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).

Powdery mildew

Pythium root and crown rot

Fusarium root and stem rot

Gummy stem 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 INFECTION

Biocontrol agent

TISSUE COLONIZATION

Biocontrol agent

GROWTH AND SPORULATION

Biocontrol agent

SPREAD AND REINFECTION

Biocontrol agent

Organic Soil

Rockwool block

Rockwool infested with Fusarium

Antagonist population

Pathogen population

Susceptible crop-plant 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 Azalea Barley Bromegrass Clover Cucumber Grape Lupine Maple Rhododendron Salmonberry Shepard’s Purse British Columbia British Columbia Denmark, Michigan, U.S.A. British Columbia British Columbia British Columbia New York, U.S.A. Japan, British Columbia, Netherlands British Columbia British Columbia British Columbia 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. minor (T. Hijwegen) T. fulvescens CBS 370.85

T. washingtonensis ATTC 96156

Tilletiopsis – _-1,3glucanase production in 5 species

20 kDa 32 kDa

T. pallescens ATCC 96155 T. pallescens ATCC 66538 T. pallescens ATCC 162.85 T. pallescens ATCC 36535

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

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 Concentrate to dryness Concentrate to dryness, dissolve in methanol TLC PTLC Bioassay (200 _g/mL) XAD-4 column Re-extract with ethyl acetate

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 )

10.0

a

Control Canola oil/lecithin Liq. Paraffin/lecithin Liq. Paraffin/Tween 80 1% canaola oil 0.1% lecithin

6

8.0

6.0

b
4.0

b

2.0

c

c

c

0.0 Amendments

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