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F. V. Hebard Research Farms American Chestnut Foundation Meadowview, VA Fred@acf

Physiology & histology of chestnut blight cankers and screening for blight resistance. F. V. Hebard Research Farms American Chestnut Foundation Meadowview, VA Fred@acf.org www.acffarms.org. Co-Authors. Bob Paris William White Shaun Yarnes Mila Wilmoth. Outline.

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F. V. Hebard Research Farms American Chestnut Foundation Meadowview, VA Fred@acf

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  1. Physiology & histology of chestnut blight cankers and screening for blight resistance F. V. Hebard Research Farms American Chestnut Foundation Meadowview, VA Fred@acf.org www.acffarms.org

  2. Co-Authors • Bob Paris • William White • Shaun Yarnes • Mila Wilmoth

  3. Outline • Physiology and Histology • Fungus Pathogenicity • Resistance Tests

  4. Physiology and Histology Initial Lesion Formation

  5. Physiology and Histology Mycelial fan is means of breakout from initial lesion.

  6. Physiology and Histology Mycelial fans advance by physical pressure. myclelial fan

  7. Physiology and Histology In American chestnut, mycelial fans form more rapidly, in greater numbers, and grow more quickly than in Chinese chestnut, which is resistant to blight. myclelial fan myclelial fan myclelial fan

  8. Physiology and Histology Cells die in advance of hyphae - living cells indicated here by diaphorase activity stained with Nitro-Blue Tetrazolium

  9. Physiology and Histology Enzyme activity in initial lesion and lignifying zone - esterase, phenolase, beta-glucosidase

  10. Physiology and Histology Canker size dynamics

  11. Physiology and Histology

  12. Physiology and Histology Ethylene levels near cankers

  13. Physiology and Histology Ethylene can be elicited by a low-molecular-weight carboxylic acid from culture filtrates on chestnut bark broth, which is not oxalate. Oxalate does not elicit ethylene.

  14. Physiology and Histology Ethylene as measure of colonization; it is elicited more relative to basal levels in American chestnut than Chinese

  15. Funguspathogenicity

  16. Fungus Pathogenicity Demonstration of statistically significant differences in canker length on American chestnut for virulent isolates of Cryphonectria parasitica

  17. Fungus Pathogenicity Unlike on susceptible American chestnut, strain SG2-3 gives qualitatively smaller cankers than Ep155 on trees with intermediate levels of resistance, such as this one.

  18. Segregation for pathogenicity in progeny of crosses of Ep155 and SG2-3 (dark regions at ends of distribution indicate progeny significantly different in canker length). Fungus Pathogenicity

  19. Resistance Tests

  20. Resistance Tests • Mean canker size in Aug & Sept after June Inoculation for various chestnut crosses in a single test

  21. Resistance Tests

  22. Resistance Tests

  23. Resistance Tests - mortality

  24. Resistance Tests - mortality

  25. Small Resistance Tests -Qualitative Rating Scheme Small, Medium and Large Cankers Medium Large The only canker type that is sunken, with abundant sporulation (this canker is verging to Medium); the other two types would be superficial

  26. Rating Strain of Cryphonectria parasitica 1 2 3 4 5 Ep155 Highly pathogenic Canker size small medium large larger largest SG 2-3 Slightly pathogenic Canker size small small small medium large Resistance Tests -Qualitative Rating Scheme

  27. Resistance Tests In 2004 or 2005, some trees that scored well in 1993 started displaying fairly severe cankers. • Was resistance breaking down (pathogen mutating)? • Was the test in error? • Or was this normal canker development for Chinese chestnut? So we went back and rated all the living trees from the 1993 test (and also isolated the blight fungus from cankers).

  28. Resistance Tests • Could not connect initial rating in 93 to anything in 06, except for American-class guys. • Could connect initial rating in 93 to survival by 8/94. • Could connect survival in 8/94 to survival in 06. Death between 94 & 06 may have been breaking connection to initial rating. • Could connect survival in 8/94 to diameter 06, but not straight percent cankered. • Could connect exposed wood to percent cankered, p<.0001, and diam, p<.02 Connecting results from 1993 to tree size and canker severity in 2006. Difficult.

  29. Is this basic test of inoculating trees in late spring and rating them that summer to early winter and the following spring capturing all the components of resistance? • What about sclerified phelloderm?

  30. Sclerified Phelloderm Initial Observation 1982 Fred Hebard noticed sclerification of wound periderm in Chinese but not American chestnut. Hypothesis: Resistant and susceptible trees also will show this difference.

  31. Sclerified Phelloderm Expectations based on hypothesis 1. Chinese but not American trees will show sclerification. 2. Sclerification will correlate with resistance measurements in backcross trees. 3. Selection for resistant trees will inadvertently be a selection for sclerification.

  32. Sclerified Phelloderm Periderm, the outer skin of bark

  33. Sclerified Phelloderm Wound periderm

  34. Sclerified Phelloderm Evidence of Sclerification of Phelloderm Crossed polarizers with a lambda plate reveal the opposite birefringences of suberized and lignified cell walls Phloroglucinol-HCl stains lignin pink suberized phellem suberized phellem lignified phelloderm lignified phelloderm suberized phellem lignified phelloderm -45 degrees +45 degrees

  35. Sclerified Phelloderm Observing chunks of bark using epifluorescence No need to make thin sections. You can look at chunks of bark.

  36. Sclerified Phelloderm Images of chunks of bark with sclerified periderm

  37. Expectation #1 Chinese but not American trees will show sclerification • Sclerification of phelloderm occurs significantly less frequently in American than Chinese chestnut. • The presence or absence of sclerified phelloderm does not definitively distinguish the two species.

  38. Expectation #2 The presence of sclerified phelloderm will correlate with resistance measurements in backcross trees. Data collection still incomplete

  39. Expectation #3 Selection for resistant trees will inadvertently be a selection for sclerification. • At this point it looks like we are selecting for sclerification in some lines

  40. Sclerified Phelloderm Patterns of inheritance in families where sclerification occurs of none+incomplete vs. complete Trees and families deemed resistant do not necessarily have the sclerification response. Comparisons to resistance data not complete yet. Sclerification appears to be controlled by a single gene in families with the response.

  41. Sclerified Phelloderm Future Directions • Test hypothesis: The sclerified phelloderm phenotype is important to durable resistance. • See whether trait maps genetically.

  42. Crew at Meadowview, 2009

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