1. Studies on the suppression of cacao diseases with Ecuadorian endophytic Bacillus spp. Rachel L. Melnick PhD Candidate, Dept. Plant Pathology The Pennsylvania State University January 23, 2008

2. Previous research Endophytic Bacillus sp. from tomato colonized cacao foliage and reduced disease

3. Hypothesis Endophytic Bacillus spp. from cacao may provide sustainable control of cacao diseases, through long-term colonization and activation of plant defense mechanisms Susceptible tissue, no bacteria Tissue with bacteria Resistant tissue, no bacteria

4. Initial PhD research Collected endophytes from elite cacao trees at INIAP plantation Screened endophytes for biocontrol potential Four endophytic Bacillus spp. could be potential biocontrol agents ▫There initially were 70 isolates

5. Field Trial

6. Research Have conducted 2o months of field trials for biological control of witches’ broom of cacao

7. INIAP Experiment Four clonal cacao genotypes  EET19 -S  A2162 - MR  A2634 - MR  CCN51 – S/T  IMC67 - Seedling Five bacterial treatments Silwet control Bacillus A20 Bacillus ET Bacillus CT Bacillus CR

8. Application Initial application May 2007 (start of dry season Sprayed Log 8.0 CFU/mL with Silwet L-77 Plants maintained in greenhouse for 3 weeks

9. Timeline May 2007 Dec 2007 Initial Application Dry Season Phytosanitation and reapplication Rainy Season Mar 2008 Reapplication Jun 2007 Planted in the field May 2008 Phytosanitation and reapplication Dry Season Dec 2008 Phytosanitation and reapplication

10. Measure endophytic colonization Removed standard area of leaf tissue Surface sterilized Triturated and plated

11. Results

12. Dry season results (2007) Bacteria survived endophytically in foliage throughout the dry season ▫Initial concentration log 6.8 CFU/cm 2 ▫Concentration after 6 months ~ log 4.8 CFU/cm 2 ▫Populations stabilized at ~ log 4.8 ± 0.3  Applied Bacillus were 1000 fold higher than total culturable background (control leaves)

13. Dry season results (2007) Low disease pressure Measured several variables ▫Swelling ▫No. infected stems

14. Dry season results – Bacteria

15. Dry season results - Genotype

16. Significant genotype/bacteria combo Combining biocontrol with resistant genotype is better for reducing disease

17. Plant Disease Management Reports:

18. Rainy season results 2008 Phytosanitation and reapplication Reapplication

19. Rainy Season Results

20. Conclusions Successful year round disease reduction by Bacillus pumilis ET Three applications a year are enough to reduce disease Combining bacteria with resistance in the plant probably increase disease suppression

21. Current & Future Research

22. Current and future research Verification study at Nestlé Farm ▫Cultivars: EET19, EET95, SIL1 ▫Treatments: Control, CT, ET Mode of action ▫Induced resistance ▫Microbial ecology ▫Antibiosis Biological Control of Frosty Pod ▫Started this week

23. Nestlé experiment Four genotypes with varying susceptibility Endophytes: CT & ET Established May 2008, rated Sept 2008 CultivarTreatment% Living Disease Severity EET19Control700 EET95Control800.22 SIL1Control500 EET19ET700 EET95ET900 SIL1ET10* EET19CT1000 EET95CT800.25 SIL1CT500

24. Induced resistance Determine if endophytes affect gene expression of plant defense genes Seedlings grown in growth chambers in double magenta boxes Quantitative Real Time PCR (Q-PCR) ▫Collaboration with USDA-ARS SPCL Potential for microarray in the future

25. Samples of data… Some graph here

26. Microbial ecology Determine if beneficial endophyte replace neutral endophytes ▫More beneficial, more disease suppression Automated Ribosomal Intergenic Spacer Analysis (ARISA) Measure species diversity and abundance in community Peak height (fluorescence units)

27. Antibiosis Conducted plate assays Will be screening Bacillus spp. for the presence of antimicrobial genes and gene products Collaboration with Dr. Dan Roberts USDA-ARS SASL

28. Biological control of frosty pod Treatments: Control, A20, ET, CR, CT 400 pods (80 reps) sprayed at INIAP on nacional ▫Hand pollinated pods 400 pods (80 reps) at Rio Lindo on CCN51 ▫Open pollinated pods

29. Overall conclusions Biological control shows potential to reduce witches’ broom of cacao Combining IPM strategies is best for managing cacao diseases Bacillus spp. impacts cacao gene expression

30. Acknowledgements ◦ Penn State  Adviser: Paul Backman  Mark Guiltinan  Megan Hayden  Seila Maximova  Sharon Pishak  Anissa Poleatewich  Ann Young USDA-ARS SPCL ▫Bryan Bailey ▫Mary Strem INIAP Carmen Suárez Karina Solis Danilo Vera  Support :  USAID  IPM-CRSP  SANREM-CRSP  USDA-ARS SPCL  Penn State University  Dept of Plant Pathology  College of Agricultural Sciences  Tag-a-long (Office of International Programs)  Competitive Grant ◦ Nestlé  Martín Alvarez CABI Jayne Crozier