1. Impact of application of endophytic Bacillus spp
Impact of application of endophytic Bacillus spp. for biocontrol of cacao diseases on native microbial communities
Rachel L. Melnick
Ph.D. Candidate
Department of Plant Pathology
The Pennsylvania State University
Department of Plant Pathology

2. Theobroma cacao L. Tropical understory perennial
Seeds processed into chocolate
Home to wide range of fungal endophytes

3. Diseases of cacao
Main limiting factor

4. 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

5. 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

6. 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

7. Biological Control: Witches’ Broom
Bacillus pumilis ET reduced overall disease severity through dry and rainy seasons

8. Biological Control: Pod Diseases
B. pumilis ET reduced cherelle wilt in genotype CCN-51, but not diseases
See poster P-714 for more information…

9. 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?

10. 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?

11. 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

12. ARISA 16S 23S Automated Ribosomal Intergenic Spacer Analysis tRNA
Extracted genomic DNA
Nested PCR with bacilli specific primers following Garbeva et al. 2003
tRNA
16S
23S
Microb Ecol :

13. Abundance & Diversity in March
Applied species

14. Abundance in May

15. Witches’ Broom
May Sampling
Reapplication

16. Conclusions
B. pumilis ET appears to decrease diversity of native microbes
Indications that application of B. pumilis ET may displace neutral endophytes

17. 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

18. Acknowledgements Penn State Paul Backman Scott Geib USDA-ARS SPCL
Bryan Bailey
INIAP-EET
Carmen Suárez
Karina Solis
Danilo Vera
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
Research Support:
USAID IPM-CRSP
USDA-ARS SPCL
Penn State College of Ag. Sci. Graduate Student Competitive Grant

19. Questions????