Biological control of Pythium aphanidermatum

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

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

6. 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 [Mitchell 2004] [Rosenbaum & Whitman 2002]

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

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

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

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

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

17. Health rating a a a a a 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. Vermicompost Extract 1:5 Water

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

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

25. Non-inoculated Inoculated Sand Sterile VC Extract VC Extract

26. Health rating a a 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? Harvest H SAND Inoculate Transplant H SAND INOC H T SAND INOC T8 H T SAND INOC T16 H T SAND INOC T24 2 3 1 4 5 6 7 Time (d)

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

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

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

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. 7d a 8h 7d a b 7d 8h 7d a b 7d 7d Non-inoculated a When does the suppressive community develop on the seed surface? (P. ultimum on wheat) Pythium inoculation Shoot height (mm) 0 30 60 90 120 150 Sand Suppressive compost Seed Microbes [Chen & Nelson 2008] 7d

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

37. Zoospore attraction assay Agar plugs on a microscope slide infused with exudates Zoospore solution 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 Predictions for vermicompost MMSE: Unmodified exudate Repellant present Attractant missing Dilution of seed exudate Regression p < 0.001

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

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 P. dissotocum on cotton [Hawes & Pueppke 1986] [Goldberg et al. 1988]

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

47. Only certain cells attract zoospores

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

49. Cucumber border cells with zoospores 7.6 x 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