Use of Trichoderma, Pseudomonas and Bacillus spp. in IPM Programs

Barry J. Jacobsen

Dept. of Plant Sciences and Plant Pathology

Montana State University

ESA, 2012

Typically used as seed, tuber, rhizome, root stock, and soil treatments Widely used in IPM CRSP activities in South Asia

Many products in both developed and developing world

Widespread acceptance by farmers after comparison to farmer practices that include pesticides- increase yield, quality, earlier harvest, profitability-100s of trials

Mechanisms for Biological Control of Plant Pathogens

• Antibiosis-Biological Control Agent (BCA) produces antibiotic substance that suppresses pathogen. Pseudomonas, Bacillus, Trichoderma

• Competition for nutrients-BCA competes for resource in short supply that pathogen needs. e.g. Fe+++ , sugars and other needed for spore germination or growth. Pseudomonas

• Niche occupation-BCA occupies infection niche

• Predation

• Parasitism. Trichoderma, Bacillus penetrans-nematode

• Alter plant physiology (transpiration, water relations, growth hormones, nutrient uptake, N fixation)

• Induced resistance. Pseudomonas, Bacillus, Trichoderma

• MOST BCAs HAVE MULTIPLE MECHANISMS

Rhizosphere – PGPR-Plant growth promoting rhizobacteria and

Trichoderma colonize Rhizosphere/endosphere

• Pseudomondas

• Enterobacter

• Bacillus

• Azospirillum

• Many others

Rhizosphere colonist produce antibiotics,etc Antagonistic to pathogens

Induce plant defense genes systemically

Colonize rhizosphere occupy infection courts Tie up critical nutrients needed by pathogens, produce growth promoting substances

15C=1.13 e5

24C=7.59e 5 7.1 e5

1.3 e5 2.1 e5

1.5 e5

2.1 e5

5.4 e5

Colonization (CFU/g ) of sugarbeet by 341-16-5 from treated seed

Many PGPR are endophytes

Growth promotion by PGPR Control of root pathogens, growth regulators, improved nutrition, induced resistance?

Damping-off

Mixing Trichoderma product in potting medium

Planting Trichoderma/PGPR colonized seedlings

Healthy seedling protected by PGPR Trichoderma

Begonias were grown in the greenhouse and inoculated with Botrytis cinerea under conditions optimal for the development of disease. Treatments left to right : untreated (Un), CaCl2, chlorothalonil (Fung), and the biocontrol agent Trichoderma hamatum T382 inoculated into the potting mix (T382). Hoitink, et al

Competition for nutrients • Many rhizosphere colonizing Pseudomonads (PGPR), Serratia, Erwinia

(Pantoea) provide disease suppression by competition for Fe+++ with pathogens via chelation by siderophores (pyroverdin, pseudobactin, pyochelin). – Fusarium wilts of flax, carnation, Take-all, Thielaviopsis, Rhizoctonia,

Sclerotium rolfsii, Erwinia carotovora, several patch diseases of turf, dollar spot of turf(Sclerotinia), melting out of turf (Drechslera-Bipolaris), DRBs, Deliterious rhizobacteria that produce HCN, - may also be SAR/ISR signaling agents

– Fireblight- • Pantoea agglomerans competes for nutrients and niche with

Erwinia amylovora, • Pseudomonas fluorescens A 506(Blight Ban)- Fe+++ allows

production of antibiotic antagonistic against E. amylovora that allows competition for site-this is used commercially

• PGPR competes for nutrients (root exudates) with slower growing

pathogens-fungistasis, chlamydospores, macroconidia, oospores

Altered physiology • Trichoderma harzianum/viridae-improve water and nutrient

uptake- many references

• Bacillus subtilis (Kodiak, other), Bacillus- pumillus GB 34 (YieldShield), Azospirillum, Pseudomonas

– Auxins- Asghar et.al.,2002, Idris et.al. 2007

– Gibberelins-Joo, et.al.2005

– Cytokinins-Garcia de Salmone, 2001, Castro, et.al. 2008, Dobbelaere,et.al. 1999.

– P uptake-Ramirez and Kloepper, 2010 (phytase activity)

– Improved N utilization-Shoebitz et.al. 2009-nitrogenase and IAA

– improved water relations

Phyllosphere/Phylloplane Biological Control

• Environment for BCA in this environment is relatively hostile compared to rhizosphere / endosphere. – Physical environment: great flux in moisture, relative humidity, UV/IR

radiation, paucity of nutrients that change with leaf age and time – Biological environment: competition with phylloplane colonists and invaders,

plant responses and exudates vary with physiological age, genetics, etc

• Majority of products are oriented to greenhouse or controlled storage situations where environment is more stabile. – Even here BCA performance has greater variability than chemicals

• Vast majority of research has focused on Botrytis, powdery mildew and fruit storage molds. – Significant markets-high value of vegetables, ornamentals and fruit – Fewer registered pesticides-fungicide resistance problems

• Lower costs to register BCAs in many countries

– BCAs considered more acceptable to greenhouse workers (reentry periods) and to consumers

Pseudomonas syringae ESC 10/11 antibiosis and niche occupation

Induced Resistance=SAR,SIR and ISR Common mechanism for

Pseudomonas, Bacillus, Trichoderma

• SAR-Systemic Acquired Resistance-SIR-Systemic Induced Resistance

– Activation of master switch via salicyclic acid pathway signal –Classical PR-Proteins-Chitinases, β glucanases, proteinases, etc

• ISR-Induced Systemic Resistance

– Activation via jasmonic acid/ ethylene pathway -no classic PR-proteins but the defense compounds

– Usually associated with PGPR(plant growth promoting rhizobacteria( Pseudomonas sp.)-insects

Induced Resistance now we know that many biological inducers induce via salicylic acid, NPR-1 gene, jasmonic acid, ethylene or combination of these pathways

Induced resistance

• Seed Treatments: Pseudomonas, Bacillus, Trichoderma-root diseases caused by fungi, nematodes-foliar diseases caused by bacteria, fungi, viruses

• Foliar treatments: Bacillus mycoides-foliar diseases caused by bacteria, fungi, viruses-Root diseases caused by Pythium

PGPR induced resistance is a state of enhanced defensive capacity developed by a plant reacting to specific biotic or chemical stimuli

Stimulus Stimulus

Stimulus

Stimulus from PGPR/Trichoderma

PGPR induced resistance is a state of enhanced defensive capacity developed by a plant reacting to specific biotic or chemical stimuli

potentiated induction of stress-related genes enhanced resistance

22

SAR/ISR-Foliar Induction

Protective effects of SAR extend to

all plant parts

Resistance is detectable 2-3 days

post induction

Peaks 5-7 days post induction

Effective for ~14-20 days or longer

Suppresses many pathogens:

fungi, bacteria, viruses

Point of induction

Trichoderma harzianum/viridae Fungal Parasite Antibiotic producer Improved water and nutrient uptake Induced Systemic Resistance Inducer

Trichoderma antibiotic deficient mutants still retain biocontrol activity

Trichoderma viridae and antibiotic deficient mutants

Trichoderma mycoparasitism deficient mutants still produce biocontrol

Mycoparasitism deficent mutant

Mycoparasitism and ISR Phase 1: high MW

Antibiotics

ho

st

Tric

ho

der

ma

CWDEs

Antibiotics

ho

st

CWDEs

Tric

ho

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receptors?

Phase 2: low MW

The pre-contact events of mycoparasitim may also activate ISR in the plant

Lorito

Cell wall degrading enzymes

PGPR- Viruses • 1996- Raupach et al. Two strains of PGPR induce ISR in

cucumber and tomato against CMV • Some strains of Pseudomonas fluorescens, Bacillus

pumilis, B. amyloliquefaciens, B subtilis, Kluyvera cryocrescens rhizobacteria reduced CMV and Tomato Mottle geminivirus infection (50-70%), reduced symptoms and lengthened period from infection to symptom development-Zehender et al, 1999

• Bacillus globisporus, Pseudomonas fluorescens, Streptomyces gibsonii-30-60% reduction of tobacco necrosis virus local lesions in bean. Shoman, et al 2003

• Pseudomonas fluorescens- Barley Yellow Dwarf Mosaic- Mysus avenae-Wheat and Barley~50% reduced disease severity. Al Ani et al.2011

BmJ Virus Disease Control mechanical transmission

Virus Latent period -days

% symptomatic plants

Virus titer Symptomatic plants

CMV-cucumber

water 6.7 75 2.37

BmJ 9.0 25 0.49

TMV-tomato

water 4.8 82 2.35

BmJ 8.3 24 1.1

28

PVY Greenhouse-mechanical transmission

Treatment % PVY Average

Dead BmJ +PVY 58.3 a

Dead BmJ 0 c

BmJ induction 5 days before inoculation with PVY + BmJ @ 14, 28, and 42 days post inoculation

26.6 b

2010 Greenhouse PVY Aphid Transmission March-May

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

24

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26

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28

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1-A

pr

3-A

pr

5-A

pr

7-A

pr

9-A

pr

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1-M

ay

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ay

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ay

Date Tested

Aphid Transmission of PVY

Distilled

Autoclaved BMJ

BMJ

No Treatment

% infection ELISA

Transferred 10 green peach Aphid/ plant from PVY infected potato- 20 replications summary of 3 experiments

Hermiston, OR- Integrated PVY Management Plots Red flags- Russet Norkotah-Mazzama Borders

Treatment 2010 % PVY total including winter test

2011 % PVY total including winter test

BmJ WP 2.0 oz/A 14 days emergence to harvest 3.5 10.4

BmJ WP 2.0 oz/A 14 days emergence to harvest- rogue out infected plants

1.5 4.7

Admire Pro 8.7 oz @ plant +BmJ WP 2.0 oz/A 14 days emergence to harvest @ 60 days post emergence Assail 1.7 oz, 67 days Fulfill 5.5 oz, 75 days Beleaf 2.8 oz, 87 days Leverage 3.8 oz-rogue out infected plants

3.0 5.3

Admire Pro 8.7 oz @ plant @ 60 days post emergence Assail 1.7 oz, 67 days Fulfill 5.5 oz, 75 days Beleaf 2.8 oz, 87 days Leverage 3.8 oz-rogue out infected plants

4.5 7.6

Untreated 10.0 10.0

Flsd 0.05 5.9 5.3

How does induced resistance reduce virus

• Direct effect on insect vector-JA? • Love, et al., 2007 showed salicylic acid pathway involved in delayed

symptoms and severity and alternative oxidase. • Ethylene/Jasmonic acid deficient mutants implicate ISR in reduced

long distance spread in plant . • Lewsey et al., 2009 showed RNA silencing and salicylic acid

mediated defense to restrict virus replication and movement. Jasmonic acid may have direct effect on aphid vector.

• Data using salicylic acid, Acibenzolar-s-methyl-(Actigard,Bion)/ CMV TMV in tobacco, squash, Arabidopsis show reduced virus movement-cell to cell (delay symptom development) and systemic movement. IR involves mitochondrial enzyme alternate oxidase and RNA dependent RNA polymerase. – Mayers, et al 2005 : Madhusudham, et al., 2008

Conclusion

• IR shown to delay symptom onset and reduce infection, disease severity, virus titer, virus movement or symptom severity for a wide range of viruses ..

• Control levels are generally in the range of 30-80% and that the mode of action or efficacy differs remarkably by biological control agent and plant species.

• May have direct effect on aphid vectors

Thank You &Happy Trails