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Impact of application of endophytic Bacillus spp. for biocontrol of cacao diseases on native microbial communities
Rachel L. MelnickPh.D. CandidateDepartment of Plant PathologyThe Pennsylvania State University
Department of Plant Pathology
Department of Plant Pathology
Theobroma cacao L.•Tropical understory
perennial
•Seeds processed into chocolate
•Home to wide range of fungal endophytes
Department of Plant Pathology
Diseases of cacao•Main limiting factor
Department of Plant Pathology
Biological Control: Pod Diseases•Cherelle wilt
▫Thought to be physiological thinning mechanism
▫Likely has biological component•Frosty Pod•Black Pod•Witches’ broom
•Focus on biocontrol for disease management
Department of Plant Pathology
Hypothesis •Endophytic Bacillus spp. may provide
sustainable control of cacao diseases, through activation of plant defense mechanisms and alteration of the microbial ecology of tree
Department of Plant Pathology
Bacterial endophytes
•Bacillus spp. isolated from pods, leaves, branches, floral cushions at the INIAP station in Pichilingue, Ecuador
•Screened for elite qualities for biocontrol agent
•Identified 4 potential biological control agents
Department of Plant Pathology
Biological Control: Witches’ Broom•Bacillus pumilis ET reduced overall
disease severity through dry and rainy seasons
Days after bacterial application
0 20 40 60 80 100 120
Dis
ea
se
se
ve
rity
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
A20 CT Control CR ET
A
AB
BBB
Department of Plant Pathology
Biological Control: Pod Diseases•B. pumilis ET reduced cherelle wilt in
genotype CCN-51, but not diseases
•See poster P-714 for more information…
Department of Plant Pathology
What is known to date…•Bacillus pumilis ET works as a biological
control agent, but how?
•Antibiosis – Reduced growth of M. roreri, M. perniciosa, and P. capsici & chitinase producer
• Induced resistance – Activates expression of cacao defense genes
•Niche displacement?
Department of Plant Pathology
Niche displacement•Thought that most endophytes are neutral
in terms of plant health
•Introducing beneficial endophytes likely displaces neutral microbes to increase benefits
•How to test?
Department of Plant Pathology
The trees…•Sprayed branches of cacao trees in
cardinal directions with 1x108 CFU/ml at beginning of rainy season (Jan) & again in end of March▫Three replicate trees per treatment
•Collected leaves from branches in March and May
•Preserved and shipped in RNALater
Department of Plant Pathology
ARISA
•Automated Ribosomal Intergenic Spacer Analysis
•Extracted genomic DNA•Nested PCR with bacilli specific primers
following Garbeva et al. 2003
16S 23StRNA
Microb Ecol. 2003. 45: 302-316
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Abundance & Diversity in March
Applied species
Department of Plant Pathology
Abundance in May
Department of Plant Pathology
Witches’ Broom
Days after bacterial application
0 20 40 60 80 100 120
Dis
ea
se
se
ve
rity
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
A20 CT Control CR ET
A
AB
BBB
Reapplication
May Sampling
Department of Plant Pathology
Conclusions•B. pumilis ET appears to decrease
diversity of native microbes
•Indications that application of B. pumilis ET may displace neutral endophytes
Department of Plant Pathology
Future work…•Statistical analysis of the data presented
today to get more exact measures of diversity & abundance
•ARISA with universal bacterial primers▫As soon as I get home!
•ARISA with universal fungal primers
•ARISA of pods
Department of Plant Pathology
Acknowledgements
• Penn State▫Paul Backman▫Scott Geib
• USDA-ARS SPCL▫Bryan Bailey
• INIAP-EET▫Carmen Suárez▫Karina Solis▫Danilo Vera
Research Support:• USAID IPM-CRSP• USDA-ARS SPCL• Penn State College
of Ag. Sci. Graduate Student Competitive Grant
Travel Support:• APS Potomac
Division Travel Award
• The Eddie Echandi & H. David Thurston Award APS Foundation Award
• Penn State College of Ag. Sci. Student Travel Award
Department of Plant Pathology
Questions????
