Microbial interactions on the

phyllosphere of plants –
applications to biological
control of fungal diseases on
greenhouse vegetable crops

Zamir K. Punja
Simon Fraser University, Burnaby, B.C.

SFU
Hydroponic culture system
 Fungal diseases on greenhouse
cucumber
Cucumber
• Powdery mildew (Sphaerotheca fuliginea)
(= Podosphaera (Sect. Sphaerotheca)
xanthi).
• Pythium root and crown rot (Pythium
aphanidermatum, P. ultimum).
• Fusarium root and stem rot (Fusarium
oxysporum f.sp radicis-cucumerinum).
• Gummy stem blight (Didymella bryoniae)
(= Phoma cucurbitacearum).
 Pythium root
Powdery mildew and crown rot

Fusarium root Gummy stem

and stem rot blight
 Disease management strategies

• Resistant or tolerant cultivars, if

available.
• Fungicides as seed treatments, drench,
foliar spray, vapours.
• Cultural control – environmental
monitoring, plant health management.
• Sanitation – removal of diseased
plants, hygiene and clean-up,
disinfectants.
• Biological control – as preventative
applications, or potentially curative,
depending on the disease.
 The greenhouse environment

• The controlled environment of the

greenhouse provides suitable
conditions for the establishment of
biological control agents.
• Paradoxically, it can also provide
conducive environmental conditions
for the build-up of fungal plant
pathogens on greenhouse crops.
• In greenhouse rockwool-hydroponic
productions systems, antagonistic
microorganisms are virtually absent,
allowing an introduced pathogen to
grow rapidly and cause disease.

• How can we establish a biological

balance (biological buffer) between
antagonists and root pathogens?
 Biological control
• Reduction of inoculum density or
disease producing activities of a
pathogen or parasite (in its active or
dormant state) by one or more
organisms.
• Can be accomplished naturally or
through manipulation of the
environment, host or antagonist, or by
mass introduction of one or more
antagonists (biocontrol agent).
 SURVIVAL Biocontrol agent

GERMINATION
AND Biocontrol agent
INFECTION

TISSUE Biocontrol agent

COLONIZATION

GROWTH
AND Biocontrol agent
SPORULATION

SPREAD
AND Biocontrol agent
REINFECTION
Organic Soil Rockwool block
Rockwool infested with Fusarium
 Antagonist
population

Susceptible
Pathogen crop-plant
population
population
Phyllosphere

Phylloplane

Rhizoplane

Rhizosphere
• Biological control agents (BCA) have to be
introduced early, before the pathogen infects
(preventative). There are only a few BCA that
show curative properties.
• There are several options for applying a
BCA:

To planting medium.

On seed surface.

Transplant drench.

To mature plants.

Via irrigation.
• More than one application is usually
required, generally at 3-6 week intervals.
 Microbial interactions in the
phyllosphere

Biological control of powdery mildew

using antagonistic yeasts in the genus
Tilletiopsis.
 Powdery mildew diseases are of widespread
occurrence on plants, particulary those
grown commercially in greenhouse eg:
cucumbers, rose, pepper.
Powdery mildew pathogen
 Tilletiopsis spp.
(Class: Basidiomycetes
Order: Tilletiales)

 Saprophytic yeast-like fungi.

 Associated with mildew or rust on
leaves.
 Some species have demonstrated
biological control activity.
 Form ballistospores, blastospores and
chlamydospores.
 Species of Tilletiopsis

 T. washingtonensis – type specimen

(Nyland, 1950).
 T. minor – (Nyland, 1950).
 T. albescens – (Gokhale, 1972).
 T. flava (Tubaki, 1952).
 T. fulvescens (Gokhale, 1972).
 T. pallescens (Gokhale, 1972).
Identification of Tilletiopsis spp.

1. Colony morphology – color, texture,

size.
2. Utilization of carbon and nitrogen
sources.
3. Comparative guanine and cytosine
ratios (Boekhout, 1991).
4. Chromosome counts in orthogonal field
electrophoresis (Boekhout, 1991).
5. Ribosomal DNA sequence (large
subunit) (Boekhout, 1991).
 Biological control activity of
Tilletiopsis
 T. minor – cucumber powdery mildew
 T. pallescens – barley powdery mildew
 – cucumber powdery
mildew
 T. washingtonensis – cucumber powdery
mildew

- Tested in Netherlands, Denmark, Canada on

semi-commercial scale.
- Limited factor is the dependence on high
relative humidity (>80%).
 Objectives
1. To recover species of Tilletiopsis from a
range of plant species.
2. To study the ecology of these yeasts on the
leaf surface (phylloplane).
3. To demonstrate biological control activity
of the yeasts against mildew diseases.
4. To study the mode(s) of antagonism of
yeasts against mildew fungi.
5. To use molecular methods to identify
species of Tilletiopsis.
Hosts and areas of origin of isolates
of Tilletiopsis
Apple British Columbia
Azalea British Columbia
Barley Denmark, Michigan, U.S.A.
Bromegrass British Columbia
Clover British Columbia
Cucumber British Columbia
New York, U.S.A.
Grape Japan, British Columbia,

Lupine Netherlands
Maple British Columbia
Rhododendron British Columbia
Salmonberry British Columbia
Shepard’s Purse British Columbia
Isolation of yeasts – spore fall method
Leaf imprint – colonies recovered
Species of Tilletiopsis and Pseudozyma
Tilletiopsis washingtonensis
72 hr-old culture – blastospores
 Biological control activity

Treated Untreated
Control leaf – no
applications
Biological control
– 2 applications
in greenhouse
Biological control – 3 applications in greenhouse
Given the dramatic reduction of
powdery mildew following Tilletiopsis
application, what interactions are
occurring between the two species?
SEM – cucumber leaf
Tilletiopsis colony – 72 hr
Tilletiopsis – spore production – 5 days
Tilletiopsis spores and mildew – one day
Tilletiopsis and mildew – 2 days
Tilletiopsis – ballistospores – 5 days
Tilletiopsis ballistospores – 6 days
Tilletiopsis ballistospores – 6 days
 Mechanisms through which biological
control agents could work in vivo

1. Production of antibiotics, toxins and lytic

enzymes.
2. Direct parasitism of hyphae or propagules
of the pathogen.
3. Competitive exclusion by occupation of
infection sites and/or depletion of nutrients.
4. Plant growth enhancement and induction of
resistance.
5. Reduction of aggressiveness or virulence
through mycovirus infection.
 29 kDa

20.1 kDa
T. pallescens
ATTC 96155
T. albescens
CBS 370.85
T. washingtonensis
ATTC 96156
T. minor
(T. Hijwegen)

T. fulvescens
CBS 370.85
species
glucanase
production in 5
Tilletiopsis – _-1,3-
20 kDa
32 kDa
T. pallescens
ATCC 96155

T. pallescens
ATCC 66538

T. pallescens
ATCC 162.85

T. pallescens
ATCC 36535
pallescens
in 4 strains of T.
Tilletiopsis – _-1,3-
glucanase production
 Modes of Action

• Hydrolytic enzymes may have a

secondary role in biocontrol activity of
Tilletiopsis against powdery mildew as
they are produced later during growth
(> 10 days).
• Are there antibiotic-like substances
produced?
 6-day-old culture filtrate

Methanol extract XAD-4 column

Concentrate Re-extract with

to dryness ethyl acetate

Concentrate to dryness,
dissolve in methanol

TLC PTLC Bioassay (200 _g/mL)

Control T. pallescens

Tilletiopsis – antifungal
compounds
 Modes of Action
• Fatty acid esters are a component of
the culture filtrate of Tilletiopsis and
demonstrate a wide spectrum of
antifungal activity.
• A number of additional uncharacterized
antifungal compounds are also present.
Pseudozyma – antifungal compounds

SOURCE: T.J. Avis and R.R. Belanger. Appl.

Environ. Microbiol. Vol. 67. Feb 2001. pp 956-960
 Applications of biotechnology to
biological control agents
1) Use of molecular markers to
characterize biocontrol strains, and to
track movement and survival.
2) Use of biochemical/molecular
approaches to elucidate mode (s) of
action.
3) Use of molecular/physiological tests
to ensure quality control, i.e. strain
efficacy.
RAPD analysis of 3 Tilletiopsis species
RAPD analysis of 6 Tilletiopsis species
• Characterization of a biocontrol agent
(molecular, physiological, biochemical)
is an important aspect for registration
purposes.
• Formulation of a biocontrol agent is the
key to ensuring product efficacy.
 Effect of Amendments on Survival (24 hr)

12.0
Tilletiopsis colonies recovered at 24 hr (x 10 )

Control
6

Canola oil/lecithin
a
10.0 Liq. Paraffin/lecithin
Liq. Paraffin/Tween 80
1% canaola oil
8.0
0.1% lecithin

6.0 b
b

4.0

2.0
c c
c

0.0
Amendments