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
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
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
der
ma
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
-…
26
-…
28
-…
30
-…
1-A
pr
3-A
pr
5-A
pr
7-A
pr
9-A
pr
11
-…
13
-…
15
-…
17
-…
19
-…
21
-…
23
-…
25
-…
27
-…
29
-…
1-M
ay
3-M
ay
5-M
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
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
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