1. Biological control of Pythium aphanidermatum Impacts of the seed colonizing microbial community on zoospore pre-infection events Allison L. H. Jack Dr. Eric B. Nelson’s research group April 8, 2009

2. Outline Background on zoospore pre-infection events Disease suppressive vermicompost and vermicompost extracts Investigations into the mechanism behind observed suppression Vermicompost use in horticulture

3. Zoospore pre-infection events

4. vegetative hyphae sporangium germinating sporangium zoosporangiumzoospores antheridium oogonium oospore Germinating oospore asexual sexual direct indirect DISEASE [modified from Matthews 1931] Pythium aphanidermatum

5. Microbial interactions in the spermosphere Gradient of seed exudates Seed colonizing microbes Pythium aphanidermatum zoospore responding to seed exudates with chemotaxis

6. [Rosenbaum & Whitman 2002] [Mitchell 2004] Eukaryotic flagella Play a crucial role in sensing the extracellular environment and transmitting signals to the cell body Oomycete zoospores have specific receptor ligand interactions as encystment cues

7. PIPK GG GPCR GG PsCAM1 PsCMK3 PsCMK4 Calmodulin dependent protein kinases [Hua et al. 2008] Chemotractant Zoospore signaling Phytophthora sojae

8. P. aphanidermatum zoospores Known chemotractants: –L-aspartate –L-glutamate –L-glutamine –L-alanine –D-mannose –Sucrose –Maltose –D-fucose [Donaldson & Deacon 1993] If the solution contains a high enough background concentration of an amino acid, then chemotaxis is abolished

9. [Liu et al. 2007] Cucumis sativum cv. Marketmore 76 Exudates contain –Carbohydrates –Organic acids –Amino acids –Many other compounds

10. Zoospore pre-infection events (chemotaxis) ?

11. Disease suppressive vermicompost

12. Brief history of disease suppression research Late 1800s: suppressive soils documented [Huber & Schneider 1982] 1930s – 1940s: Link made between composts and soil health [Howard 1942] 1959: Biological nature of suppression documented [Menzies 1959] 1970s - 1980s: Extensive work done on suppressive composts [Hoitink & Kuter 1986, Weltzein 1989]

13. Vermicompost Separated dairy manure solids Hot composted for 5 days under forced aeration Fed in thin layers to continuous flow through worm beds Harvested out the bottom after 65 days Highly controlled process leads to a material with consistent properties Can vermicomposted dairy manure consistently suppress Pythium damping off?

14. Height of water column determines matric potential in growing media

15. Sterile glass fiber filter Sand or Sand/compost mixture Cucumber seeds Sand or Sand/compost mixture

16. Sand Sterile Batch 3 Batch 1 2006 Batch 2 2007 Batch 3 2008 Non-inoculatedInoculated

17. Health rating a aaaa a a b c d

18. Total seedling stand

19. Conclusions Suppression of disease caused by P. aphanidermatum is relatively consistent from batch to batch Suppression is dependent on a biological factor

20. Compost extracts Traditional agricultural practice Extensively studied in Europe in the 1980’s [Weltzien 1989, Trankner 1992] Recent literature exists [Scheuerell & Mahaffee 2004, 2006] Most published methods use 1:5 – 1:10 ratios of compost to water

21. Compost extracts provide soluble nutrients, especially when plug size limits compost amendment in certified organic systems

22. Water Vermicompost Extract 1:5

23. Chemical characteristics A.1 week extracts, B. 2 week extracts DO = dissolved oxygen in ppm EC = electrical conductivity in mS cm -1

24. Non-aerated vermicompost extract 1:60 ratio of vermicompost to water (by mass) Circulation for 5 min 2 x per day Strained through 4 layers of cheesecloth sump

25. Sand Sterile VC Extract VC Extract Non-inoculatedInoculated

26. Health rating aa a b c c

27. Seedling stand

28. Future directions Lyophilize the extract –Reconstitute –Use as seed treatment Consider adding as a treatment for follow up experiments with seed colonizing microbial community

29. How are zoospores prevented from infecting the seeds?

30. When do P. aphanidermatum zoospores reach the seed surface? H H H H H Inoculate Harvest Transplant 1 23 4567 Time (d) SAND SAND INOC SAND INOC T8 SAND INOC T16 SAND INOC T24 T T T

31. 8 hours after sowing in sand Proportion of 10 seeds with Pythium present in specific sections

32. 16 hours after sowing in sand Proportion of 10 seeds with Pythium present in specific sections

33. 24 hours after sowing in sand Proportion of 10 seeds with Pythium present in specific sections

34. Conclusions and next steps Pythium is present on most seeds within 24 hours Surface sterilize to distinguish between presence and infection Add seeds sown in vermicompost as a comparison – will this change the timing of zoospores reaching the seed surface? Confirm results with qPCR once time frame is worked out in detail

35. Shoot height (mm) 0 306090120150 [Chen & Nelson 2008] Pythium inoculation SandSuppressive compost 7d a a 8h7d b 8h 7d b Non-inoculated a 7d When does the suppressive community develop on the seed surface? (P. ultimum on wheat) SeedMicrobes a 7d

36. 24 hr incubation in: water Seeds rinsed in sterile water Seeds removed, exudate sterile filtered Microbially modified seed exudate (MMSE) Zoospore attraction assays with microbially modified seed exudates (MMSE) 24 hr germination in: Sand Vermicompost (40%) Sterile water & filter paper Hypothesis: Seed colonizing microbes modify exudates which alters zoospore behavior.

37. Zoospore attraction assay Zoospore solution Agar plugs on a microscope slide infused with exudates Slides are removed after 30 min, imaged and encysted zoospores are counted 19x

38. 40% v:v amendment of vermicompost

39. Are lower numbers of encysted zoospores due to the presence of a repellant, or the absence of an attractant?

40. Dose – response curve Regression p < 0.001 Dilution of seed exudate Predictions for vermicompost MMSE: Attractant missing Repellant present Unmodified exudate

41. Chemotaxis – The zoospore maze Imaging the zoospores as they respond to exudates in real time Perfusion chamber Entire chamber filled with 275uL zoospore suspension

42. 27 2 3 Unmodified exudate Vermicompost MMSE Water (no seed) Short videos taken after 5 minutes

43. Are additional stages of zoospore pre-infection behavior affected by seed colonizing microbes?

44. Zoospore pre-infection events (chemotaxis) ?

45. Interaction with plant cells: Root border cells [Hawes & Pueppke 1986] [Goldberg et al. 1988] P. dissotocum on cotton

46. Time lapse of interaction with a single root border cell 19x T = 0 T = 50 m

47. Only certain cells attract zoospores

48. Root border cell viability: Fluorescein diacetate staining [Larkin 1976] 7.6 x

49. Cucumber border cells with zoospores 7.6 x

50. Conclusions Zoospore attraction appears to be affected by seed colonizing microbes from vermicompost which may account for the observed suppression of disease Whether this is due to an attractant missing or the presence of a repellant remains to be determined Time frame of when zoospores reach the seed and the nature of their interactions with root border cells need to be refined

51. Burning questions Which microbial taxa / functional genes are present on the seed surface during the critical time frame when suppression is expressed? How exactly are these seed exudates being modified?

52. Cabbage transplants 19 DAP, Grower’s mix (A.) with bloodmeal (B.), 10% vermicompost (C.), 10% vermicompost & bloodmeal (D.), Cornell base mix (E.) with bloodmeal (F.), 10% vermicompost (G.), 10% vermicompost + bloodmeal (H.). Treatments D and H had the highest transplant biomass of all treatments tested. Horticultural applications

53. Acknowledgements Nelson Lab: Mary Ann Karp Eric Carr Hillary Davis Monica Minson Liang Chen Sarah Arnold Dave Moody My committee: Eric Nelson (PPPMB) Anthony Hay (MICRO) Anu Rangarajan (HORT) Kathie Hodge (PPPMB) Scott Peters (EDUC) Financial support: Department of Plant Pathology and Plant Microbe Biology USDA BARD Knight Institute for Writing in the Disciplines New York Farm Viability Institute NYSTAR Center for Advanced Technology & USDA SBIR Phase I (with RT Solutions) Organic Farming Research Foundation Organic Crop Improvement Association Andrew W. Mellon Fellowship The “Worm Guy” Tom Herlihy – RT Solutions “Boo Boo” Steffen Jack Kent Loeffler – photo credits