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
Colonization (CFU/g ) of sugarbeet by 341-16-5 from treated seed
15C=1.13 e5
24C=7.59e 5
7.1 e5 2.1 e5
1.3 e5 1.5 e5
Many PGPR are endophytes
2.1 e5
5.4 e5
Growth promotion by PGPR
Control of root pathogens, growth
regulators, improved nutrition, induced
resistance?
Healthy seedling protected by PGPR
Trichoderma
Damping-off
Mixing Trichoderma product in
potting medium
Planting Trichoderma/PGPR
colonized seedlings
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 (DrechsleraBipolaris), 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
periods) and to consumers
(reentry
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, virusesRoot 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 from PGPR/Trichoderma
Stimulus
Stimulus
Stimulus
PGPR induced resistance is a state of
enhanced defensive capacity
developed by a plant reacting to specific
biotic or chemical
stimuli
potentiated induction of stressrelated genes
enhanced resistance
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
Point of
induction
 Effective for ~14-20 days or longer
 Suppresses many pathogens:
fungi, bacteria, viruses
22
Trichoderma
harzianum/viridae
Fungal Parasite
Antibiotic producer
Improved water and nutrient
uptake
Induced Systemic Resistance
Inducer
Trichoderma viridae and antibiotic deficient mutants
Trichoderma antibiotic deficient mutants still retain biocontrol activity
Mycoparasitism deficent mutant
Trichoderma mycoparasitism deficient mutants still produce biocontrol
Mycoparasitism and ISR
Phase 1: high MW
Phase 2: low MW
CWDEs
receptors?
host
Trichoderma
Antibiotics
host
Trichoderma
Antibiotics
CWDEs
Cell wall degrading enzymes
The pre-contact events of
mycoparasitim may also
activate ISR in the plant
Lorito
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
6.7
75
2.37
BmJ
TMV-tomato
9.0
25
0.49
water
4.8
82
2.35
BmJ
8.3
24
1.1
CMVcucumber
water
28
PVY Greenhouse-mechanical
transmission
Treatment
Dead BmJ +PVY
Dead BmJ
BmJ induction 5 days
before inoculation
with PVY + BmJ @
14, 28, and 42 days
post inoculation
% PVY
Average
58.3 a
0c
26.6 b
2010 Greenhouse PVY Aphid
Transmission March-May
Aphid Transmission of PVY Transferred 10 green peach
Aphid/ plant from PVY
infected potato- 20 replications
summary of 3 experiments
% infection
ELISA
45%
40%
35%
30%
Distilled
Autoclaved BMJ
25%
BMJ
20%
No Treatment
15%
10%
5-May
3-May
29-…
27-…
25-…
23-…
21-…
1-May
Date Tested
19-…
17-…
15-…
13-…
11-…
9-Apr
7-Apr
5-Apr
3-Apr
1-Apr
30-…
28-…
26-…
0%
24-…
5%
Hermiston, OR- Integrated PVY Management Plots
Red flags- Russet Norkotah-Mazzama Borders
Treatment
2010 % PVY total 2011 % PVY total
including winter including winter
test
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 harvestrogue 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 3080% 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