for 271 integrated pest control 188,279,
mortality rates and 271 300,316
mycorrhizal 277 Intercept 190
protection of 325--6 international programmes 318
rhizobiaI 265-71 international unit (IV) 88
peat-based 268 Intrawite products 71, 77, 359
soil 225, 236-53 inundation 206
inoculation, seed 256-65, 269-71 Invade 320, 325
Inopus rubriceps, see sugarcane soldier ion exchange 247, 249
fly see also calcium; potassium; sodium
inositol 68,163,353,360 ionic gradient 305
insect body temperature IPRI-DF-l cells 78
adjustments 20, 174 iron 157, 160
insect cuticle iron oxide 353
adhesion to 137, 147, 315 irradiation, see gamma irradiation
charge on 172 irrigation 209, 299
chemicals on 21 drip 199,238,299
infection through 132-3, 179 irritants 174,331
insect debris 38, 44, 73, 75, 98, 329 isolates, new 253,300,305
insect growth/moult 137,173 isoleucine 80, 351
insect growth regulators 301 iso-octyl p-aminobenzoic acid 68,
insect gut 53, 77-9, 80, 82, 84, 86, 360
173,301, 328 iso-octyl p-dimethyl aminobenzoic
dormancy termination acid 360
within 323 iso-octyl-phenyl ether 218
flora of 100 Isopar products 46,336,371
Na/K transport in 80 isoparaffins 97
oxidative/alkaline activity in 102 isophorone 47, 371
pH in 78 isopropanol 44, 199
insect haemocoel 290 iso-propanol 217,348
insect larvae, virus production 4-isopropyl-dibenzoyl methane 356
with 38-110 isostearyl alcohol 97
insect/plant interactions 78, 103 itch grass 219
insects
aquatic 19,87-9 Japanese beetle 290
biological seed control of 272 Javelin 54
boring 301 Jetcote 270
ecology of 41 jimsonweed 217,219,219,220,350
foliar-feeding 276, 299, 300, 301 Johnson grass 219
food intake of 62, 78 Jordan clay 154, 337
foraging/nesting 151
formulations for control of 33-109 kaolin 95, 207, 208, 218, 224, 224,
fungal pathogens on 133 225,226,262,262,269,338,344
hemimetabolous / Kaolin China Clay 52
holometabolous 303 kaolinite 154, 157, 197, 337, 338
humidity around 319 Karion F 164,339
peroral control of 31-109 K-carrageenan, see carrageenan
soil-dwelling 104, 149, 276, 301, Kelgin products 195, 216, 339, 342,
325 348
target species 301 kelp 195
see also larvae; individual species/ Keltose 45, 64, 342, 362
genera Kelzan 47, 153, 156, 342, 348, 353
 Index 397
kerosene 90, 161, 162, 169, 336 leaf cuticle 213, 315 lignosulphate 23, 342
deodorized 336, 371 conidia distribution on 139,315 lignosulphonate 39, 61, 70, 75, 76,
odourless 159 leaf extract 63 77,295,362
Kitchen Aid products 246, 250 leaf feeders, undersurface 41, 45, lime 57
Klebsiella 54 140 as seed coating 269,270,270,271,
KLX 343 lea f rollers 41 272,273
Kodiak products 190,193,242,272, leaf spot 214 see also calcium carbonate
273,279 leaf surfaces 377 limestone, ground 269,270,338
Kollidon 249 alkaline 66, 76, 77, 78 linoleic acid 161, 163, 336
Kolomona weed 203 chemicals on 20, 26 linseed oil 161,216,336
Kraft pine 342, 343 lipophilic/hydrophobic 137 Liparol 97
Krausella amabile, see grasshoppers upper /Iower 67, 100, 368, 373, lipid content 207,291-2,299
374 lipophilic barriers 213
labelling 319 leaf wetness 315,319,325 lipophilic surfaces 137
Labrafil 157, 353 leaf whorls 142 lipophilic surfactants 147
Lacanobia oleracea 41,57 leafy spurge 227 liquid paraffin 161, 336, 353
lactamide-B-mercaptoethylamine, see leaves Liqui-Prep 240, 271
LAMEA frictional drag on 209 loading time 104
lactic acid 53, 161, 322, 336 oil absorption by 143, 146 locust bean 61,215,348
lactose 23, 37, 51, 63,95, 155-6, 163, penetration of 222 locusts 35, 132, 135, 137, 138, 142,
197,339,353 pick-up of spores from 172 148, 151, 159, 164, 166, 172,
lactose-acetone co-precipitation 37, spore survival on 173 313, 318, 350, 370
40 spray spread on cryptic parts Lodige high-shear mixer 250
Laetisaria arvalis 194, 265, 349 of 57 looper larvae 41,42
Lagenidium giganteum 154 water repellency of 147 Lovo 192 57,345
lag period 140,166,239,240 see also foliage LUB1LOSA programme 318
l.a.i., see leaf area index lecithin 97, 164, 165,219,220,343 lubricants 249,341-4
LAMEA 214-15,214,343 Lecithin II-S 343, 353 Lubritab 249, 343
laminarin 262 Lecithin IV-S 353 lucerne 84,87,268,269
Laponite 274, 339 leek seed 260, 262 see also alfalfa
larvae legumes 190 LV, see spray, low volume
aggregation of 95 Rhizobium symbiosis with 236, Lymantria dispar, see gypsy moth
behaviour of 40-2,41 240,266 lyophilization 240-1,242
boring 301 seeds of 236, 257, 260, 261, lysine 61, 68, 80, 351
breathing of 96, 97 269-70,271 Lysine KKL 348
cryptic 59 Leonardite shale 264, 273, 339
lethal dose for, measurement Lepidoptera 35, 79, 86, 87, 108, 133, machinery
of 48-9 301 application 253
looper 41, 42 Leptinotarsa decemlineata, see Colorado batch 258
multi-instar populations of 99 potato beetle macroconidia 218, 223
neonate 41 lethal dosage 45,47, 48-9, 313, 316 macrocysts 247
noctuid on cotton 373,373 lettuce 54, 63, 65, 66, 151, 189 macrospores 250
stress in 78 lettuce seed 257, 260, 262, 262, 263, Macrotermes michaelseni 152
vomiting by 48 264,276 magnabentonite 65
larval extract 61, 348 leucine 80, 249, 343, 351 magnesium 20, 77, 78
larvicidal powder 92 Leucophor products 71-2,77, 199, magnesium aluminium silicate 344
LarvXSG 92, 105 359 magnesium chloride 81, 94, 351
Laspeyresia pomonella, see codling LIFT 240 magnesium silicate 159, 362
moth lignaceous shale 338 see also talc
Later's surfactant 57,345 ligneous shale 263, 264, 276 magnesium stearate 249
latex 46, 65, 95, 347 lignin 62, 98, 101, 103, 292, 294 magnesium sulphate 81, 158, 248,
Latron CS-7 348 cross-linked 61 351,353
lauric acid 82, 351 as harmful to maize 42,107,190, 196,236
leaching 322 microorganisms 250, 338 maize dextrin 52
leaf allelochemicals 55, 66, 84, 324 kraft 53, 61, 70, 342, 343 maize oil 336
leaf angle 315 lignin sulphonic acid 362 maize seed 257, 258, 262, 270
leaf area index 367--8 lignite 195, 197, 268, 339 maize starch 151
leaf assays 80-3 lignite fly ash 142, 343 Malacosoma, see tent caterpillar
leaf attachment 59 Lignosite AN 65, 66, 362 Malacosoma neustria 61
398 Index
maleic acid 78 meniscus 262, 262 temperature and 164, 171, 174,
co-polymer 250 Merbromin 357 175,326
malic acid 82, 351 Mercurochrome 69, 357 UV susceptibility of 143
malt broth 215, 353 metabolic gradient 305 water activity and 137
malt extract 159, 171 metabolic products, nitrogenous 71, wetting of 147
maltose 63, 155, 164, 164, 170, 197, 75 young cultures of 172
248,353 metabolic wastes 174 methacrylate polymer 342
Malva neglecta 242 metabolites 247,249, 252 methacrylic acid 250
Malva pusilla, see round-leaved absorption of 159 methanol 18,45,345
mallow release of 162 methionine 80, 351
Mamestra brassicae 41,85 metalaxyl 193,302 methyl alcohol 218
Mamestrin+ 85 Metamucil 216, 343 3-(4-methylbenzylidene)
Manduca sexta, see tobacco homworm Metaquino 135 camphor 356, 362
manganese 20 Metarhizium spp. (M. anisopliae, M. methyl cellulose 61,194,197,249,
mannitol 243, 244, 248 flavoviride) 144-5, 148, 153, 250,343,348
Manoxol()T 57,345 158,326 in seed coating 273-7, 279
Manucol products 164, 215, 215, additives affecting physical methyl~thyl ketones 47
348,353 properties of 341-4 methyl-p-hydroxybenzoate 83, 351,
Manugel products 215, 215, 348 against termites 135 353
manure 150 bag method for 169 methylene bisdiamyl phenol
Manutex products 215, 215, 348 in baits 152 ethoxylate 346
markets 228, 253, 295-8 blastospores 158, 164 5-methylresorcinol 357
size of 109,170,172,317 carriers for 336-40 Metribuzin 212
specialized 312 conidia 133,141 Mexenone 145,357
marumerizers 252, 379 concentrations of 17, 165 microcapsules 39,77,96,101-2,200
masking 102-3,152,331 in emulsions 139 living 107
Mason jar 239 fresh, pre-soaked 142 methods of forming 26
matric potential/tension 136,136 on millet 169 microfiltration 40
Matricap 93 submerged 170 microgranules 152, 188
matrices 22, 43, 53, 77, 379 conidial chains 175 Micronair emission system 65
controlled-release 94 features of 133 microporous film 168
encapsulation 102, 329 fermentation, two-step 170,171 microsclerotia 207, 227
floating 93 frozen 153,155 microsponging 93
formulated 95 germination of 162,165 Micro-Ulva sprayer 139, 368
immobilization of nematodes granules against mosquito microwave 251,252
in 291 larvae 152 milk 62
mucilaginous 207 humidity and 156, 175 as dust sticker 22
polymer granular 93 in methyl cellulose 276 evaporated 269,343
wheat gluten 225 moisture content of 155, 156, 166, peptonized 72, 360
matriconditioning 263 321,330 skimmed 45,51, 61, 72, 98, 141,
matrix priming, see seed, solid-matrix in oil 138, 146, 159-60, 160, 161, 141, 146-7,153, 156, 163, 165,
priming 166, 169, 216, 312 336,348
maturation in storage 174,175 oxygen and 158,162 as membrane stabilizer 240,
Maximillion Brilliant Flavine/ in powders 159, 166, 168 242,248
Pink 357 pre-drying 158 as sunscreen 75, 76
Maywood surfactant 57, 345 rainfastness of 146 whole 61,348
Mazola oil 336 rehydration of 166, 323 milk powder 159, 351
MCPA 212 shelf-life of 174 Miller Nufilm 147,345
mecoprop 212 soil percolation by 149 millet 168, 169
melanin 71, 75, 98, 103, 329-30 spore number fluctuation in milling, wet 25, 55
genetic engineering and 106 time 149 milling qualities 169
from Streptomyces lividans 362 spread of 150 mineral oils 46,51, 141, 161, 162,
Melanoplus spp., see grasshoppers stabilizers for 353 163,216,249,252, 336-7, 351,
Me/olontha melolantha, see cockchafer surfactants/ wetters for 346 353
melt congealing 252 survival 150, 154, 162, 165 mineral soils 268
membrane inoculation 169,172 in drying 155, 156 Miragel 43, 339, 362
membrane protection 305 in field 149 Mirasperse 53
membrane stabilizers 155, 240, 242, in storage 136,319,330 Mishagi's liquid medium 262
247-8 synergists 350 misting 220,299,368

mites 133
miticides, chemical 302
mixers 22,42,92, 167, 196,220, 246,
250
see also individual mixers
mixing 250,258,263,314
wettable powder problems
with 23-4, 52
models 99,320, 321, 325, 330
mode of action 1, 2
of contact pathogens 129-285
of peroral pathogens 31-109
moisture content
equilibration of 9
optimum low 326
of soil types 136
in storage 55, 228, 320
see also water; individual
organisms
molasses 102,159,197,268
in bait 151
as binder 249
as carrier 39, 339
as feeding stimulant 21, 44, 59,
63-4, 66, 67, 351
in forest/field tank mixes 65-6
as nutrient 250, 354
as preservative 50, 102, 161
as sticker 21, 59, 61, 62
as sunscreen 20, 71, 75, 76, 77,
362
as thickener 21, 47, 341, 345
see also Dar-mol; De-mol; Dri-mol;
Mo-mix; ProMo
molasses, cane 275
molasses of peat 61, 71,348,362
mole cricket 299,301,303,305
molecular biology 228
molybdenum 241
Mo-mix 339, 351, 354, 362
Monilia laxa 191
monoacyl glycerol 343
monoculture 62, 219
monoglyceride 220-1,220
monomolecular layers
(monolayers) 96-7, 106, 109,
380
Monoxi 97
Montanox 80 52, 57
montmorillonite 151, 154, 197, 337,
338
calcium 151, 269, 337
Morrenia odorata, see stranglervine
mortality
additive 304
complementary 96
synergistic 300
Morton mixer 42, 52
Morwet 57, 92
mosquito control 23, 35, 87, 89,
90-2,96,97
mosquito larvae 19,87-8,90-1,91,
92,94,95,104-6,109
aggregations of 95
container-inhabiting 90, 92
feeding habits of 19,87,88,89-90,
316
as fungi host 133, 152
habitat 93
moth larvae 41
motor oil 216,336
moulting inhibitors 173
mowing 218
Mowiol 141, 348
mucilage 329
muck soil 194, 276, 339
Mucor pyroformis 189,193
mulberry 60, 78
Multifilm Buffer X 57,345
Musca domestica, see house fly
muscadine 133, 142
mushroom 189, 192
mushroom spawn bags 167, 168
Muson mixer 22, 92
mutagenesis 143
Mycar 141
mycelial fragments 141,152,259
mycelial homogenate 224, 224, 225
mycelial mat/pad 142,155
mycelium 132, 135
aqueous suspension of 204
biomass of 133
conidiating 151
dry-period survival of 155
entrapped (granules) 22~
fermentation of, mechanized two-
step 170, 171
growth optima of 136
harvesting of 206, 224
oxygen demand by 207
pelletized 208
preservation method for 170
as slurry 208
vacuum-filtered 170
Mycocentrosporum acerina 214-216,
217, 221-2, 222
mycoherbicides 2,131-185,198,
199,313
mycoinsecticides 2,131-176,134,
198
mycoparasites 250
mycorrhizae 236, 237, 277, 380
Mycostop 190,193,242,272
Mycotal 135, 140
Mycotrol 135,172,241
Myrothecium verrucaria 188, 190
Myverol products 220, 221, 343
Myzus persicae 140,141
Index 399
Nacrylic products 60, 348
Nalcontrol 47, 343
naphthenic oil 336
naphthol 336
heavy aromatic oil 161,336
Naturalis 135
nectarine 191
Nectria ditissima 191, 203
neem oil 161,162,351
Neemazal-T 82,351
N-gel 216, 343
nematicides 302
nematodes
applications with 295, 299-300,
301
combined 300, 304
extraction time from
carrier 295
foliage 299-300
future technology of 300-5
strategies 290
binders for 344, 352, 354
biochemistry of 292
carriers for 338
chemicals, compatibility
with 302, 304
containers for 298
cryopreservation of 298
desiccated 246, 292, 294-5, 305
desiccation of 291, 299, 300
tolerance 293, 305
droplets containing 294
drought-resistant 247
energy utilization by 293
entomopathogenic 199,287-306
entomophilic 2, 236, 246
environmental impact of 306
foraging strategies of 293, 304-5
formulations 289-306
considerations in 298-300
development of 291-8,292
future research on 300-5
granules with 246, 294-5, 295,
295-8
harvesting 291
infective juvenile
activity of 293
immobilization of 291, 294, 295
lipid content of 291, 292, 293,
298,299
mobility of 290
oxygen needs of 292, 293, 295,
298,320
partial desiccation of 294, 298,
298, 299, 322,323
phagostimulants and 350
refrigeration of 291, 293, 298
survival of 330-1
temperature and 291-2,294
400 Index
nematodes (cont.)
life cycle 289-90
market acceptance of 295-8, 299
membrane protection 305
metabolism 291
moisture levels 294
mortality 293
nictation 293, 304, 380
packaging ?98,305
pathogenicity 298
persistence 300
in pesta 300
physiology 305
and plants 188, 189, 190
production 290-1,298,298,305
costs of 290, 291, 295, 300
quality of 290, 298-9
shelf-life of 290,291-5,293,294,
298-300,304,305
stability of 9,298, 299, 300, 305,
306
storage of 298, 298, 299
strain differences in 291
symbiotic bacteria 290
target insects 301
traps with 303, 306
UV light and 299
water requirements of 20
see also individual nematode
genera
Neodiprion lecontei 65
Neodiprion sertifer, see pine sawfly
Neodiprion swanei 65
Neosyl 51, 66, 339
nicotine 78
nicotinic acid 68, 360
see also vitamin B
nictation 293, 304, 380
Nigrosin 70, 357
Nilaparvata lugens, see brown plant
hopper
Nipasorb 357
Nitracoat 269, 343
Nitragin Gold 242, 267
see also Pelgel
nitrifying ability 317
Nitrigum 269,343
nitrogen fixation 107,199,224,236,
23~265-6,268,269,277,278
nitrogenous compounds 352,360-1
nitrogenous metabolic products 71,
75,76
NMD (number mean diameter), see
droplets, size of
No. 9209 emulsion 139
No.2 fuel oil 46, 336
No.2 furnace oil 58
nodulation 250,265-6,268,269,270,
277,278
Nogall 190,193
Nolo Bait 64
Nomuraea 133, 135
Nomuraea rileyi 67,139,149
non-oxynol surfactants 214,217
Nonoxynol 345
nonylphenol ethoxylate 345, 346
nonylphenoxy polyethoxy
ethanol 211,345
noodles 246, 252
Norbac 84C 190, 193
Norpar products 46, 336
northern jointvetch 204, 220
Nosema spp. 35, 35, 42
Nosema locustae 64
Novemol 57, 345
nozzles 99, 251, 315
air-shear 45
angled 209
blocking of 23,36,53,62, 168, 169,
299,314
exhaust 156
flat fan 65, 92, 373
flow rate of 238, 369
hydraulic 23, 18, 49
upwardly directed 45, 140, 373,
374
NPPL HiStick 267, 268, 271
nuclear polyhedrosis virus
(NPV) 35,45-6
additives for 47, 57, 60-1, 63--4,
336-52,354-62
aqueous 61
carriers for 336--40
for cotton larvae control 373, 373
encapsulated 53
multiple-embedded (MNPV) 53
oils with 46
solvents with 47
sunscreens, effects on 68-73
synergists and 82, 84, 85, 86, 102,
351-2
tank mixes, forest/field 65-6
as technical powder for maize 42
see also individual host insects
NuFilm 60-1,141,348
number mean/median diameter
(NMD), see droplets, diameter
of
nut trees 41, 190
nutrient balance/ratio 171,176,
314
nutrient granules 166, 168, 169, 170,
174,224,225
nutrient medium, solid 167
nutrient substrates 168
nutrient wash 171
nutrients 323, 353--4
absorption by fungus 168
as additives for specific
organisms 219,253,266,317,
325
agent-specific 174
bacteria survival and 330
composition of formulation 198,
324
exotic 199
leaching of 162,165,166
in mycoinsecticides 140,141-2,
141,146,172
in plant-disease control 196,
197
slow-release 176
in soil applications 225,250
Nutri-Link 245, 277
Nutrisoy 65
oak 41,60-1
defoliation of 84-5, 102-3
red 62
oat flakes 151
oat grains 208,339,354
oatmeal 198, 275, 339
occlusion bodies, see viruses
octyldodecylamine
hydrochloride 345
octyl-p-methoxycinnamate 358
octylphenol ethoxylate 344, 346,
349
octyl-salicylate 357
Odina oil EL 337, 368, 371
odour S15
oil-flowable products 172
oil seed 257
see also individual oils
oil sprays 45, 137-9, 149, 159, 169,
216-22,324,327,368
oil suspension, dormant 240-1
oils 10, 16, 17,20,46,51, 144-5, 151,
169, 172,216, 336-7
absorption by leaf cuticle 146
adverse effects of 95, 198
essential 336
and evaporation 324, 369
and feeding inhibition 104
and genetic engineering 106
as granule coating 227
handling properties improved
by 322
and hydrophobic structures 324
as nutrient 324, 325-6
organism movement in drops
of 45
oxidation in 160
palatability of 46, 173
purity of 322
rancidity of 162
specific gravity of 324

oils (cont.)
and spore carriage over/into
leaf 137-9
spreadability of 145
see also conidia, storage in oil;
emulsions; individual oils
oilseed rape 142,260
olefinic polymers 347
oleic acid 344
oleic acid monoethoxylate 97
oleyl alcohol monoethoxylate 97
olive oil 161, 216, 337
onchocerciasis programme 90
on-farm equipment/
techniques 258,265,270,271
onion seed 257, 260, 263, 264, 277
oospores 133,135,154,274,275
optical brighteners 71-2, 77, 82, 84,
143,144,145,199,359,380
see also individual compounds
orange, dried 152
orchards 133, 150
Orcin 70, 357
orcinol 357
organic powder 154
organisms ,
qualities of 2
size of 320
organochlorine 148
organophosphates 65, 148
organosilicone 56,57,58, 104, 174,
218,325,327,346
ornamentals 169, 190, 191, 193
ornithine 79,80,351
Ortho X-77 348
Orzan LS 65, 70, 76, 77, 362
osmoconditioning 263, 264-5, 380
osmotic potential/pressure/
tension 136, 136, 322
osmotic protectants/regulators 142,
157, 339, 342, 353, 354
osmoticum 208
Ostrinia nubilalis, see com borer
Otiorrhynchus sulcatus, see vine
weevil, black
Output 140, 141,351
oviposition sites 62
oxamyl 304
oxidase activity 103,352
oxidation 160
oxybenzone 144,146,357
oxygen 158,160,169,207
absence/exclusion of 156, 157,
174
consumption of 293, 320
depletion of 162,175
and dormancy 321
free radicals 77
tension, dissolved (DOT) 207
Index 401
oxygen-absorber 353 see also noodles; pesta
oxygen sink 327 Pasteuria penetrans 325
oxysorbic 345 pathogen production 97-9
oxysorbic polyoxyethylene sorbitan pathogens, human 38
monolaureate 211 pea plants 87, 189, 191
pea seed 193,262,262,265,276
PABA, see p-aminobenzoic acid peach 189
packaging peanut 189,191,195,271,272
Collego 207-8 see also groundnut oil
costs of 91 pear 41,189,193
flexible impermeable 158 peat 159,194,197,225,242,245,246,
heat-sealable 39, 239 247,260, 266-8, 269, 279, 291,
instruction labelling of 319 339
moisture-proof 321 alternatives to 267, 268
for nematodes 298 drying of 266
one-use 319 granular 244, 244, 251, 273
permeability of 322 neutralization of 267
twin 319 in pelleting (seed) 261
water vapour-proof 38, 39, 55 rhizobial survival in 268
see also bags; containers; storage sterilization of 267, 268
paddle mixer 42, 52, 220 toxicity of 267
Paecilomyces 133, 156, 236, 241 peat-based inoculants 260, 270, 271,
Paecilomyces !arinosus 136,142 273
Paecilomyces jumosoroseus 135, 139, pecan weevil 150
143, 155, 169 pectin 44, 219
blastospore survival 159 fruit 219, 351
rehydration of 166 PEG 151, 164, 166, 171, 173,343,354
young cultures of 172 see also polyethylene glycol
palatability 8, 46, 97, 151 Pelgel 193-4,269,274,276,349
palm oil 161, 337 see also Nitragin Gold
palm olein 50 pelleting 260-1,270,271
palmitic acid 96 pellets 22, 105, 142, 151, 188, 246,
Pandoraddphac~ 142,340,354 252,380
Pandora gloeospora 142 agglomeration of 93, 261
Pantorhytes plutus, see cocoa weevil alginate 151, 208, 224-5
pantothenic acid 68,360 baits with 301
-paper 63 colourless 156
paraffin compost 150
liquid 157, 161, 336, 353 conidial 224
long-chain 339, 343 mycelial 150,170,208
paraffin oil 46, 144, 161, 164, 211, seed 260-1,270,271,273,274,275
219,220,220,336,337 silica gel 156
paraffin wax 219,220,220,239,339, size grading of 261
343 slow / sustained-release 93, 95,
Parsol products 144-5,146,358 315
particles see also granules
behaviour of 370,371 penetration of leaf 213, 222
carried in water 88,89,90,104 Penicillium 188, 189, 193, 195, 198,
density, net 313 236,237
distribution among droplets 373 Penicillium bilaii 236
entrapment, filtration from Penicillium oxalicum 275
water 89 Penicillium urticae 150
floating 104-5, 152 Peniophora gigantea 194
release of 91 pentachloronitrobenzine 193
sinking 89 pepper seed 194,260,263
size of 38, 45, 51, 88, 89--:90, 104, pepper seedlings 205
109,174,197,243,266 pepper-pot shakers 13
texture of 95 peppers 199
pasta maker 226 peptides, water-soluble 360
402 Index
peptone 163,171,218,354
Perfekthiom EL Oil 371
performance of organisms in
field 204, 205, 320
perlite 92, 197, 244, 245,252,261,
339,342
peroral mode of action 31-127
peroxidase 73,360
peroxide radicals 74, 76, 362
peroxides 360-1
persimmon tree 203
persistence 48,59,108,210
environmental 19-21,98
persistence ratio 56
pesta 151,199,225-7,226,300,301,
380
pesticides, chemical 191,205,259,
305,317
application techniques 11
compatibility with
organisms 147-8, 212, 300,
302,304,314,325
co-formulation with
biologicals 279
shelf-life of 9
water-soluble 58
see also individual pesticides
pest generation asynchrony 327
pest population dynamics 330
pests
food store 104
soil 104
Petrol AG 57, 345
petroleum distillate 47
petroleum extracts, crude 159
petroleum-based oil 161
Petro Morwet EFW 57, 345
Pfr 97 135
PGPR, see rhizobacteria, plant
growth-promoting
pH 9,54,55, 170, 171
buffers for 157, 163, 249
leaf surface 20, 76, 77, 78
manipulation of 98,167,168,322
optima 50, 249, 268, 273, 276
in storage 314-15
see also major organisms
Phaedon cochleriae 142
phagocytes 82
phagostimulants 21,52,55,59,
62-7,63-4,75,83,98,99,105,
315,331,336,350-2,380
as masks 102
and non-target fauna 328
synergizing 79
see also major organisms
Phagus 190
Pharmamedia 63
phase separation 26
Pheast 63, 351 toxin-producing 86
phenolic compounds 83, 352 transgenic 36,86, 103, 106-7
phenylacetic acid 83, 351 plasmids 199
phenylalanine 68, 80, 352, 360 Plasrnopara viticola 191
2-phenylbenzimidazole-5-sulphonic plaster, moulding 93
acid 356 plastics, UVB penetration of 19
phenylthiocarbamide 73, 361 plasticizers 92, 380
phialides 175 Pleurotus spp. 190
Phlebia gigantea 191, 193 Plodia interpunctella 107
Phorna aquilina 215 Plurafac A-24 57, 345
Phorna exigua 214 Plutella rnaculipennis 107
Phorna proboscis 217 Plutella xylostella, see diamondback
Phornopsis spp. 190,193 moth
Phorwite products 72, 77, 199,359 Plyac 57,60-1,147,346,349
phosphate buffer 77, 248 Poa annua, see annual bluegrass
phosphates 236, 266, 271, 277 POE, see polyoxyethylene
phosphatidylcholine 353 Poisson distribution 373
photoinactivators 67 polyacrylamide 94, 216, 245, 247,
photoprotectants, see sunscreens 267, 268, 273, 292, 338, 339,
photoreaction 143 341,343
see also sunlight; ultra violet polyamide 339
radiation polycarboxylic acid 341
Photorhabdus 290, 291 polyethylene 96, 158, 167
phthalic glyceryl alkyd resin 346 A-C 346,349
Phthorirnaea opercullela 42 polyethylene glycol 57, 142, 198,
phylloplane bacteria 54 249,250,263,343,354
phylloplane chemicals 20, 77 see also PEG
Phyllosticta 224 polyethylene glycol oleate 347
Phyllotreta cruciferae, see crucifer flea polyethylene monostearates 249,
beetle 343
Phytophthora spp. 189,190,191,193 polyflavonoid, natural 362
Phytophthora palrnivora 204, 207 Polygandron 190,193
see also DeVine polyglucine 61, 71
phytotoxicity 160 polyglycosamine 54
PIB, see polyhedral inclusion bodies polyhedral inclusion bodies 59,75,
picloram 212 373,374
Pieris brassicae 41, 53, 57 see also viruses, occlusion bodies
Pieris rapae 41 polyhedrin 55
pigments 143,329,330, 362 polyhydroxyl alcohols 276
see also melanin polymer binder 194
pine forest weed control 223 polymerizers 338-40
pine lignin 343 polymers 245, 247, 258, 275
pine needles 59, 60 anionic powder 341
pine sawfly 59,61 aromatic 347
pineapple 195 in encapsulation 26
pink bollworm 63 hydrophilic 216
Pinolene 1882 57, 345 olefinic 347
Pinopsida 48 synthetic 24
pinto bean 267 water-soluble 343
Pinus radiata seed 277 Polynox products 194,349
piperonyl butoxide 103, 352 polyol content 140, 171, 176
Pitsulin 58, 345 Polyox 276
plant disease, control of 187-200 polyoxyethylene (POE) 24, 217
plant extracts 21, 62, 67, 78, 350 polyoxyethylene glyceryl
plant nutrition 236 monooleate 157
plant residues as carriers 22 polyoxyethylene sorbitan
plant secretions 20 monolaurate 211, 214
plants polyoxyethylene sorbitan
growth habits of 209 monooleate 214
 Index 403
polyoxypropylene 24 coarse 278 trends in 320-1
polyphenol oxidase 79 dry 172,242 yield-optimized 326, 329
polyphenols 78 wettable 241-2 Proflo 63
Polyplasdone 249,343 moist 242-3, 243 proline 68,79,80,159,352,361
polypropenoic acid 94 size grades of 261, 266 ProMo 65
polypropylene 92,93,341, 343 spore, pure 168 Promot 188
polypropylene carbonate 343 storage of 321 propagules 198,206-8,217
polypropylene microporous see also dusts; wettable powders antagonist on leaf surfaces 199
film 168 powdery mildew 140, 188, 189 see also conidia; spores; etc.
polysaccharides 215, 249, 262,276, PRECEP 189,193 propanil 212
339,341-2,348,353 precipitation, post-spray 48 prophylactic treatment 151
Polysuif 274,339 Precirol 249,343 proprionate 50, 354
Polysurf-e gel 194, 349 pre-drying 156, 158, 159, 160 propylene carbonate 49
polythene blackouts 140 preservatives 50, 98, 102, 240, 322, propylene glycol 161,339
Polytran 274,339 351, 352, 35~ propyl gallate 73, 77, 361
polyvinyl acetate 64,258 bacteriostatic 354 Propyltex 92, 343
polyvinyl alcohol 45,46,58,66, 141, see also acidification; anti- protease 54
274,343,346,348,349,362 microbials; individual types alkaline 55,98
polyvinyl sticker 61, 62, 349 pre-soaking 142, 173,323 inhibitors 79, 80, 83
polyvinylpyrrolidone (PVP) 65, presporangia 154 Protec 65, 71,362
249,250,260,269,343,349 pressure damage 218 protein hydrolysates 361
polyvinylpyrrolidone/vinyl acetate prills 96,169,195, 195--6,262,274, protein solubilizing reagents 83
(PVP/VA) 343 380 proteinases 98
pome fruit 188, 189, 193 see also alginate prills; granules proteins 312-13,353
Popilia japonica, see Japanese beetle Primojel 249,343 anti-viral 78
poplar bark 197,339 processes, unit 205 conidial 207
post-application life 109 processing methods 250-3 denatured 351
post-harvest disease 194--5 products 380 precipitation of 78
potassium 24 aqueous 101 stabilization of 164,305
transport of ions in gut 79,80 characteristics of 239 as sunscreens 72,75,76,329,360-1
potassium carbonate 79, 81, 84, 85, combination 241 proteolysis 79
352 commercial 135, 188-93 protoxins 79
potassium-channel blocker 79 complementary 316 Protozoa 35,35,54,98,99,108,242,
potassium chloride 38,163,164, development costs of 253 247
166,354 dry 22-4,97,98 additives with 56, 6~, 361, 362
potassium dihydrogen efficacy and application Prunus serotina 223
. phosphate 81, 83, 163, 354 methods 237 Pseudaletia unipuncta, see armyworms
potassium hydrogen carbonate 81, failure of 135 Pseudomonas 54,197,236,237,240,
352 liquid 98 245,262,273,279
potassium nitrate 263 multipurpose 109,228,316 carriers for 337-9
potassium phosphate 83, 158, 263 proprietary 108 stabilizers for 353
potassium polyacrylamide 94,198 spore-free 100 Pseudomonas cepacia 54,190,193
potassium polyacrylate 94 spore quality and 175 Pseudomonas fluorescens 189, 193,
potassium sorbate 37,48,354 types of 377-81 194,262,273
potassium tartrate 82, 352 see also individual products artd Pseudomonas putida 194,199,261,
potato 189, 249, 276 groups 262,273
pests of 41 production Pseudomonas solanacearum 191
. transgenic 86 costs of 24, 98, 176, 204, 241, 253, Pseudomonas syringae 189,193
potato dextrin 348 266,291,295,317 Pseudomonas tolassii 189, 190, 192,
potato de,.;trose agar 141,352 of fungi 166-72 193
potato seedpieces 193,194,197,273, industrial flow lines for 134 Pseudoperonospora subensis 191
275 liquid fermentation 170-2 pseudoplasticity 342, 348, 353, 380
potato starch 246, 249, 344 mass 236,261,290-1 Psorophora columbiae 91
polato tuber stores 42 of mycelium 170 PSSOL 191
potency 37, 49, 88, 96, 320 optimal 36-40 pteridine 76
powder balls, unwetted 23 run-length in 175 Pteridium aquilinum, see bracken
powders 159,241-3 semi-automated 259 published information, lack of 3
adherence to leaf by 315 solid-substrate 166-70, 176 pumice 95, 168
application of 259 of technical powder 167 pumping problems 90
404 Index
pumps 12,13,137,172
putrefaction 105
PVP, see polyvinylpyrrolidone
PVP/VA, see polyvinylpyrrolidone/
vinyl acetate
PVP /VA-S-630 269, 343
pycnidial parasites 188
pycnidiospores 153, 156,380
Pyrax 154,242,275,338
pyrazosulfuron ethyl 213
pyrethrins 103
pyrethroid insecticide 350
pyridoxine 68, 361
pyrimidine dimers 74
pyrophyllite 154, 193, 195-6, 196,
197,225,275,338,354
pyrrolidone 258
Pythium 188,189,190,191, 193,194,
245,250,264,273,274,276
Pythium oligandrum 190,193,194,
261,274-5,275
Pythium ultimum 190,194, 199,273,
274,276
quality control 174,305,320-1
quality standards 268, 279, 298, 299
quantification, in-field 173
quantification units 56
Quantum 4000 242, 272
quartz flour 194, 275
radish seed 265, 276
raffinose agar 63
rain 209, 217
simulated 59,60-1
see also wash-off
rainfastness 53,60-1,101, 146-7,
315,327
rainsplash, dispersal by 206
Ramularia rubella 218
rancidity 25, 322
rapeseed oil 46,139,141,216,221-2,
221,222,223,336,337,347,350
ratios 56, 75
Raymix products 70-1,76,362
Reax 907 70, 343
reflectors 20, 72, 359
refrigeration 10, 135,239, 239, 291
refugia 107
registration 188, 268, 317
rehydration 166,173,174,242,305
relative humidity 138, 153, 158,217,
227,319,324,326,380
equilibrium 13~, 153
see also humidity
release kinetics 93
reliability trials 318
research
analytical 324
basic 328-31 Rhizoctonia 189, 191, 245, 265, 272,
costs of 200 274
future 97-109, 172-6, 199-200, see also damping-off
227-8,253,277-9,300-6, Rhizoctonia solani 188, 189, 190, 191,
311-331 194,195,199,274,275,276
joint/partnership 228,319 Rhizogen luteolus 277
resins 21, 59 Rhizo-Kote 270
resistance 78,86,106-8,209 rhizomes 209
resorcinol 83,352 rhizoplane 237
respiration 162,225,295 Rhizopus stolonifer 191
as measure of energy use 320-1, rhizosphere 237,238,278,380
326 Rhodamine B 70, 269, 358
in store 322, 326 Rhoplex products 60-1,65,349
retention of spray 21 riboflavin 68, 76, 361
Rex clay 154,338 rice 102, 107, 135, 141, 147,154, 168,
rhinoceros beetle 108 171, 191, 204, 249
rhizobacteria as bait 152
herbicidal 227 rice fields/paddies 90,91,216
plant growth-promoting 194,245, rice flour 225, 339
272,273,277 rice hulls 342,353,360
rhizobia rice starch 249, 344
in broth cultures 266 Richard's medium 207
carriers for 266-8 Ringer's solution 164, 164, 171, 354
coated 260 Risella oil 46, 337, 371
in gypsum granules 245 rodlet layer 137
harvesting of 240 root dips 188, 194
nutrients for 323 root exudates 278
pre-inoculation of 261 root growth, inhibition of 250
production of 240-1,243,246 root knot 190
and seed inoculation 269-72,318, root rot 189, 193, 245, 274
325 RootShield 188,191
shelf-life of 240 rootworms 299,301
standardization 269 Rose Bengal 196,248,354
stickers for 268-9 roses 193
storage of 240 Rotstop 191, 193
survival of 268, 269, 278 Rottboellia cochinchinensis,see itch grass
temperature and 20 round-leaved mallow 212, 242
rhizobial inoculants 259,260,262, Rumex spp., see docks
265-71 run-off 208, 210, 315, 327
oil-based 267 rutin 78
Rhizobium 87,197,198,224,236,242, rye 64
243, 244, 245, 247, 248, 250, ryegrass seed 273,276
267,268
binders for 342-4, 349 Sabouraud dextrose agar 141, 146,
carriers for 337, 338-40 352
dormant 240-1 Sabouraud dextrose broth 163, 165,
early commercial production 354
of 238,240 Sabulodes aegrotata 41
nitrogen fixation and 236 Saccharomyces 198
sunscreens for 358 safety 108,317,319,324
symbiosis with legumes 236, 240, sales 188,204
266 salicylate 199,317
synergists 350 salicylic acid 82, 352
Rhizobium japonicum 243,244,245, Salmonella 358
246, 269, 337 salts, synergistic 81-2, 82
Rhizobium leguminosarum 197, SAN-285-WPG 6 352
266 sand 90, 92, 105, 159, 247, 261, 301,
Rhizobium meliloti 266, 269 303,338
Rhizobium phaseoli 267 blasting 91, 92
 Index 405
sand-and-oil granules 96 storage of 279 shale 194, 339
Sandovit 57, 346 transgenic 107 see also Leonardite shale; ligneous
Sandoz products 40, 65 treated shale
sandwich technique 260 as bait 325 Sharples A5-16VB centrifuge 39
SAS 90 oil 140,141,346 user-friendly 325 shear 10, 49, 49
saturation, soil-water 136 treatments for 193-4 sheets, crop-cover 140
Saturn Yellow 358 chemical 261 shelf-life 9, 50, 52, 90, 94, 204-5
sawdust 152,242,245,246,247,339 for insect control 272 carriers affecting 197
sawfly 65 weight increase of 260 of dormant suspensions 240
sawfly NPV 46 yields of 269 dry vs liquid 99
scale insects 133 see also matriconditioning; of epiphytes 107
scale-up 36,37, 170, 175, 266, 291, individual seeds/crop types of flowables 101, 109
317 seed coating 242, 243, 247, 258-61, of fungi 154-6,158,159,172,
Scapteriscus vicinus, see mole cricket 269, 276, 277, 380 174--6,228
scarab beetles 304, 320 compounds for 22, 95, 227, 242, humectants and 198
see also individual species 247,250 of monolayers 106
Schistocerca gregaria, see locusts lime 270 of mycelia 206
sclerotia/microsclerotia 265,380 machinery for 15 of nematodes 305
sclerotial homogenate 250 multi-layered 258, 278 perlite and 245
Sclerotinia 189 seed dressing 87 in pesta 227
Sclerotinia homeocarpa 191 seed encapsulation 262-3 production factors, influence
Sclerotinia minor 189,274 seed extracts 63, 79,83 on 326-7
Sclerotinia sclerotiorum 189, 191, 250, seed filmcoating 276, 380 of suspensions 104
275 binder system for 261,274 of wettable powders 101
sclerotinia wilt 276 particle grade for 278 see also storage; individual
Sclerotium rolfsij 191, 194, 198, 265 pelleting and 273,274 organisms / formulations
screening 168,169 techniques for 258-60 shellac 250
screens, thermal 140 side-vented drum 259 Shell Risella oil 337
seal oil 46 spouted-bed 258-9 Shellsol products 46,47, 139, 159,
search, power of 287-308 seed pelleting 194, 260, 270, 273 161,337,356,358,359,368,
Seaton products 46,337 seed priming 173,194,263-4 370,371
seawater 94 bio-priming 194 showy crotalaria 219
sedimentation 24, 25, 51 drum 263 sicklepod 217,219,220,225,226,
see also settling solid-matrix 194, 263, 264, 264, 226
seed 273,276 silage inoculants 240
application of microorganisms seedling emergence 270 silica 23, 154
to 247, 255-279 seedling necrosis 217 amorphous 295
costs of treatment 257 seedling pathogens 191 fumed 97,152,227,295,343,349
as delivery vehicle 238, 241, 247 seedlings, pregerminated 265 in granule formulations 292, 294
germination of 263, 265 semolina 226 synthetic 51,339
humidification for 264, 273 sentering 252 silica dusts 22
hydration of 264 separator, vibrating 39 silica gel 9, 153, 159, 174, 198,218,
immersion techniques for 263-5 Sepiret 260 224, 336, 340, 344, 350
impregnation of 270 serine 79,80,352 indicator grade 156
inbibition damage to 174 Serratia 320 pellets 156, 158, 159, 160, 161
incubation of 263 Serratia entomophila 273, 338, 341 silica powder 42, 91, 157, 157, 168,
in-furrow applications of 237,238 sesame oil 216, 337 218,339,340,341,344
inoculation of 269-71 Sesbania exaltata, see hemp sesbania silica zerogel 159, 167,339,342
mycorrhizal 277 Setaria spp., see foxtail silicate 20
rhizobial 265-71,318 sethoxydim 212 silicobenzone 75, 346
techniques for 256-65 settling silicon anti-foams 23
machinery for 256-65, 271, 278 during storage 50, 104, 172,314 silicon dioxide
on-farm application at sowing 271 in spray tank 24,44,45,50,314 fumed 249,341
as phagostimulants 62 in water after application 88, 89- synthetic amorphous 342
pre-inoculation of 269-70, 271, 90,105 silicone oil 152
318 see also sedimentation silkworm 41,60,78,142
quality parameters for 257 sewage 92 Siloid 344
steeping of 265 Shade 60, 63-4, 65, 71, 74, 75, 76, 77, Silverson mixer 48
sterilization of 265 362 Silwet products 58, 147, 214, 346
406 Index
Simulium spp., see blackfly
sinking, see settling
Sipernat 22 92, 344
Sitona spp. 87
size enlargement 251-2
skinning 224
slime mould 247
slow / sustained-release
formulations 19,224,225,
227,250,344,353,360
for use in water 91-5,97, 106
slugging 252
slurries 37,98,261,269, 272, 276,
278
ex-fermenter 50, 100
inoculation of 271
mixing of 250
mycelial 208
storage 165, 175
SMA-2625A 77, 340, 343, 349, 362
smectite clay 341, 344
smell, termite rejection for 331
snap bean seed 264, 272
soap 23, 24, 342
sodium, gut transport of ion 79, 80
sodium acetate 82, 352
sodium acrylate 94
sodium alginate 164,166,195,195,
197,215,220,224,224,262,
262, 322, 340, 344, 348, 349,
353
see also alginate
sodium alkyl aryl sulphonate 94
sodium ascorbate 73, 159, 165, 361
sodium azide 248, 354
sodium benzoate 50, 83, 249, 344,
352,354
sodium carbonate 79,81,352
sodium carboxymethylcellulose
249,338,339,341, 347
sodium caseinate 269,340
sodium chloride 163,354
sodium citrate 293, 294
sodium CMC 340
sodium 2,2'-dihydroxy-4,4'-
dimethoxy-5-
sulphobenzophenone 358
sodium dioctyl
sulphosuccinate 345, 346
sodium dodecyl sulphate 80, 84,
352
sodium formate 82, 352
sodium glutamate 157, 163, 164,354
sodium glycerophosphate 83, 84
sodium hydroxide 48, 244
sodium lauryl sulphate 344, 352
sodium lignosulphonate 362
sodium nitrate/nitrite 81,352
Sodium Omadine 40
sodium polyacrylate 341
sodium salicylate 82, 352
sodium salts 342
of alkylosulphonated alkylate
of polycarboxylic acid 341
of polypropenoic acid 94
sodium silicate 163,276,354
sodium starch glycolate 249, 339,
343
sodium stearate 24
sodium sulphate 22 . sorghum silage 349, 354
sodium tetraborate 21, 85
sodium thioglycolate 79, 83, 352
soil
accidental movement of 236
application of agents to 149-52,
197,225,227,236-53,324-5
fungal control of pests in 174
as mechanism for spread of plant
disease 236
moisture content of 136
penetration of 325
spraying of
direct 299
droplet size for 368
sterile ISO
subsurface of 40, 325
'suppressive' 236
water relations in 136
waterlogged 151
soil biota
action of 150
competitive 325
population dynamics of 278
Soil Implant 244
soil injectors 15
soil inoculants 210,236-53
basic formulations for 237-47
as granules 243
ingredients for 247-50
placement of 237
processing methods for 250-3
subsurface 325
soilborne crop diseases 189-92
SoilGard 188, 191,245,249
Solanum ptycanthum, see black
nightshade
Solenopsis invicta 301
solid-matrix priming, see seed
priming
solvents 47
Sorba Spray Zip 77, 354
sorbic acid 50, 82, 248, 352, 354
sorbitan monolaurate
ethoxylate 345
see also Tween 20
sorbitan monooleate 340
sorbitan monooleate ethoxylate, see
Tween 80
sorbitan monopalmitate
ethoxylate 347, 352
sorbitan monostearate
ethoxylate 347, 352
sorbitan trioleate 346
sorbitan trioleate ethoxylate 347
sorbitol 47, 60-1, 164, 164, 166, 214,
215,219,248,339,344,349,354
sorghum 191, 195
sorghum powder 142,340,354
sorghum straw 223, 340
Sorghum halepense, see Johnson grass
Southern blight 265
see also Sclerotium rolfsii
Sovix 346
soya (soybean) flour 62,63,65-6,
198,225,340,352,354
soya hydrolysate 68, 361
soya meal 250
soya (soybean) oil 46,63,65,
159-60, 161, 162, 216, 219,
220,220,337,343
soya polysaccharide 342
soybean 103,189,191,204,216,240,
243,246,248,259,262,264,
265,269,270,274,353
soybean capsules 102,262
soybean lecithin 97,220,220
soybean seeds/seedlings 193,227
Soy-Dex 219, 344
space-planting 260
Span products 46, 57-8, 65, 340, 345
Speswhite china clay 51, 66, 338,
344
sphagnum moss 264, 340
spheronization 252
Spicaria 133
spindle oil 46,336
spiny cockleburr 217
split-pill process 275
Spodoptera 41,79,84,86
Spodoptera exigua 57-8, 107, 300,
304,319
see also beet armyworm
Spodoptera Jrugiperda 42, 84, 166
Spodoptera littoralis 45,46,46, 47, 55,
58, 63, 66, 84, 85, 373, 374
GV 86
~PV 39,51,59,61,65-6,84
spoilage, autolytic 55
sponges 22, 291, 292
spores
additive toxicity to 200
adhesion of 215
aerial 241
ageing 176
allelochemicals and 103
annular aggregates of 209

spores (cont.)
aqueous suspensions of 205
centrifuged 157, 206
clumping 209
concentration of 162, 165,207,
208,313,322
count of 61
'dead' 106
dehabilitating metabolism of 162
dehydration of 272
delicate 135,176
deterioration of 175
dispersion of, in water 218
dormant 240
endogenous 323
dusty 166
environmental resistance of
204-5
fission type 207
floating 152
flocculation of 23
free water and 205
frozen, survival of 165
germination of 153, 162, 163, 218
delayed 166
inhibition of, 79, 162, 164,
211-13,214,215-16,217,248,
325
in oil 223
optimal 136
premature 55,169,322,323
speed of 140,165,173
stimulation of 54,142,172,216
storage effects on 161, 165-6
water content for 221
half-life of, dry fungal 164
harvesting of 241
humidity and 205
hydrophobic 56, 165, 218
irradiated 106
irritants and 174
on leaves
growth 140
pick-up from 139, 172
lipophilic fungal 25
moisture content of 155
mutagenesis of 143
number fluctuations of 149, 373
nutrients and 174
in oil 137, 216, 241
pigmentation of 143
preservation of 211
pre-soaked 140
production of 133,175-6
water activity and 136
quality of 175-6
rehydrated 173
resting 133, 170
separation of 206
Index 407
shelf-life of 168, 174,207 retention of 17,327
soil penetration by 149 to run-off 205, 210
stability of 164, 207 spore-free 219
sunlight effects on 74 standing before use 55
survival of 145, 146, 155-6, 159, timing of 209
162, 163, 165, 173, 251 ultra low volume (VLV) 18, 45, 47,
synergists and 103 48,90, 100-1,104,137,140,
thick-walled 242 160,170,241,313,315,316,
VV radiation and 172, 205 324,327,368,369,371,373
viability of in storage 53 underleaf coverage 46
vigour, optimizing 171-2 very low volume (VLV) 18,140,
virulence 166 368,374
yield of 207, 225 viscosity of 21, 172, 372
see also blastospores; conidia; fungi; see also viscosity
individual species/genera volumes, optimum 46-7, 221, 299
spore-free mutant 100, 104, 106 see also drift; droplets; oil sprays
Sporidesmium 250 spray drying 37,37,39-40,98, 100,
sporulation 150 101,153,156,241-2,250-1,
induced 208 258,259
submerged 206 for encapsulation 53, 102
spouted-bed drying 258-9 Spray Oil 435 147,346
spraying/sprays sprayers 11,12-13,219,323
accumulation on leaf 208 accuracy of 11
aerial 90, 100, 172, 299 aerosol 210
application 223 air-assist 223
factors for 205, 206 airblast 13, 368, 373
fluid suspensions for 237-41 atomizer, electronically charged
of fungal 137-48, 149 rotary 141
interaction with boom 12, 238, 313
formulation 228 tail 46
parameters of 205,206,223, centrifugal disc 138
228,315,367-374 compressed air 12
rates of 206, 367-9 drop-leg 209
to water bodies 89 efficiency of 327
applicators, coating 259 electrostatic 45, 209, 299
aqueous 139,142,151,169,210, exhaust-nozzle 156
216-22,259 fan-assisted 374
dormant 240 flow rate from 369
bulk 100 hose-end 12
climate effects, dry 137-42 HV 44
coarse 97 hydraulic 12, 239
controlled droplet (CDA), see knapsack 12,46,373,374
droplets Micro-Viva 139
costs of 50, 139 rapid loading of 172
coverage factors 16 spinning cage, 12, 18
deterioration of 24, 55 spinning disc 17, 46, 369, 370,
dilution of 164 373,374
dispersal of 313 syringe/piston/stirrup pump 12
driers 37 trigger pump 12
early 140 Turbair 374
formulation trends for 99-102 VLV 13,17
high volume (HV) 44, 101, 137-S, see also nozzles
140, 313, 327, 368 Spreader 57
impaction of 16,17 spreaders 141, 238, 347-9, 381
for insect control 44-51,99-102 Spreadite 57, 346
low volume (LV) 45,47,101,140, spring-loaded pellet hammer 203
313,368 spruce budworm 41, 47, 48, 49, 50,
penetration of leaf tissue by 222 60,65,356
ready-to-use 172 spurred anoda 217,219,224
408 Index
stabilization 9-10 for mycoinsecticides 141, 146--7, strawberry 189,191,193
aqueous emulsion 48, 50 151 Streptomyces 197,236, 242,261, 339,
baculovirus 38-40 for plant-disease control 151, 353
fungal spore 207 193-4, 197 Streptomyces griseoviridis 190, 193,
membrane 242,247-8 for seed formulations 268-9, 271 272,279
problems of 8 see also individual types Streptomyces lividans 103, 362
protein 164 sticking, preventing in cereal streptomycin 265
semi-solids 36 grain 168 streptomycin sulphate 354
virus 98, 100-1 stilbene derivatives 20 stunt virus, see Helicoverpa armigera
stabilizers 336, 353-4, 381 stilbene optical brighteners/ styrene maleic anhydride half-
enzyme 156 sunscreens 71-72,77,84,85, ester 53, 340
food-grade 55 144, 145, 173,359 succinate 252, 340
Staganospora sp. 221,223 stimulants, feeding, concentrations sucrose 53,60,63,66--7,70-1,93,99,
standardization 319,320-1 of 62 152, 155, 157, 163, 164, 167,
see also quality control stolons 209 171,208,219, 225, 227, 248,
starch 98, 211, 218, 227, 249, 292, storage of bulk food 40, 42 248,250,301,344,352,354
.294,295,322,340 storage of formulated products 134, Sudan IV 60
in encapsulation 26,53,103,227 154-66,167,174-5,314 sugars/sugar solutions 47,155
gelatinized 199,340 bacterial activity in 90 as dispersants 52
microcapsules 101 cold 153 in encapsulation 53
as multifunctional additive 102 costs of 10 for freeze-drying 156
palatability of 66--7 deterioration in 54-5 in granules 24
pre-gelatinized 249, 340, 344 dry 154,155,156,198,224,227, humectant action of 219
as storage protectant 164 326 as nutrients 323
as UV blocker 146 dry products vs liquid in 99, as phagostimulants 63-4, 66
see al$o com starch; potato starch; 321-2 as preservatives 50
rice starch; wheat starch fermentation during 243 as stickers 54
starch granules 64, 99 high-temperature 326 as stimulants of feeding 54
starch matrices 77 humidity during 136, 175, 176 in storage water 164
starch polymers 22 maturation in 174 sugar-starch buffers 78
Star-Tab 340 moisture content during 228, 326 sugarbeet 190, 191, 193, 194, 257,
Sta-Rx 1500 340 in oil 152, 159-62, 160, 160, 161, 258, 260, 261, 262, 262, 263,
steam, see sterilization 169,227 265, 273, 274, 275
stearic acid 96, 249, 344 on-farm 271 sugarcane soil 150
steeping 265 problems of 8, 204 sugarcane soldier fly 150
Steinernema 289-90, 292, 293, 293, refrigerated 135 sugarcane stem borer 54
301 with silica 168 sulisobenzone 71, 77, 362
Steinernema carpocapsae 291, 292, surfactants and 25 sulphated alkyl carboxylate 94
293, 293, 294, 294, 296, 297, survival in sulphates, fatty alcohol 24
298,300,302,303,304-5 doubled by formulation 317 sulphonated alkyl naphthalene 94
Steinernema feltiae 291, 292, 293, 360, temperature during 9,154-5,158, sulphonated kraft pine lignin 343
361 159, 164, 244 sulphuric acid 48
Steinernema glaseri 291, 292, 293, trends in 321-2 sunflower 189, 191
293,294 vacuum 157, 158, 167 sunflower oil 161, 216, 337
Steinernema riobravis 291, 292 virulence and 165-6 sunflower seed 257,274
Steinernema scapterisci 292,293,299, in water 51, 162-5, 163, 164, 175 sunlight
303,305 see also bags; containers; packaging; artificial/simulated 64,74,143,224
sterculia tree 348 individual formulations types damage by 67, 315
sterility 158 and organisms inactivation by 19-20,93
sterilization 252-3 strain slants 171 mode of action of 74,143
electron beam 252 strain specificity 172 protection from 143
heat 225-6 strains 279 see also sunscreens
steam 239,243,247,252,265,267 with different temperature test conditions for 74-5,100
Sterotex 249, 344 ranges 174 in water 89
stickers 21,22,59,60-1, 315, 327-8, selection of 211,314,327,329-30 wavelengths 19,67, 143
347-9,381 variation between 253,272,277-8, see also ultra violet radiation;
for herbicides 215-16,215 318 individual organisms
for insect control 52, 55, 59-62, variability of 172 sunscreens 20, 44, 55, 315, 355-62,
92,97-8,102 stranglervine 204 381

sunscreens (cont.)
cosmetic 68-9,391, 355-8, 378
costs of 76, 101
droplet size and 17
effects of different 75-7
encapsulation and 26, 53
in floating granules 105
fungi and 142-6, 144-5, 173
insect control and 65,67-77,68-73
limitations of 8
multifunctional 102, 103
oil-soluble 145
synergists and 84
in traps 108
trends in 328, 329
in water bodies 89,145
Sunspray Oil 139
Superfloc 37
Supernat 22 52
supernatant, cloudy 37
superoxide dismutase 73,361
Supersorb products 93, 344
superspreaders 56, 58
supersurvivors 327
super-wetters 104
supplement, compatible salts 158
suppliers of formulants 363-5
suppressants
contaminant 240, 248
growth 240, 248
Supresivit 188, 191
surface tension 23, 56, 76, 166, 209,
216, 218, 315, 362
surfactants 23,24-5,52,57-8,92,
104,301,314,345-7
amphoteric 24
anionic/cationic 24
compatibility of 56
deterioration, as causes of 54
evaporation and 209
for hydrophobic spores 165
ionic 25, 242
lipophilic 147
low-foam 52
in mycoherbicides 213-14,214,
216,217-18
in mycoinsecticides 141,147,166,
170, 173, 174
non-ionic 24,25, 171, 213, 214
non-oxynol 214
potential harm from 175
with sunscreens 75
see also detergents; emulsifiers;
wetters
Surfino products 346
Surfynol products 48, 52, 58, 157,
346
suspenders 10,48,314,341-4,381
suspensibility 104
Index 409
Sutro 61, 349 in spray drying 10,55, 156,251,
swelling 36, 169, 197, 249 259,266
Syloid 249 tolerance of 245, 245
symbionts 2, 277, 290, 329 insect body 174
Synacril White 72, 359 light inactivation and 74
synergism 213, 300, 314, 325 low 175,239
synergists 21, 341-4, 381 nutrients and 168
allelochemical 103 optima for organism
costs of 84 activation 20,50
fungal 147-8, 173 ranges for different strains 174
in insect control 54, 55, 56, 78-86 relative humidity and 136
mode of action of 80-83 shelf-life and 154
multiple 102 soil 150
research, future 102-3 during spraying 315
transgenic plants and 86 storage 52-3, 153, 154-5, 158, 159,
trends in 328, 331 160,164,204,244,268,326
viral 84 survival and 268
System 3 191, 193 ultra violet light and 143
systemic action 10, :40, 86-7, 106-8 virus inactivation and 74,329
Tenac 57,349
T-22G 188, 191 Tenn K clay 338
T-22HB 188, 191 tent caterpillar 45, 139
T-400-100 oil 161, 337 termites 331
tablets 95, 218, 246, 249, 252 terpene 141,345,348
effervescent (rapid-release) 91, terpolymers 258
249 Terraclor 193
see also pellets testing
take-all disease 189, 194, 197, 236 reliability 318
Talaromyces flavus 193, 194, 197, 275, safety 317
338 statutory, rhizobia on seed 271
talc/talcum powder 20,42, 71, 74, toxicity 317
93, 154, 159, 194, 197, 242, 249, Tetrahymena pyriformis 105
273,338 Texas blast sand 92
tallow amine ethoxylate 211, 345 Texas gourd 198,225
tank mixes 1,56,57,84-5,314,327 Thelaviopsis basicola 197
chemical compatibility in 302, Thermospora curvata 346, 347, 352,
304 354
economics of 101,318 thiamine 68,361
forest/ field 65-6 see also nicotinic acid
technology research on 109 thickeners 10, 21, 47, 47, 59, 220,
tanks, bulk transport 50 314,315,341-4,381
tannic acid 79, 85, 352 thidiazuron 213,352
tannins 78, 84, 103 thinners 314
taste 315 see also diluents
technical concentrate/material/ Thiodan CO oil 371
powder 23, 37, 37, 49, 52, 53, thiram 264, 265
91, 92, 100, 101, 105, 167, 169 Thixcin 152, 154, 340
see also concentrates thixotropic gel 329, 381
Technical Protein Colloid 90014 92 threonine 80, 352
Teepol 56,57-8,65-6,346 Thuricide 57, 60
teflubenzuron 148 Tinopal LPW 66,72,77,144,145,
Teknar 90 146,199,359
temperature feeding inhibition by 102
in agar cultures 238-9 synergism of NPV 82, 84-5, 102,
deterioration caused by 54 331
dew and 209 Tinopal products 65,72,76,359
growth and 171 Tinuvin products 145,359
high titanium dioxide 72, 77, 359
and disease curing titanium oxide 20
410 Index
tobacco 41
tobacco budworm, see Heliothis
virescens
tobacco hornworm 78
a-tocopherol 164,165,353
tolerance 198-9,206
toluene 47
Tolypocladium cylindrosporum 133,
142, 158, 159, 162, 163, 164,
165, 166
tomato 41, 188, 189, 190, 191, 193
ortho-hydroxy phenolics 78
tomato seed 194,260,262,262,
263,264,264,265,273,275,
277
Top oil 46,337
Topwet 61,349
toxicity, additives 210-16,333-362
toxicity assessment 211
toxicity testing 317
toxin genes 86, 87, 106-8
tragacanth 249
transgenic algae 107
transgenic plants 11, 86, 106-8
see also genetic engineering
transport 50,91
traps 15, 108
nematode 300,301,306
sound 301,303
see also baits
tree inoculation 15,216
tree wounds 189
trefoil 270
trehalose 171, 176, 247-8, 305, 330,
354
Trematoda 108
trends 97-108,309-331
see also individual subjects
tributyl citrate 163, 354
Trichoderma 188,189,191, 194, 197,
236,241,250,261,264,265,
275-6, 279, 326
Trichoderma 2000 188
Trichoderma hamatum 199
Trichoderma harzianum 188, 189, 191,
194,195,197,198,199,276,
337,339,343,349
Trichoderma koningii 276
Trichoderma polysporum 189
Trichoderma viride 188,191
Trichodex 188, 191
Trichodowels 188, 191
Trichoject 188, 191
Trichopel 188, 191
Trichoplusia ni 41, 64, 86, 304
Trichoseal 188, 191
3,4,5-trihydroxybenzoic acid 351
3,7-trimethylxanthine 350
Tri-philizer freeze-dryer 39
Triton products 56, 57-8, 65, 346
Triton X-100 56,146,147,165,323,
344
Tropaeolin 00 357
tropical conditions 20,55,67,74,96,
97,153,210,268,328
trypsin inhibitors 79, 83
tryptophan 68, 74, 75, 76, 79, 80,
352,361
excited moieties 74
tumble-drying 224
turmeric 355
turnip moth 304
tussock moth NPV 53
Tween 20 52,57,147,171,211,214,
214, 217, 346
Tween 40 80,214,216,217,221,222,
347,352
Tween 60 79,80,214,217,347,352
Tween 70 147, 347
Tween 80 56,58, 79, 80, 137, 138,
139,146,147, 164, 165, 171,
214,214,217,323,347,352,354
Tween 85 214, 217, 347
tyrosinase gene 103,361
tyrosine 68, 75, 76, 80, 352, 362
ultra violet (UV) radiation 19-20,
67, 68-73, 75, 77, 85, 98, 357
baculovirus and 329
blockers of 145, 146
entomopathogen protection
from 199
fungi and 143-5,144,172,173
nematodes and 299-300
protectants, mixtures of 328
spore inhibition by 205
viruses and 98
in water 89, 96
see also sunlight
ULV, see spray, ultra low volume
VIva sprayer 373
Univul D49 145
Uranine 358
urea 71, 83, 199, 340, 352, 361
uric acid 71, 82, 361
user, cost savings to 316-17,318
user education 319,323,324,327
user flexibility 109
user-friendliness 98,100, 102,318,
322-3,326,327
Uval 362
UVA/UVB, see ultra violet radiation
Uvinal 358
Uvinul 65, 69, 358
Uvitex 65, 69, 358
vacuum drying 155,251
vacuum filtering 170
vacuum storage 157, 158, 159, 167,
321
vacuum suction 241, 270
Vairimorpha necatrix 67
valine 79, 80, 85, 352
Valmic 168
VAM, see vesicular-arbuscular
endomycorrhiza
van der Waal's forces 59,247
vapour pressure 136
VatsolOT 347
Veegum 48,344
vegetable extracts 63
vegetable oils 25, 46, 51, 90, 93, 95,
104,139,152,161,162,163,165,
211,211,216,217,247,249,268,
336, 337, 343, 344, 350, 351, 362
vegetable pests 41,189,190
vegetable seed 265
see also individual vegetables
velvetleaf 213, 219, 226
ventilation of packaging 322
vermiculite 36,150,154,168,194,
196,197,198,208,224,225,
242, 243, 244, 245, 246, 246,
247,248,248,252,261,267,
268,274,291,292,338
Vertalec 135,140,141
Vertal products 154,338
Verticillium 133, 135, 140, 148
Verticillium biguttatum 194,276
Verticillium dahliae 197,275
Verticillium lecanii 151, 173,329
additives for 336,347-9,350,352
blastospores 135,142,155,162,
163,164,165
conidia 133, 146, 147, 153, 164,
170, 172,329
growth on leaf 140
hosts 133
mycelium 174
naturally occurring 173
nutrients for 140, 141
production of 133
wettable powder 140,141
vesicular-arbuscular
endomycorrhizae
(VAM) 236, 245, 277
Vespula spp., see yellowjacket
Vesuvin 355
vetch seed 270
viability, measuring 320
Vicchem EOP 141,349
Victus 189, 192, 193
see also Conquer
vine weevil 146,150
black 304
vinyl acetate 271, 276
see also PVP IVA
 Index 411
vinyl chloride polymer, wall formation on capsules 26 for plant disease control 187,
colloidal 60, 348 wallpaper glue, see glues 194-5
vinylpyrrolidone 271, 343 walnut oil 216, 337 pre-soaked 142
vinylpyrrolidone/styrene 269,338 wash-off 44, 169,215, 327 production of 51, 52, 157
see also Aatara 430 water for soil inoculations 241-2
Viola arvensis, see field pansy applications to 19, 23, 104-9 storage of 156-9
virions 35,35,55 bacterial 87-97 user-friendly 100
virulence fungal 152-4 wetters 55,56-9, 147,241,242,314,
effects of additives/formulations availability of 20,198,322,381 325-7,345-7,381
on 152, 164, 219 buffers in 140 concentration and effects 57
effects of storage on 165-6 chlorinated 23, 44 mixing problems 323
nutrition and 197 deionized 164, 164 non-ionic 56
system-dependent 330 distilled 165 super- 327
viruses free 54, 140,205, 209, 210, 218, see also organosilicones
additives for 350-2 221, 241, 305, 324 see also surfactants
on blackfly 105 polluted 96 wetting 371
carriers for 336-40 retention of 228 wetting agents 24, 157, 295
harvesting 321 soft 314 WGS 72
humidity and 20 sunlight and 20, .89, 96 Wheast 352
inactivation water activity (aw ) 136, 137, 146, wheat 189, 208
by leaf alkali 77 171,176,198,259,272,298, as bait 21
over time 373 324,380 wheat bran 36,64, 142,196,197,
for insect control 2, 20, 21, 35-6, water bodies 87,104-5 198,223,225,244,245,246,
35 insect targets in 87-9 247, 249, 250, 340, 352, 354
leaf adhesion by 59,331 larval targets in 19 wheat fields, fallow 151
liquid produ~ts 100-1 small 93,96,97, 105 wheat flour 42, 88, 95, 225, 226, 250,
nematodes combined with 304 water environment 340,354
occluded, in water 51 problems of 87-9 wheat gluten 72, 196,225,361
occlusion bodies 35, 38, 44, 56, 77, water hyacinth 209 wheat leaves 222
98,101,321,331,380 water potential 263, 326, 381 wheat seed, coated 194
oils and 51 water relationships 135-6, 136, 293 wheat starch 249,344
phagostimulants 55 water reservoirs, domestic 95 wheat straw 340, 354
production methods for 98 water tension 136, 136 wheatgerm 63,250,352,354
shelf-life of 99, 109 water-soluble pesticides 58 .wheatgerm oil 161, 162,337
stabilizers and 353-4 wax moth 41 wheatgrass 145,146
stickers for 59, 347-9 waxes see also crested wheatgrass
storage bees 56,57 whey 57,247,266
pH in 98 epicuticular 59 whitefly 135, 140, 148
deterioration in 55, 355 paraffin 219,220 whitening agent, see optical
sunscreens for 77, 355-62 waxy foliage 327 brightener
surfactants 345-7 waxy targets 315 white rust disease 141
synergism 84, 102,331,350-2 WOC, see granules, water-dispersible Wiley mill 243
thickeners 341-3 weather, formulation for 142, 324 wilt 189, 203, 276
transformed 108 weathering resistance 65 wilting point 136, 136
wetters for 57 weeds wind 18,21, 43, 59, 91, 96, 105, 241,
see also baculovirus control of 203-38 315, 328, 369
viscometer 17 see also herbicides dispersal by 206
viscosity 17,49,51,53,139,162, 172, defence mechanisms in 319 Witconol H-31A 57, 347
209, 220, 221, 313, 369-72, 370, regrowth of 209 wood chips 154, 340
371,372 Welgro 147 wood decay 189
measuring 90 Wessalon S 42,52,91,157,340,344 wood derivatives 362
vitamin B 68, 75, 76, 353, 360-1 wettable powders 23-4,314 wood fibres 261
vitamin C 350, 353, 360 fungal 135, 140, 141, 142, 156-9, wood inoculation 203-4
VK2 carrier 18,45 169,170 wood-rot fungi 188
Volck Spray Oil 46 herbicidal 218 woody weeds 203, 211, 223
volume median diameter (VMD), see improved 101 wounding 218
droplets, diameter of for insect control 51-53,89-90,
vomiting, larval 48 93,104 xanthan, see gums, xanthan
vortex mixing/washing 146,220 xanthine 71,292,361
412 Index
Xanthium spinosum 210, 217, 218, xylol 50, 103 Z-blade mixer 42, 52
221 zein 250
see also Bathurst burr; spiny yeast 93,95,96167,171,242,245, zeolite 197, 227, 338
cockleburr 351 zerogel, see silica zerogel
Xanthomonas 240 brewer's 71,360 zinc 79
Xanthomonas campestris 218, 342, yeast extract 142, 266 zinc oxide 20
348 yeast-mannitol broth 243, 244, 248 zinc sulphate 79, 81, 84, 352
xanthopterin 68, 76, 361 yellowjacket 301,303 zinc sulphite 79, 82, 352
Xenorhabdus 290, 291 yield optimization 176,320 zinnia 199
X-Link 2873 60, 349 YpSs agar 154 zoochromes 360, 361
xylene 354