Pflanzenschutz-Nachrichten Bayer 1/00, 1 7293

1 Introduction
The development of biological products
based on beneficial micro-organisms
can extend the range of options for
maintaining the health and yield of
crops. Targeted research into the prin-
ciples of biological control of microbial
pathogens began in the early twentieth
century (Cook and Baker 1983). As early
as 1897 a bacteriological fertilizer for
the inoculation of cereals was marketed
under the proprietary name Alinit by
Farbenfabriken vorm. Friedrich Bayer
& Co. of Elberfeld, Germany, todays
Bayer AG. The product was based on a
Bacillus species now known by the taxo-
nomic name Bacillus subtilis. According
to contemporary literature sources the
use of Alinit raised yields by up to 40%.
In the mid-1990s in the USA, Bacillus
subtilis started to be used as a seed
dressing, with registrations in more than
seven crops and application to more
than 2 million ha (Backmann et al. 1994).
This was the first major commercial
success in the use of an antagonist. In
Germany, FZB24

Bacillus subtilis has

been on the market since 1999 and is used
mainly as a seed dressing for potatoes.
Although activity and effects have been
reported for a number of antagonits, the
underlying mechanisms are not fully
understood. This deficiency in our knowl-
edge often still hinders attempts to opti-
mize the biological activity by employ-
ing tailored application strategies.
Accordingly, the present article sum-
marizes current knowledge about the
mode of action of FZB24

Bacillus
subtilis and the biotic and abiotic
environmental factors that influence its
action.
2 Microbiological activity of soils
Bacteria are the most abundant micro-
organisms in the soil, with an average of
6 x 10
8
cells per g of soil and a weight of
approximately 10,000 kg/ha. Bacterial
mass thus accounts for approximately
5% of the total organic dry weight of
soils. The number of bacteria depends
strongly on the season, the type of soil,
the moisture content, the oxygen supply
in the soil, as well as the tillage and fer-
tilization of the soil, and also on the
penetration of the soil by plant roots and
the depth from which the soil samples
were taken. The micro-organism popu-
lation density and the make-up of the
population in terms of species can vary
by up to a factor of 50 only as a result
of soil tillage or organic fertilization
(Scheffer and Schachtschabel 1979;
Lynch 1983).
Next to the genera Pseudomonas,
Arthrobacter, Clostridium, Achromo-
bacter, Micrococcus, and Flavobacterium,
Bacillus species are the most common
72
FZB24

Bacillus subtilis mode of action of a microbial agent

enhancing plant vitality
M. Kilian, U. Steiner, B. Krebs, H. Junge, G. Schmiedeknecht, R. Hain
Pflanzenschutz-Nachrichten Bayer 1/00, 1 73
types of bacteria isolated from soil
samples (Darbyshire and Greaves 1973;
Hallmann et al. 1998) and can account
for up to 36% of the bacterial popu-
lations. Like the total number of micro-
organisms, this amount varies according
to environmental factors, plantation, type
of fertilization, and the other factors
mentioned above (Lynch 1983, Mahaf-
fee and Kloepper 1996; Darbyshire and
Greaves 1973).
Bacteria having the ability to form anti-
fungal metabolites can be isolated easily
from soil samples. However, there have
been only little systematic studies of the
abundance of such micro-organisms as a
percentage of the total population. Leyns
et al. (1990) and Lievens et al. (1989)
found that about 30% of all bacteria
isolated from soils were able to produce
antifungal inhibition zones in vitro.
About 3% of these isolates were assign-
ed taxonomically to the genus Bacillus.
The rhizosphere, which comprises the
region close to the surfaces of roots,
and the root surface itself, the rhizo-
plane, are colonized more intensively by
micro-organisms than the other regions
of the soil. Rhizobium bacteria, pseudo-
monads, and mycorrhiza fungi are
among the best-known colonizers of this
region. Many micro-organisms from the
rhizosphere can influence plant growth
and plant health positively, and are
therefore often referred to as plant
growth promoting rhizobacteria (Schip-
pers 1992). However, their effects must
be seen as the complex and also cumu-
lative result of various interactions be-
tween plant, pathogen, antagonists, and
environmental factors (Schippers 1992).
The various effects produced by Bacillus
subtilis and the mechanisms proposed
for these effects as well as the inter-
actions between them will be discussed
in more detail below (Fig. 1).
Fig. 1: Overview of the modes of action of FZB24

Bacillus subtilis and the interaction between

the bacillus, the plant, and the pathogen
Pflanzenschutz-Nachrichten Bayer 1/00, 1
3 Competition through colonization
of the rhizosphere and the rhizo-
plane by Bacillus subtilis
Micro-organisms in the rhizosphere and
the rhizoplane live on discarded and
dead epidermis cells and root hairs and
as well from metabolites such as assi-
milates and amino acids excreted by the
roots. In total, up to 20% of the energy
gained through assimilation in the leaves
of a plant may be lost again via its roots
(Martin 1971, Lynch 1983). Especially
the so-called border cells which are
eliminated into the surroundings from
the periphery of the root above the root
cap are significant for plant microbe
interactions (Hawes et al. 1998). Under
controlled conditions, border cells and
the metabolites formed by them ac-
counted for 98% of the carbohydrate-
rich material released by the plant as an
exudate (Griffin et al. 1976). They have
chemotactic effects on micro-organisms
and stimulate their sporulation and
growth.
Though Bacillus subtilis is generally
characterized as less competitive in the
rhizosphere than e.g. pseudomonads,
colonization of the root by various
strains of this species has been found.
Relatively high population densities
have been isolated from root surfaces
and from the rhizosphere (Curl and
Truelove 1986, Leyns et al. 1990; Berger
et al. 1996; Hallmann et al. 1998). As
for other bacteria that colonize the
rhizosphere, for Bacillus subtilis, colo-
nization of the roots and co-growth
during their further development ap-
pears to require the presence of a thin
film of water on the root surface
(Bowen and Rovira 1976, Liddel and
Parke 1989).
Antagonists for the control of plant
diseases are also selected according to
their ability to colonize the rhizosphere
(Parke 1991).
As a result of the colonization by the
applied antagonist, the naturally occurr-
ing micro-organisms are faced with a
competition situation, both for space
that offers favorable possibilities for
development, such as attachment sites
or regions into which plant exudates
emerge, and for nutrients and essential
growth factors. Roots evidently have
only a limited capacity to provide a cer-
tain population size and a certain species
of micro-organism (Handelsmann and
Stabb 1996).
3.1 Colonization of the rhizosphere
and the rhizoplane by FZB24

Bacillus
subtilis
The ability of FZB24

Bacillus subtilis
to colonize roots has been demon-
strated in in vitro experiments, in which
tomato seeds that had been treated with
FZB24

were cultivated on Gelrite-

Murashige and Skoog-medium. The
absolutely clear medium also made
it possible to observe root growth as
well as bacteria development. FZB24

Bacillus subtilis colonized the root from

the treated seed and closely followed
its growth in the rhizosphere region,
so that a 0.4 0.8mm thick film of
bacteria was formed around the root
(Figs. 2, 3).
It was also possible to confirm the
colonization of the roots with the aid of
scanning electron microscopy. The close
association of Bacillus subtilis with pea
roots was shown by the fact that the
bacteria attached themselves directly to
the rhizodermis (Fig. 4).
74
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Fig. 2+3: Tomato roots (cv Minibell) on gelrite medium colonized by FZB24

-Bacillus subtilis.
Application of the bacteria was done by seed dressing (Photo: Dr. Thomzik, Bayer AG)
Fig. 4: Scanning electron micrograph of a pea root with adhering FZB24

Bacillus subtilis cells

(Photo: Dr. Schmiedeknecht, Humboldt University Berlin)
grain
Bacillus subtilis
radicle
Bacillus subtilis
root tip
Bacillus subtilis
Bacillus subtilis
Pflanzenschutz-Nachrichten Bayer 1/00, 1
3.2 Population development of FZB24

Bacillus subtilis in the soil and on the

root
The highest and most durable coloni-
zation rates of Bacillus subtilis in the
rhizosphere were attained in artificial
substrates or if the substrate had been
sterilized before the application of the
bacteria (Batinic et al. 1998; Krebs et al.
1998; Grosch et al. 1996, Zimmer et al.
1998a). In all investigations, the number
of FZB24

Bacillus subtilis bacteria as a

percentage of the total micro-organism
population showed a distinct decrease in
the course of time. The colonization of
the root and of the rhizosphere was
influenced by a number of environmental
factors, such as plant species, soil type,
and application technique. This was
shown by pot experiments with maize in
various substrates (Table 1).
(Zimmer et al. 1998b). The root tips,
which are the most physiologically active
part of the root and release the greatest
amounts of root exudate, are preferenti-
ally colonized by Bacillus subtilis (Fig. 6).
The different factors that influence the
colonization of plant roots by micro-
organisms may therefore also be respon-
sible for the differences in the re-isolation
rates of FZB24

Bacillus subtilis, which

are of the order of 1x10
3
to 1x10
7
CFU/g
of fresh root weight. However, it has not
always been possible so far to demon-
strate a clear relation between the inten-
sity of colonization and the effects on
plant health and plant productivity (Bull
et al. 1991, Handelsmann and Stabb
1996, Tutzun and Kloepper 1994).
Investigations on the harvested crop
have also confirmed the decrease in the
population of FZB24

Bacillus subtilis
a few weeks after application. Potatoes
76
Table 1: Population development of FZB24

Bacillus subtilis on maize roots and in

the soil after seed treatment.
Number (10
x
) of spores and cells/g after
Soil type isolation site
7 days 21 days 40 days
Clayey humus
root 11 3.2 0.1
substrate 0.4 0.4 0.2
Loamy sand
root 16 3.3 1
substrate 0.2 1.7 0.09
The metabolic activity of cells increases
with rising temperature, and this increase
is often accompanied by a higher multi-
plication rate. This leads to a distinctly
increased colonization of the root by
Bacillus subtilis, as has been shown in
model experiments with peas (Fig. 5).
Clear differences in the colonization of
different parts of the root were found
from three practical field trials in 1998
were harvested to determine the num-
bers of Bacillus sp. present. At the end of
the vegetation period, the counts of
Bacillus sp. found on the potatoes from
the plots in which the seed tubers had
been treated with FZB24

Bacillus subtilis
were not higher than on those yielded
from untreated seed tubers.
Pflanzenschutz-Nachrichten Bayer 1/00, 1 77
Fig. 5: Re-isolation of FZB24

Bacillus subtilis from pea roots as a function of the temperature

and the application technique. Model experiment with peas, sterile quartz sand substrate,
30 days after sowing and treatment. (Seed treatment: Dip treatment of seeds in suspension of
1g FZB24WG/l. Soil treatment: Drench treatment with 1,2x10
7
spores /ml substrate)
Fig. 6: Re-isolation of FZB24

Bacillus subtilis from pea roots as a function of the re-isolation

location and the application technique. Model experiment with peas, 20C, sterile quartz sand
substrate, 30 days after sowing and treatment. (Seed treatment: Dip treatment of seeds in
suspension of 1g FZB24WG/l. Soil treatment: Drench treatment with 1,2x10
7
spores/ml sub-
strate)
Pflanzenschutz-Nachrichten Bayer 1/00, 1
4 Formation of antibiotic metabolites
Various antibiotics can be produced by
Bacillus subtilis; of these, bacilysin is
regarded as taxonomically relevant for
the group because of the regularity of its
occurrence (Loeffler et al. 1990). In liquid
cultures, FZB24

Bacillus subtilis also

produces iturin-like lipopeptides such as
those described by Krebs et al. (1996).
The efficacy of purified lipopeptides of
this type against various phytopathoge-
nic fungi is in the range of 5100g/ml,
which is similar to that of fungicidal
agents.
The formation of secondary metabolites
by micro-organisms in synthetic culture
media and the quantity and composition
of these secondary metabolites depends
strongly on the culture conditions and
the growth phase of the culture (Loeffler
et al. 1990; Krebs et al. 1996, Krebs et al.
1998; Gupta and Utkede 1987), which is
also true in the case of Bacillus subtilis.
For the production of large quantities of
these metabolites it is therefore neces-
sary to optimize the growth conditions
and culture media during the fermenta-
tion process. Additionally the production
of such metabolites also depends on the
stage of development of the bacteria.
The lipopeptides formed by Bacillus
subtilis are released into the medium
only at the time of endogenous spore
formation during the stationary phase of
the culture (Loeffler et al. 1990).
Investigations have been carried out to
determine the significance of the meta-
bolites being effective against fungi in
vitro, especially of the lipopeptides, for
the efficacy of FZB24

Bacillus subtilis.
Maize seedlings were planted in sterile
quartz sand in small pots and drenched
with 10
7
spores per ml of substrate, cor-
responding to the recommended appli-
cation rate for horticultural crops. Appli-
cations of lipopeptides added directly to
the substrate were used for comparison.
Whereas the added lipopeptides could
be partly re-extracted and quantitatively
determined, no lipopeptides were detec-
ted in the substrates and roots treated
with FZB24

Bacillus subtilis. The definite

proof of the formation of these antibio-
tically active metabolites in non-sterile
humous substrates was impossible be-
cause of the complex matrix and the
metabolic activity of the accompanying
microflora. The assignment of the lipo-
peptides found to a distinct organism is
hindered by the fact that organisms that
produce lipopeptides of this type occur
very widely in soil and plant samples
under natural conditions, as was shown
by Lievens et al. (1989).
Moreover, it is probable that due to the
competition between the micro-organisms
in the soil, only very small amounts of
free nutrients are present, so that second-
ary metabolites of the type in question
are formed only in extremely small
quantities. Where lipopeptides were
actually detectable, their concentrations
were less than the minimum inhibitory
concentration for phytopathogenic soil
fungi.
Further confirmation that the formation
of antifungal metabolites does not con-
tribute significantly to the effect emer-
ges from a comparison of different iso-
lates of Bacillus subtilis. No correlation
is found between e.g. the ability to form
metabolites that are effective against
Fusarium oxysporum on various media
in vitro and the observed effects on the
course of the Fusarium wilt disease in
greenhouse experiments with ornamen-
tals (Grosch et al. 1999).
78
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It is therefore difficult to judge whether
the lipopeptides play any part in the
reduction of the incidence and severity
of plant diseases achieved by the ap-
plication of FZB24

Bacillus subtilis. In
contrast the formation of compounds
possessing antibiotic activity appears to
be a more basic factor for the effectivity
of pseudomonads. The importance of the
antibiotics, like phenazine, formed by
these micro-organisms, for the suppres-
sion of plant diseases has been demon-
strated with mutants deficient for phena-
zine-production (Pierson and Thomas-
how 1992). Moreover, the antibiotic was
detected on roots in the soil (Mazzola et
al. 1992), and a quantitative dose-effect
relationship between antibiotic forma-
tion and the disease-suppressing effect
of Pseudomonas fluorescens against
Pythium sp. has been demonstrated in
cucumbers (Maurhofer et al. 1992).
5 Plant resistance induced by
Bacillus subtilis
All plants have evolved defense mecha-
nisms against pathogens. The efficacy of
these resistance reactions is modified as a
function of the ontogenetic development
of the plants and the influence of biotic
and abiotic environmental factors. Thus,
contact with non-pathogenic micro-orga-
nisms or limited infections leads to a de-
crease in the susceptibility of the plants.This
increased resistance due to exogenous
factors with no alteration of the plant
genome is known as induced resistance.
Induced resistance can be triggered both
by pre-inoculation with non-pathogens,
pathogens, symbionts, and saprophytes
and by application of so-called abiotic
inducers such as salicylic acid or microbial
metabolites (Schnbeck et al. 1993).
The induction of resistance has frequently
been described and discussed in the lite-
rature as an ability of micro-organisms.
Established phytopathological tests for
induced resistance are based on the se-
paration in space and/or in time between
the application of the inducing agent
and the inoculation of the plants. Biotro-
phic fungal pathogens such as powd-
ery and downy mildews or Phytophthora
infestans are better controlled with resi-
stance inducers.
It is assumed that the enhanced resist-
ance of the plants is due to altered gene
expression. In many cases the induction
of resistance is accompanied by induc-
tion of various so-called PR proteins
(pathogenesis-related proteins). Some
of these are 1,3--glucanases and chiti-
nases having the ability to lyse fungal
cell walls. Other PR proteins are less
well characterized or exhibit antimicro-
bial activities (van Loon and van Strien
1999). On the one hand, PR proteins are
regarded as markers of induced resist-
ance, while on the other, these proteins
themselves appear to be involved in the
increased resistance of the plants.
5.1 Changes in the gene expression of
the plants after application of FZB24

Bacillus subtilis
A test system for the identification of
resistance inducers, based on the follow-
ing principle, was developed by Hain et
al. (1995). Genes in plants are combined
with promoters that regulate their gene
activity. The genes responsible for de-
fense reactions in plants were regulated
also with promoters, which can be swit-
ched on by various stimuli (inducible
promoters). Transgenic tobacco plants
with the gene for herbicide resistance (in
Pflanzenschutz-Nachrichten Bayer 1/00, 1
this case BASTA

= phosphinothricin
N-acetyltransferase = pat) combined with
various inducible promoters were there-
fore cultivated for the test system. Pro-
moters that switch on the genes involved
in defense reactions against pathogens
were used for this purpose. The promo-
ters used were the following:
prp1 promoter of the proteins that
accompany pathogenesis develop-
ment from potatoes
chit2a promoter of a chitinase gene
from peanuts
Vst1 promoter of the stilbene synt-
hase gene from vines
When the promoter is activated by
the treatment of the test plants, the gene
for herbicide resistance is expressed as
a consequence. 15 days after the ap-
plication of an inducing agent, the
plants are insensitive to a spray treat-
ment of 515l of Basta/ha. Effective
resistance inducers lead to plants with
no herbicide damage. Resistance induc-
ers described in the literature, like
Na-salicylate and the commercial prod-
uct Oryzemate were detected with this
test system.
FZB24

Bacillus subtilis was tested in

repeated experiments in this test screen-
ing system. All three promoters were
activated by the treatment with FZB24

,
though partly with different intensities.
The prp1 promoter responded partic-
ularly strongly (Fig. 7). Both soil treat-
80
Fig. 7: Transgenic tobacco plants with the prp1/PAT gene fusion for identification of resistance
inducers. Plants 120 h after treatment with FZB24

(left) or with blank formulation (right). Test

plants were treated with 15l of Basta/ha. Photograph taken 9 days after herbicide treatment.
The activation of the prp1 promoter by FZB24

is clearly demonstrated by the reduction of

herbicide damage on the left of the photograph
Pflanzenschutz-Nachrichten Bayer 1/00, 1 81
ment by drenching and leaf treatment
by spraying resulted in the activation of
the promoters. These experiments pro-
vide direct evidence of the involvement
of resistance-inducing mechanisms in
the biological efficacy of FZB24

Bacillus
subtilis.
The experiments show that the bacteria
quickly trigger a signal that can be
systemically translocated within the
plant, so that an altered gene expression
and hence herbicide resistance were also
induced in the above-ground parts of the
plant. It has not been established whether
the resistance-inducing metabolites them-
selves act as signals, or whether they
trigger the formation of yet unknown
systemically translocatable signals. No
systemic colonization of the plants by
FZB24

Bacillus subtilis was found.

As well as the induction of PR proteins,
the application of plant growth promoting
rhizobacteria were also followed by ac-
tivation of other defense genes in plants.
Podile and Lami (1998) demonstrated a
systemic increase in the phenylalanine
ammonium lyase (PAL) activity in pigeon-
pea seedlings after treatment of the seeds
with the Bacillus subtilis strain AF1.
5.2 Demonstration of induced resistance
by FZB24

in phytopathological tests
Many plants, such as tomatoes, beans,
and tobacco produce pathogenesis-
related proteins, which are described as
markers of induced resistance. Rauscher
et al. (1999) were able to show that the
application of resistance inducers was
followed by the formation of PR-1 pro-
teins inhibited the differentiation of
infection structures of bean rust in the
apoplastic space of bean leaves. The
roots of various plants were treated with
FZB24

and infected with fungal patho-

gens on the leaves. Five days after the
application of FZB24

Bacillus subtilis
to the roots, tomato plants showed
distinctly less attack by Phytophthora
infestans and by Botrytis cinerea (Fig. 8).
Disease severity by P. infestans was
found to be reduced by up to 50% in
laboratory tests. The infestation of
B. cinerea is generally much more diffi-
cult to reduce by induced resistance.
Only with a higher concentration of bac-
teria a reduction of 20% was achieved.
The application of the abiotic resistance
inducer salicylic acid reduced the infec-
tion density of P. infestans by 30 %,
whereas it was found to be ineffective
against B. cinerea.
The use of a reference strain of Bacillus
subtilis did not lead to any changes in
the susceptibility of the plants.
Further evidence of increased resistance
of the plants came from experiments
that showed a reduction of 25% of
disease severity of powdery mildew on
wheat (Fig. 8).
A number of metabolites of bacteria are
under consideration as triggers of indu-
ced resistance; among others, these in-
clude lipopolysaccharides (Newmann et
al. 1995), enzymes (Palva et al. 1993),
and siderophores (Leeman et al. 1996),
and also salicylic acid (Meyer and Hfte
1997). The resistance systemically induc-
ed in tobacco by extracellular pectinases
and cellulases of Erwinia carotovora is
probably due to the release of cell wall
fragments as signals for the activation
of defence genes (Palva et al. 1993).
Bacillus subtilis forms mainly serine-
specific endopeptidases (Kula 1982).
From plant cell walls, proteases cleave
mainly hydroxyproline-rich glycoproteins
(Showalter 1993). These are derived
Pflanzenschutz-Nachrichten Bayer 1/00, 1
from the most important cell wall
protein extensin, which is present in all
higher plants (Showalter 1993). It is pos-
sible that through these interactions
with the cell wall, the proteases released
by FZB24

Bacillus subtilis also cause

the release of fragments that act as sig-
nal substances inducing resistance.
In contrast to biological control of
pathogens, which is based only on com-
petition or antibiosis, the protection of
plants by induced resistance can be
effective even when the inducing bacterial
population has already decreased. The
reason may be that defense mechanisms,
once activated, increase the defensive
capacity of the plants against various
pathogens for a long time, or that even
low population densities act continuously
as signal sources.
6 Promotion of root growth
A larger and healthier root system, such
as has been observed in a number of
greenhouse and field experiments with
FZB24

Bacillus subtilis, also leads to

improved uptake of water and nutrients.
In a greenhouse experiment with kohl-
rabi plants, the soil was drenched imme-
diately after sowing and again 4 weeks
later with 0.2 g FZB24

WG/l water at a
rate of 2 l/m
2
. The treatment led to a 5%
increase in the dry root weight (Fig. 9).
In addition to an improved germination
of the seeds, the yield of the plants at the
end of the cultivation time was up to
12% higher, depending on the variety.
The root development of potato plants
was determined in a field trial in 1998.
The potatoes were planted in mid-May
82
Fig. 8: Induced resistance by FZB24

Bacillus subtilis against leaf diseases in wheat and

tomatoes in laboratory experiments, in comparison with another strain of Bacillus subtilis and
the resistance inducing agent Na-salicylate
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and treated during planting with a liquid
seed treatment in the recommended do-
sage of 10g of FZB24

WG/100 kg of seed
potatoes. With the beginning of tuber
formation in early August, the root fresh
weight of the plants treated with FZB24

was 6% higher than that of untreated

plants. The yield of the plants after treat-
ment with FZB24

in this experiment
was increased at harvest time in Septem-
ber by about 8%.
Enhanced root formation of infected
plants has been described by Garett
(1956) as a disease escape mechanism.
Increased root growth enables the plant
to grow out of contaminated regions of
the substrate and to replace infected
root sections more easily, and at the
same time enables the plant to reach
earlier growth stages in which it is less
susceptible. The intensified root forma-
tion after application of FZB24

may
therefore also be a reason for a reduc-
tion of plant damage due to infections
of Rhizoctonia solani or Fusarium oxy-
sporum.
7 Effect on plant growth and yield
Another reason that has been proposed
for the promotion of plant growth by
bacteria that colonize the rhizosphere
is the production of phytohormones
and phytohormonally active metabolites
(Kloepper et al. 1991).
Dolej (1998) was able to show that the
growth-promoting effect of culture fil-
trates of FZB24

Bacillus subtilis is not

due to lipopeptides having an antibiotic
action. This is supported by investigations
with Bacillus subtilis mutants that no
longer had the ability to form antibio-
tics, but still led to increased yields from
peanut plants (Backmann et al. 1994).
Fig. 9: Promotion of root growth of kohlrabi (cv Rogli) by two drench treatments with FZB24

WG. Left: untreated; Right: plant treated with FZB24

Pflanzenschutz-Nachrichten Bayer 1/00, 1

The phytohormonal activity of the meta-
bolites formed by FZB24

Bacillus
subtilis in liquid cultures has been demon-
strated by biological test methods. Com-
plex culture filtrates and fractions derived
from them led, like cytokinines, to enhanc-
ed growth of radish cotyledons, and like
auxins, increased the elongation of the
cells of wheat coleoptiles. These effects
appear to be initiated by mixtures of se-
veral proteins, while further separation
and purification of the culture filtrates led
to the loss of the effects (Alemayehu
1998). According to Tang (1994), a num-
ber of Bacillus subtilis isolates have the
ability to form phytohormones such as
zeatin, gibberellic acid, and abscisic acid.
A culture filtrate of a Bacillus subtilis
isolate that was used as a resistance indu-
cer against biotrophic fungal plant patho-
gens has also been found to contain the
cytokinins zeatin and zeatin riboside. The
senescence-delaying effect of them could
be a cause of the reduced damaging effect
of pathogens and the increased yield of
plants in which resistance has been in-
duced (Steiner 1990).
Enhanced root growth is often accom-
panied by increased branching and a
higher number of root tips. Their meri-
stems are the most important sites
for the synthesis of free cytokinins
(Torrey 1976). These are presumably
transported into the shoot via the xylem.
Intensified and prolonged synthesis of
these phytohormones may be regarded
as a cause of delayed senescence and
improved yields (Mengel 1973).
Since the application of Bacillus subtilis
leads to stronger root growth, there may
also be an increased synthesis of plant
cytokinins, which also cause delayed
senescence and higher yields, as described
above. These effects on the phytohor-
mone balance of the plants can explain
why increased yields are found even for
plants that show no visible attack by
soil-borne or root diseases.
An increase in the yield was also achiev-
ed by leaf applications of FZB24

. In
three field trials in 1998 with potatoes, a
leaf treatment was carried out in com-
bination with the application of leaf
fungicides to control P. infestans. Four
applications of FZB24

with a dosage of
0.4% were carried out at intervals of
10 to 14 days beginning in mid-June. The
effects were compared with a dry seed
treatment with FZB24

. Because of the
fungicide treatment, the plants were
largely protected against attack by
P. infestans. The application of FZB24

WG to the leaf did not produce any

visible improvement in the protection
against the leaf pathogen, but led to an
increase in the yield of 8.5%, which was
higher than the yield increase achieved
with the dry seed treatment in these
experiments (Fig. 10).
In addition to the effects on the phyto-
hormone balance of the plants, an im-
provement of the tolerance of the plants
may also contribute to increased yields.
Tolerance is defined as the ability of the
plant to survive attack by pathogens or
the action of abiotic stress factors with
smaller losses of viability and produc-
tivity than another plant subjected to the
same exposure intensity (Aust et al.
1991). Possible factors that lead to to-
lerance of plants towards pathogens
(according to Clarke 1986) are:
reduced sensitivity of the plants to-
wards toxic metabolites produced by
the pathogens,
ability of infected and uninfected
parts of the plants to compensate
84
Pflanzenschutz-Nachrichten Bayer 1/00, 1 85
through increased metabolic activity,
e.g. photosynthesis,
delayed senescence,
less influence on sink-and-source
relationships.
Tolerance was described to be influenc-
ed e.g. by culture filtrate fractions of
FZB24

Bacillus subtilis having cyto-

kinin-like and auxin-like activities.
In vitro tests with callus cultures of to-
matoes showed increased stress toler-
ance towards the Fusarium toxin fusaric
acid, with the result that the browning
symptoms were reduced (Alemayehu
1998).
Culture filtrates of a Bacillus subtilis isol-
ate, used as a resistance inducer against
biotrophic leaf pathogens, induced chan-
ges in the sink-and-source relationships
in barley after attack by powdery mil-
dew. This was related both to the trans-
port of assimilates from the host to the
pathogen and to the translocation of
assimilates in the whole plant. It could
be proved that the application of culture
filtrates led to increased movement
of assimilates from the flag leaf into the
ears, which then also had a higher starch
content (Kehlenbeck et al. 1994).
8 Conclusions
Numerous mechanisms seem to be in-
volved in the effect of FZB24

Bacillus
subtilis. These mechanisms can con-
tribute in different degrees to the re-
duction of disease and the enhance-
ment of yields, depending on the plant,
the environmental conditions, the appli-
cation form, and the time of application.
The most obvious effects of the bacteria
on diseases have been found in the case
of attack by root pathogens. This could
be due to the fact that all of the mecha-
nisms mentioned, i.e. competition, anti-
biosis, resistance induction, and disease
escape as a result of growth effects, are
able to operate in the root area, whereas
only resistance induction can operate in
the leaf area, since no translocation of
the bacteria takes place.
Fig. 10: Effect of FZB24

Bacillus subtilis on the yield of potatoes after seed tuber treatment

(20g FZB24 DS/100kg) and leave applications (0,4% FZB24 WG)
Pflanzenschutz-Nachrichten Bayer 1/00, 1
In contrast with biological methods for
controlling pathogens, which are based
only on competition or antibiosis, the
protection of plants by resistance in-
duction can be effective even when the
bacterial population that triggered these
effects has already declined. This may
be because defense mechanisms, once
activated, increase the defensive capa-
city of the plant against various patho-
gens over a long period, or because even
low population densities function contin-
uously as inducing agents.
However, the observed biological
effects are also due to changes in the
physiology of the plant. In the first
place, the tolerance towards abiotic and
biotic stress factors is improved because
the root system of the plant is strength-
ened, and hence also the uptake of
water and nutrients. In addition, many
results indicate that the application of
Bacillus subtilis changes the phytohor-
mone balance in the plant in such a
manner that greater quantities of reserve
substances are incorporated into stor-
age organs.
The large number of mechanisms invol-
ved may be one reason why FZB24

Bacillus subtilis can be used for a wide

spectrum of crops with their different
culture conditions. However, none of the
observed mechanisms has any curative
effect, so that early treatment, prefer-
ably right from the beginning of culti-
vation, is advisable.
9 Summary
The present article summarizes current
knowledge about the mode of action of
FZB24

Bacillus subtilis and the biotic
and abiotic environmental factors that
influence its action.
Bacteria are present in the soil in an aver-
age content of 6x10
8
cells/g of soil, and
with a live weight of about 10,000kg/ha,
they are the most abundant micro-orga-
nisms in soil samples. However, the num-
bers of bacteria vary by up to a factor of
50, depending on biotic and abiotic en-
vironmental factors. Bacillus species are
among the most common organisms
isolated from soil samples. Within this
microbiological context FZB24

Bacillus
subtilis must temporarily establish itself in
the rhizosphere of the cultivated plant.
A number of mechanisms that could
contribute to increased yields and
reduced attack by pathogens following
the application of Bacillus subtilis have
been described in the literature. The
following mechanisms and effects have
so far been demonstrated experimen-
tally for FZB24

Bacillus subtilis:
Competition by temporary coloni-
zation of the rhizosphere and
rhizoplane.
FZB24

has the ability to form anti-

biotic metabolites in vitro. However,
this depends very strongly on the
culture medium. It was not possible
to confirm the formation of these
metabolites on the root and in the
substrate in vivo.
Induced resistance by activation of
defense genes in plants, which has
been demonstrated both by mole-
cular-biological methods and by
phytopathological tests.
Promotion of plant and root growth.
The formation of substances and
mixtures having cytokinin-like and
auxin-like effects by B. subtilis has
been demonstrated in vitro. Howe-
ver, the enlarged and more highly
branched root system of the plant
86
Pflanzenschutz-Nachrichten Bayer 1/00, 1 87
also alters the endogenous phyto-
hormone balance. The stronger root
system ultimately also leads to an
improved uptake of water and nutri-
ents, and hence to faster growth and
greater dry stress tolerance. Moreo-
ver, the growth promotion leads to
the possibility of disease escape,
since the plants can grow out of sen-
sitive stages more quickly and en-
hanced root growth allows a better
compensation for diseased parts of
the roots.
Zusammenfassung
FZB24

Bacillus subtilis Wirkungs-

weise eines mikrobiellen Pflanzenstr-
kungsmittels
In dieser Arbeit wird der aktuelle Wis-
sensstand zur Wirkungsweise von FZB24

Bacillus subtilis und zu den biotischen

und abiotischen Umweltfaktoren, die
die Wirkung beeinflussen, zusammenge-
fasst.
Bakterien kommen im Boden im Mittel
mit 6x10
8
Zellen/g Boden vor und sind
mit einem Lebendgewicht von ca.
10.000 kg/ha die hufigsten Kleinlebe-
wesen in Bodenproben. Die Bakterien-
zahl variiert um den Faktor 50 in Abhn-
gigkeit von biotischen und abiotischen
Umweltfaktoren. Bacillus-Arten gehren
zu den hufigsten Gattungen, die aus
Bodenproben isoliert werden. In diesem
mikrobiologischen Umfeld muss sich
FZB24

Bacillus subtilis temporr in der

Rhizosphre der Kulturpflanze etablie-
ren.
In der Literatur sind eine Reihe mg-
licher Wirkmechanismen beschrieben,
die zu den immer wieder beobachteten
Ertragssteigerungen und Reduktionen
des Befalls mit Pathogenen nach einer
Bacillus subtilis Anwendung beitragen
knnen. Fr FZB24

Bacillus subtilis
konnten bisher die folgenden Mechanis-
men und Effekte experimentell demon-
striert werden:
Konkurrenz durch vorrberge-
hende Besiedelung der Rhizosphre
und Rhizoplane durch Bacillus
subtilis.
FZB24

ist in vitro in der Lage, anti-

biotische Stoffwechselprodukte zu
bilden. Dies ist aber stark von der
Zusammensetzung der Nhrmedien
abhngig. In vitro konnte eine Bil-
dung dieser Stoffwechelprodukte an
der Wurzel nicht besttigt werden.
Resistenzinduktion durch Aktivie-
rung von Abwehrgenen in Pflanzen,
was sowohl mit molekularbiologi-
schen Methoden wie auch mit phyto-
pathologischen Tests nachgewiesen
werden konnte.
Frderung des Pflanzen- und Wur-
zelwachstums. In vitro konnte die
Bildung von Substanzen und Sub-
stanzgemischen mit cytokinin- bzw.
auxinartigen Wirkungen durch B.
subtilis festgestellt werden. Aber auch
das vergrerte und strker ver-
zweigte Wurzelsystem der Pflanze
verndert deren endogene Phytoh-
ormonbalance. Das strkere Wurzel-
werk fhrt letztlich auch zu einer
verbesserten Aufnahme von Wasser
und Nhrstoffen und damit zu
schnellerem Wachstum und grerer
Trockenstresstoleranz. Darber hin-
aus ermglicht die Wachstumsfrde-
rung ein disease escape, indem die
Pflanze schneller empfindlichen
Stadien entwachsen kann und
kranke Wurzelteile besser kompen-
siert werden.
Pflanzenschutz-Nachrichten Bayer 1/00, 1
Rsum
FZB24

Bacillus subtilis Mode dac-

tion dun stimulateur microbiologique
de vigueur vgtale
Ce travail rcapitule les connaissances
actuelles relatives au mode daction du
FZB24

Bacillus subtilis et aux facteurs
environnementaux biotiques et abioti-
ques qui influent sur cette action.
On trouve des bactries dans le sol, en
moyenne raison de 6.10
8
cellules/g, et,
avec un poids vif denviron 10 000 kg/ha,
elles reprsentent les microorganismes
les plus frquents dans les chantillons
de sol. Mais le nombre de bactries varie
dun facteur de 1 50, selon les facteurs
environnementaux biotiques et abio-
tiques. Les espces de Bacillus apparti-
ennent aux genres les plus frquemment
isols des chantillons de sol. Dans
cet environnement microbiologique, le
FZB24

Bacillus subtilis doit stablir

titre provisoire dans la rhizosphre de la
plante cultive.
Dans la bibliographie, de trs nombreux
mcanismes daction potentiels sont
dcrits, qui, aprs une utilisation de
Bacillus subtilis, peuvent contribuer aux
phnomnes toujours et encore obser-
vs daugmentation de rendement et de
rduction de lattaque par les microor-
ganismes pathognes. A ce jour, on a
pu montrer titre exprimental les
mcanismes et effets suivants du
FZB24

Bacillus subtilis:
Comptition par colonisation provi-
soire de la rhizosphre et des rhizo-
plans par Bacillus subtilis.
Le FZB24

est, in vitro, mme de

former des mtabolites antibio-
tiques. Mais cette production dpend
beaucoup des milieux nutritifs.
Aucune formation de ces mtaboli-
tes na pu tre confirme sur les raci-
nes.
Stimulation des dfenses naturelles
des vgtaux par activation de leurs
gnes de dfense, ce qui a pu tre
mis en vidence tant par des mtho-
des de biologie molculaire que par
des essais phytopathologiques.
Promotion de la croissance vgtale
et radiculaire. In vitro, on a pu con-
stater sous leffet de B. subtilis la for-
mation de substances et de mlanges
de substances ayant une action ana-
logue celle de la cytokinine ou de
lauxine. Cependant, laugmentation
du volume et des ramifications du
systme racinaire des plantes modi-
fie galement leur bilan phytohor-
monal endogne. Enfin, lappareil
radiculaire plus dvelopp permet
aussi une meilleure absorption de
leau et des substances nutritives, et
donc une croissance plus rapide et
une plus grande tolrance au stress
hydrique. En outre, la stimulation de
la croissance permet une fuite en
avant (disease escape), la plante
pouvant crotre plus vite pour dpas-
ser les stades sensibles, en assurant
une meilleure compensation des
parties racinaires malades.
Resumen
FZB24

Bacillus subtilis Mecanismo

de accin de un vigorizante para plantas
En este trabajo se expone el estado
actual de conocimientos cientficos
sobre el mecanismo de accin del
FZB24

Bacillus subtilis y sobre los

factores medioambientales biticos y
abiticos que influyen en dicha accin.
88
Pflanzenschutz-Nachrichten Bayer 1/00, 1 89
Las bacterias estn presentes en el suelo
en una concentracin media de 6 x 10
8
clulas/g de suelo y son los microorga-
nismos vivientes ms frecuentes en las
muestras de suelo, cifrndose su peso en
unos 10.000 kg/ha. Las concentraciones
bacterianas pueden oscilar incluso en un
factor de 50, en funcin de los factores
medioambientales biticos y abiticos.
Las especies Bacillus son las que se
aslan con mayor frecuencia de las mue-
stras de suelo. En este contexto micro-
biolgico, FZB24

Bacillus subtilis debe

establecerse temporariamente en la
rizosfera de las plantas cultivadas.
En la bibliografa tcnica se describen
mltiples mecanismos potenciales de ac-
cin que, despus de una aplicacin de
Bacillus subtilis, pueden contribur a los
incrementos de rendimientos de cos-
echas observados repetidamente y a la
reduccin de la incidencia de patgenos.
Hasta ahora se han podido demostrar
experimentalmente los siguientes meca-
nismos de accin o efectos de FZB24

Bacillus subtilis:
competencia basada en la coloniza-
cin transitoria de la rizosfera y rizo-
planos con Bacillus subtilis
FZB24

in vitro es capaz de gene-

rar productos metablicos antibiti-
cos. Sin embargo, esto depende de
los caldos de cultivo. No se ha po-
dido confirmar in vitro la forma-
cin de estos productos metablicos
en la raz.
Resistencia inducida por activacin
de genes defensivos en plantas,
detectada tanto por mtodos de
biologa molecular como por en-
sayos fitopatolgicos.
Estimulacin del crecimiento de
planta y raz. Se ha observado in
vitro la formacin de sustancias
y mezclas de sustancias debida a
B. subtilis, cuyos efectos son simila-
res a los de la citoquinina y auxina.
Tambin el sistema radicular mas
amplio y ramificado de las plantas
cambia su equilibrio fitohormonal
endgeno. A fin de cuentas, un
sistema radicular mas vigoroso con-
duce a una mejor absorcin de agua
y nutrientes del suelo y, por tanto, a
un crecimiento ms rpido y una
mayor tolerancia a condiciones de
sequa. Por otro lado, la estimulacin
del crecimiento permite a la planta
escapar de las enfermedades, ya
que puede superar con mayor rapi-
dez los estadios crticos y compensar
mejor las partes enfermas de sus
races.
Posmuo
Hpuuqun e1nun uupu-
uue pe1na n e
peneuun pa1euu FZB24

Bacillus subtilis
B pa1e pa1 nuueae1n uuuem-
uu peeub suauu npuuune
e1eun FZB24

Bacillus subtilis, a
1axe u1uueux u au1uueux
a1pax pxake peu, asu-
eakuxn ua e1euu s1 npe-
napa1a.
Hs eex VuppauusVe e npax
uauee ua1 e1peuak1n
a1epuu: ux peuee ue1e 1a-
ene1 6x10
8
e1 ua paVV nueu, a
xue ee . 10 000 /a. ua,
e saeuuV1u 1 u1uueux u au-
1uueux a1pe pxake
peu uue1e a1epu Vxe1
pasuua1bn e 50 pas. a1epuu
Pflanzenschutz-Nachrichten Bayer 1/00, 1
eue Bacillus 1un1n uauee
ua1 eueneVuV us nueeuuux np
paV VuppauusVe. B 1aV
VupuuueV pxeuuu FZB24

Bacillus subtilis xeu epeVeuu

pasVea1bn e pusepe b1p-
uux pa1euu.
B u1epa1pe nuueae1n ueub
Vu npenaaeVux VexauusVe
e1eun, 1pue npu npuVeueuuu
Bacillus subtilis V1 n1eea1b
penpu uakaeVV eeuue-
uuk pxau1u u uuxeuuk
npaxeuun esu1enVu esue.
B uae FZB24

Bacillus subtilis
ab ux np snepuVeu1abu
npeVu1pupea1b ekue
VexauusVu uu se1u:
upeuun e pesb1a1e epeVeu-
u saeeuun pusepu u
pusneepxu1u a1epunVu
Bacillus subtilis.
FZB24

Bacillus subtilis Vxe1 in
vitro paseuea1b au1uu1uue-
ue Ve1au1u. ua, s11
npe ubu saeuu1 1 nu1a1eb-
uux pe. In vitro ue ab
n1eepu1b paseauue 1aux
np1e Ve1ausVa ua punx.
Huun pesu1eu1u1u
aapn a1ueauu sau1uux
eue e pa1euunx, u1 ab
asa1b a Ve1aVu Ve-
npu uuu, 1a u u1-
na1uueuVu unu1auunVu.
e1eue p1 pa1euu u
pue. B eunx in vitro ab
1auea1b paseauue a1e-
punVu Bacillus subtilis 1auu u
Vee 1auu, e1ekux
nu u1uuuu u auu.
HexuV ua 1Ve1u1b, u1
eeuueuuan u ee pasee1-
eeuuan pueean u1eVa pa1e-
uu 1axe usVeune1 ux u1p-
Vuabuu aau. B ueuuV
u1e ee Vuan pueean
u1eVa umae1 neuue
eu u nu1a1ebuux eee1e u,
eea1ebu, pne1 p1 u
neumae1 sax1uue1b.
pVe 1, e1eue p1 e -
neuueae1 disease escape, 1.e.
peuue npxxeuue uau-
ee ue1eu1ebuux 1au
paseu1un pa1euu, u umeuue
Vneuauu npaxeuuux ua1e
pun.
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Dr. Ulrike Steiner
Institut fr Pflanzenkrankheiten
Universitt Bonn
Nussallee 9, D-53115 Bonn, Germany
phone: 0049 228 733341
fax: 0049 228 732442
email: U-Steiner@uni-bonn.de
http://www.uni-bonn.de/pflanzen-
krankheiten/
Dr. Michael Kilian
Dr. Rdiger Hain
Bayer AG
Landwirtschaftszentrum Monheim
Geschftsbereich Pflanzenschutz/
Forschung
D-51368 Leverkusen
Tel.: O2173-383210 (Dr.Kilian)
email: Michael.Kilian.MK@Bayer-
AG.de
Tel.: O2173-384382 (Dr.Hain)
Dr. Helmut Junge
Dr. Birgit Krebs
FZB Biotechnik GmbH
Glienicker Weg 185, D-12489 Berlin
Tel.: 030-67 057 0
Fax: 030-67 057 233
email: dr.junge.fzb@t-online.de
Dr. Gunter Schmiedeknecht
Schsische Landesanstalt fr
Landwirtschaft
FB06 Integrierter Pflanzenschutz,
Referat 61, Stbelallee 2,
D-01307 Dresden
Tel.: 0351/4408326
Fax: 0351/4408325
email: Gunter.Schmiedeknecht@
fb06.lfl.sml.sachsen.de
Manuscript received: 14.6.00