NE140: Biological Improvement of Chestnut and Management of the Chestnut Pathogens and Pests

(Multistate Research Project)

Status: Inactive/Terminating

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[12/20/2000] [11/25/2002] [11/26/2003]

Date of Annual Report: 12/20/2000

Report Information

Annual Meeting Dates: 10/18/2001 - 10/20/2001
Period the Report Covers: 10/01/2000 - 10/01/2001

Participants

Sandra Anagnostakis - NE-140 Chair, CT Agricultural Experiment Station;
Pam Kazmierczak - NE-140 Chair-Elect, University of California-Davis;
Michael Gold - NE-140 Secretary, University of Missouri;
John Anderson - NE-140 SAES A.A., CT Agricultural Experiment
Station;
Clarissa Balbalian - Virginia Department of Agriculture;
John Carlson - Pennsylvania State University;
Adam Dale - University of Guelph;
Mark Double - West Virginia University;
Michael Gold - University of Missouri;
Dennis Fulbright - Michigan State University;
Lynn Geletka - University of Maryland biotechnology Institute;
Phillip Gordon - New York Botanical Garden;
Fred Hebard - The American Chestnut Foundation;
Bradley Hillman - Rutgers University;
Steven Jakobi - Alfred State University;
Andy Jarosz - Michigan State University;
William MacDonald - West Virginia University;
Colin McKeen - Canadian Chestnut Council;
Timothy McKechnie - University of Massachusetts;
Jimmy Maddox - Tennessee Valley Authority;
Carmen Medina-Mora - Michigan State University;
Donald Nuss - University of Maryland Biotechnology Institute;
William Powell - SUNY-ESF;
Charles Rhoades - University of Kentucky;
Chris Root - University of Maryland Biotechnology Institute;
Pamela Sletten - Connecticut Agricultural Experiment Station;
Terry Tattar - University of Massachusetts;

Brief Summary of Minutes

The business meeting was conducted by Chair Sandra Anagnostakis.

John Anderson, NE-140 Administrative Advisor was present and reminded the group that the current project terminates September 30, 2003.
A committee was formed to begin rewriting the new proposal that will be due for review October, 2002, with a letter of intent given to John Anderson by February 2002. Anderson reminded committee members that the renewal re-write should include these major objectives:
1. The need for research as indicated by stakeholders.
2. The need to include cutting edge research.
3. Importance of the work, and what the consequences will be if the work is not done.
4. The technical feasibility of the work.
5. Advantages of doing the work as a multi-state effort.
6. What the likely impacts will be from successfully completing the work.
7. How cooperation between participating groups enhances the overall research.
The project should not exceed 15 pages of text, excluding bibliography. Committee members include Michael Gold, Bradley Hillman, Bill MacDonald, Fred Hebard, and Don Nuss. The first task for the committee will be to write a request to write a proposal. This request is not to exceed 4,000 characters. It should be done by February and sent to John Anderson.
NE-140 now has a web page completed by Ruby Meiz. The URL is: www.agnr.umd.edu/userforms/nera/projects Ruby has set the home page up to receive all project submissions, reviews etc.
Bill MacDonald reported on the deaths of two former NE-140 members, Dale F. Hindal from West Virginia University and John E. Elliston (Connecticut Agricultural Experiment Station).

Accomplishments

<ol> <li> To better understand the interactions and ecology of the host/pathogen/parasite system at the molecular, organismal and environmental levels in order to develop effective biological controls for chestnut blight. <ol type="a"> <li> McKeen reported that hypovirulence in Ontario is not prolonging the life of chestnut trees due to the use of a hypovirulence type that is not aggressive, and timing of its application. <li> Fulbright has continued work on characterizing the nature of the hypovirulence caused by plasmid DNA in the mitochondria of C. parasitica Some mutants exhibit reduced or abnormal growth and reduced production of conidia. Others enhance senescence and reduce sporulation or attenuate virulence. Pathogenicity tests are underway to explore the potential of 2 specific mutants, a mitochondrial insertion, InC9, and a plasmid residing in the mitochondria, pCRY1. <li> Median-Mora sampled cankers on American chestnuts in Hamden, CT, Rockford, MI, and West Salem, WI and tested C. parasitica isolates for the mt insertion InC9 and the mt plasmid, pCRY1. She reported a high incidence InC9 and pCRY1 in the CT isolates. The plasmid pCRY1 has not been found at either Rockford or West Salem, but the prevalence of InC9 at Rockford, MI leads her to believe it may have originated there. <li> Tattar has treated killing chestnut blight cankers with slurry mixtures containing spores of Bacillus megaterium and soil. Many trees are still surviving, but the success rate is not 100%. <li> Hillman has reported that the virus SR2 may be the most common Cryphonectria hypovirus species in North America. It is unique, but is most closely related to the GH2 type dsRNA, CHV3. It is present in a much lower titer than other hypoviruses, and has little effect on pathogen virulence. He also reported on Crypt2, a class III transposon found in all C. parasitica isolates examined so far. He has also found that C. nitschkei occupies the same habitat as C. parasitica and contains the same genetic elements. <li> Powells group has developed American chestnut stem-expression sequence tags. Fifty-five have been sequenced and sent to GeneBank. They have developed a tissue culture system for American chestnut as well as transformation protocols, and field testing of non-transgenic somatic embryo derived plantletts has begun. <li> Hebard reported that the AFLP mapping has been completed. Working with support from a TACF grant, Steve Rogstad at the University of Cincinnati has developed a new method for extraction of DNA from chestnut and is working with mini satellite DNA. Based on Hebards continuing observation of naturally occurring hypovirulence, he believes that where disease remission is occurring in the U.S., it may ultimately reach European levels. <li> Anagnostakis reported on a CT chestnut plot treated between 1983-1986 with Ep747, a CHV1 containing C. parasitica strain. Many of the larger chestnuts in the plot have died as a result of a drought in 1999-2000 but many remaining trees are infected with blight strains similar to the original Ep747. <li> Nuss reported that the CHV1-Ep713 transgenic strains used in the Meshomasic State Forest study are ecologically unfit. The cDNA derived hypovirus used at that site can be transmitted through ascospores, but has not spread outside to the control plots. A new field trial is underway in West Virginia that will test more ecologically fit transgenic C. parasitica strains. <li> Jarosz reported on the evaluation of C. parasitica and the effect of hypovirus introduced in plots in MI in 1992. The trees have been GPS mapped, but the fungus is so prevalent that it is impossible to sample every canker. New plots were established in 2001 to better assess the spread of the fungus and the hypovirus. <li> MacDonald reminded the group that there will be a symposium at the West Salem, WI chestnut site during the APS meeting in July of 2002. TACF is reimbursing the owners of the land for preserving this site, where CHV1 continues to be the most common hypovirus, but CHV3 still persists. Hypovirus spreads most easily from infected cankers to uninfected on the same tree. New vegetative compatibility types have also been discovered. <li> Double reported on the long-term dissemination studies in cleared and cut-over areas. Conclusions: hypovirus recovery was best from trees in non-cleared plots that received hypovirus inoculum. CHV3 reduced canker growth more than CHV1. Hypovirus was most easily recovered from the outer growth ring of cankers in April. <li> Kazmierczak reported that deletion of the mf1-1 pheromone of C. parasitica results in male sterility. Expression studies show that the pheromone it is not expressed in conidia, but is enhanced under conditions of starvation. She also reported evidence that symptom expression of viral infection of C. parasitica is caused by perturbation of protein secretion. </ol> <li> To improve chestnut trees for timber and nut production, and determine the cultural requirements of chestnut seedlings in nursery and natural settings. <ol type="a"><li> Gold has determined that Willamette?and Qing?are good cultivars for commercial chestnut production, producing good size nuts with an early ripening date. A new orchard was established in MO in October 2001 to examine pruning treatments, irrigation and fertilizer regimes, and tolerance to blight in 3 cultivars: ‘Peach,‘ ‘Qing,‘ and ‘Willamette.‘ <li> Maddox reported that tillage had no significant effect on American chestnut tree survival, tree height, or root length after four years. Using cover crops, survival and growth of trees was better with crimson clover than alfalfa. Yellow sweet clover grown as a cover crop reduced survival and growth. Nursery source did not generally effect survival after 3 years. <li> Rhoades examined establishment of chestnut seedlings in clear-cuts, shelterwood harvested areas, and midstory sites with both mesic and xeric conditions. He found that insect herbivory was heaviest in the midstory. He also found that conditions for seedlings were best on high, light xeric sights and that ectomycorrhizae had little effect except on poor sandy soils. <li> Carlson is working on American chestnut silviculture in conjunction with Tim Phelps and Kim Steiner. Their objective is to restore blight resistant American chestnuts to forest settings. He has developed an in situ hybridization protocol for selection against Chinese chestnut genetic backgrounds in the ACF backcross program. <li> Dale reported that Canada is starting a chestnut restoration project that will assess the current situation in the Province of Ontario. They have begun a blight resistance breeding program, hoping to breed a blight resistant chestnut suitable for Canadas conditions. <li> Gordon presented his thesis that the current research will do little to restore the American chestnut, as it will probably recover on its own. In order to hasten this process, he suggests that we institute widespread planting of American chestnut in riparian zones. <li> Anagnostakis reported that BC2 and BC3 hybrids, American, and Japanese chestnuts were dug up, their heights and roots were measured, and the hybrids were planted in 2000 in two forest clear-cuts in central CT, and in a nursery. Two additional plots were established in 2001, in sites with a mature canopy. She reported good growth, even at a site where the soil pH is 3.6. A 6th plot will be established in 2002 in a partially harvested forest area. She has found no correlation between soil and bark manganese levels and blight resistance. </ol></ol> Impacts: The group determined that it has accomplished one of its proposed milestones for the year 2001, namely new forests plantings in Connecticut, Tennessee, North Carolina, Pennsylvania, Illinois and New York. Underway, but not yet completed, is the characterization of putative genetically transformed American chestnut embryogenic cultures and the field evaluation of confirmed transgenic chestnut trees containing single-transgene constructs.

Publications

<ol> <li> Anagnostakis, S.L. 2001. American chestnut sprout survival with biological control of the chestnut-blight fungus population. Forest Ecology and Management 152:225-233. <li> Connors, B.J., Maynard, C.A., and Powell, W.A. 2001. Expression sequence tags from stem tissue of American chestnut. Biotechnology Letters 23:1407-1411. <li> Dawe, A.L. and Nuss, D.L. 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu. Rev. Genet. 35:1-29. <li> Groome, P.C., Tattar, T.A., and Mount, M.S. 2001. Bacillus megaterium: a potential biocontrol agent against chestnut blight. J. American Chestnut Foundation 15:45-49. <li> Linder-Basso, D., Foglia, R., Zhu, P., and Hillman, B.I. 2001. Crypt1, an active Ac-like transposon from the chestnut blight fungus, Cryphonectria parasitica. Molecular Genetics and Genomics. 265:730-738. <li> Rhoades, C.C. 2001. Pre-blight abundance of American chestnut in Kentucky. J. of the American Chestnut Foundation 15:36-44. <li> Van Heerden, S.W., Geletka, L.M., Preisig, O. Nuss, D.L., Wingfield, B.D., and Wingfield, M.J. 2001. Characterization of South African Cryphonectria cubensis isolates infected with a C. parasitica hypovirus. Phytopathology 91:628-632/. <li> Yuan, W. and Hillman, B.I., 2001. In vitro translational analysis of genomic, defective, and satellite RNAs of Cryphonectria hypovirus 3-GH2. Virology 28:117-123. </ol>

Impact Statements

Date of Annual Report: 11/25/2002

Report Information

Annual Meeting Dates: 10/24/2002 - 10/27/2002
Period the Report Covers: 09/01/2001 - 10/01/2002

Participants

(*NE-140 members)

*Kazmierczak, Pam (NE-140 Chair), U. CA Davis; Van Alfen,Neal , U. CA Davis; Wilk, Debra, U. CA Davis; Roberts, Warren, U. CA Davis; Golino, Deborah, U. CA Davis; Lamb, Carole, U. CA Davis; Hoenisch, Richard, U. CA Davis; Lee, Judy, U. CA Davis; DeKleine, Arthur, CA Poly. Inst.; Wooley, Robert, Wilson Nursery, CA;
Ireland, John, Fowler Nursery, CA; Grunder, Lucienne, Owl Creek Ranch, CA; Titzel, Daniel, Owl Creek Ranch, CA; Corriea, Harvey, W. Chestnut Growers Assoc.; Canfield, Suzette, CA;Anagnostakis, Sandra, CT Agricultural Experiment Station;*Rhoades, Chuck, Univ. of KY;*Nuss, Donald, Univ. of MD; Geletka, Lynn, Univ. of MD; Root, Chris, Univ. of MD;*Fulbright, Dennis, MI State Univ.;Hunt, Ken, Univ. of MO;*Hillman, Bradley, Rutgers Univ.;Churchill, Alice, Boyce Thompson Institute;*Carlson, John, PA State Univ.;*MacDonald, William, WV Univ.; Double,Mark, WV Univ.;Garrath, John, Univ. of Guelph, Ontario, Canada

Brief Summary of Minutes

The business meeting was conducted by Chair, Pam Kazmierczak. John Anderson, NE-140 Administrative Advisor, was not present, but sent a report on the process for rewriting the NE-140 proposal. Committee members working on this new proposal include Michael Gold, Bradley Hillman, Bill MacDonald, Fred Hebard, and Don Nuss. The request to rewrite was accepted by the Northeastern AES Directors at their meeting in March 2002. The project will be submitted for review by 1 January 2003.

Anagnostakis nominated Chuck Rhoades for the position of Chair-Elect, Kazmierczak seconded the motion, and the group voted unanimously to elect Rhoades.

The NE-140 web page maintained by Ruby Meiz (URL: www.agnr.umd.edu/userforms/nera/projects) continues to function well.

Next year‘s meeting will be in Columbia, Missouri (Chair, Michael Gold), 18 - 20 September 2003.

Accomplishments

1. To improve chestnut trees for timber and nut production, and determine the cultural requirements of chestnut seedlings in nursery and natural settings. a) Corriea discussed the history of chestnut growing in California, the limited number of cultivars available, and the few diseases and insect pest problems that they have. b) Grunder reported on her evaluation of the eight cultivars which she grows, and her experiments with Ethyphon to synchronize nut drop. c) Golino explained the function of the Foundation Plant Material Service, and their plan to work with the western chestnut growers to import more cultivars. d) Cunningham reported that they have planted seedlings for grafting new material as it becomes available. e) Anagnostakis reported on her on-going root study with four hybrids planted in forest sites and a nursery in CT. Soil and leaf samples from three sites are being analyzed for total mineral content. Ca and P levels were 2X higher in nursery leaves than in forest leaves. f) Hunt outlined the project in MO to develop a chestnut management guide, and work on chestnut business and marketing. Their best cultivars so far are ‘Willamette‘, ‘Qing‘, and ‘Peach‘. g) Gerrath discussed the American chestnut recovery plan in Ontario, and his work with allozyme electrophoresis to look at genetic variability. He presented Adam Dale‘s report that 317 nuts were produced from their crosses in 2002. h) Carlson is working on silviculture to prepare for reforestation projects. Both American chestnuts, and seedlings from a group of BC2 trees were planted and are being monitored. He is also trying a GISH procedure to hybridize chromosomes with labeled probes to determine the amount of Chinese genetic material present in hybrids. i) Rhoades continues to study soil factors and their influence on root disease. He used ectomycorrhizae and soil amendments to look for effects. He is also starting to analyze soil and site factors in the West Salem, WI site with naturalized American chestnut trees. j) Fulbright is continuing his cultivar trial, and looking for optimum pollinator positions within orchards. He also conducted a restaurant trial with nuts from several cultivars. Mich. State Univ. has purchased a chestnut peeler to help the chestnut co-op with their marketing. He reported on the trees from C. dentata seed irradiated and planted in the 1950‘s, and seedlings of the original trees which have been planted in various locations. He feels that some of them do have increased resistance to chestnut blight disease. k) Anagnostakis presented a report from Schlarbaum on large Ozark chinquapins in the Ouachita National Forest in south eastern OK. Seed from the trees have been planted in a GA nursery for propagation and distribution for studies. 2. To better understand the interactions and ecology of the host/pathogen/parasite system at the molecular, organismal and environmental levels in order to develop effective biological controls for chestnut blight. a) Anagnostakis reported on her 2002 examination of the two sites in CT where transgenic hypovirulent strains of C. parasitica were released, working with Nuss. The canopy-covered site (single release) had few living stems. The clear-cut site, repeatedly treated by Nuss over 3 years, had many large, living chestnut stems, with more in the treated plot than in the control plot. C. parasitica was isolated from cankers on living stems in the fall of 2002. Two isolates from the first study, and 10 from the clear-cut study (3 from the treated plot, 6 from the control plot, and 1 from outside the plots) were all orange in culture. All contained dsRNA, but none were transgenic. All of these hypovirus-containing strains will now be examined by Nuss to confirm that the virus came from the original transgenic strains used. b) MacDonald presented an update on the West Salem, WI site of naturalized American chestnuts. This year 721of the 1330 cankers were sampled. Hypoviruses CHV1-EURO 7 and CHV3 COLI1-1 were both still found. There are now 12 vegetative compatibility types present in the C. parasitica population. c) Double reported on the effect of vegetative compatibility on canker control with hypovirus treatment. Tree longevity was greater after 5 years when vegetatively compatible inoculum was used, and spring treatment was marginally better than fall treatment for tree response. d) Fulbright is still studying the 973 bp mitochondrial insert found in C. parasitica strains from MI, which reduces virulence and sporulation. The insert was found in the first isolate from the West Salem trees. e) Hillman is working on viruses and transposons of C. parasitica while on sabbatical at Okayama University in Japan. All are cytoplasmic and transmissible by anastomosis of the fungus, most have no coat protein and their dsRNA is in host-derived lipid vesicles, they have no extracellular stage, have widely varying effects on the fungus, and are phylogenetically diverse. Only the largest dsRNA band is genomic. The most closely related other virus is Barley Yellow Mosaic Virus. f) Churchill is cloning C. parasitica genes in the pathway for orange pigment biosynthesis. She hopes to use gene tagging by insertional mutagenesis and do targeted PCR amplification. g) Nuss continues to study the genomic organization and expression strategy of Hypovirus CHV1-Ep 713. The p40 coding domain clearly has a significant effect on fungus morphology. He also reported that they trapped 3000 insects in 2001 from the second transgenic-release test site (clear-cut plots reported in 2.a. above). All were checked, and none were carrying transgenic hypovirulent strains. Also in 2001, none of the isolates of C. parasitica from the control plot or outside the plots contained dsRNA. (2002 isolate results in 2.a. above). h) Van Alfen reported on his studies of the affects of hypovirus on C. parasitica. The genes that are Kex-2-processed code for Cryparin, Laccase, and MF1-1 pheromone. Kazmierczak is using GFP-Green fluorescent protein to study virus transport within the mycelium of the fungus. She found vessels for Cryparin secretion in pycnidial initials but not in young hyphae. Most of the viral particles, however, were located in the hyphal tips.

Publications

1. Anagnostakis, S. L. 2001. The effect of multiple importations of pests and pathogens on a native tree. Biological Invasions 3:245-254. 2. Campbell, F. T., and Schlarbaum, S. E. 2002. Fading Forests II. Trading Away our Natural Heritage. Healing Stones Foundation, paper and compact disc. 128 p. 3. Dawe, A. L., and Nuss, D. L. 2001. Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annual Review of Genetics 35:1-29. 4. Gold, M., and Hunt, K. 2002. Establishment of a chestnut industry to enhance the viability of small farms in Missouri. IN: P. L. Byers, ed., Proc. 2002 Missouri Small Fruit and Vegetable Conference, 18-20 February 2002, Springfield, MO, p 11-26. 5. Hunt, K, Gold, M, and Reid, W. 2002. Growing Chinese Chestnuts in Missouri. Bulletin, University of Missouri Center for Agroforestry, Agroforestry in Action #4-2002, 12 pp. 6. Parsley, T. B., Chen, B., Geletka, L. M., and Nuss, D. L. 2002. Differential modulation of cellular signaling pathways by mild and severe hypovirus strains. Eukaryotic Cell 1:401-413. 7. Sasaki, A., Onoue, M., Kanematsu, S., Suzaki, K., Miyanishi, M., Suzuki, N., Nuss, D. L., and Yoshida, K. 2002. Extending chestnut blight hypovirus host range within Diaporthales by biolistic delivery of viral cDNA. Molecular Plant-Microbe Interactions 15:780-789. 8. Suzuki, N., and Nuss, D. L. 2002. The contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation. Journal of Virology 76:7747-7759. 9. Taylor, D. R. 2002. The evolution of hypovirulence in the chestnut blight system: implications for management options. Pp286-296 IN: Adaptive Dynamics of Infectious Diseases: In Pursuit of Virulence Management. U. Dieckmann, J. A. J. Metz, M. W. Sabelis, and K. Sigmund, eds. Cambridge Studies in Adaptive Dynamics.

Impact Statements

The group has acomplished both of its proposed milestones for the year 2002.
Dormant twig samples of chestnuts were collected by cooperators at 22 sites across the native range (ME, Ont-CANADA, MA, NY, CT, PA, MD, WV, VA, KY, NC, SC and GA). Kubisiak (MS) isolated DNA from these and found that a cline in allele frequencies exists along the Appalachian axis, and that C. dentata still exists as a highly variable species, even at the margins of its range.
Transgenic hypovirulent strains of C. parasitica effect biological control of chestnut blight disease and allow hypovirus spread from the treatment site (details above).
Not likely to be completed (due to a lack of funding for Powell's NY research) are two 2001 goals: characterization of putative genetically transformed American chestnut embryogenic cultures and the field evaluation of confirmed transgenic chestnut trees containing single-transgene constructs.

Date of Annual Report: 11/26/2003

Report Information

Annual Meeting Dates: 09/11/2003 - 09/13/2003
Period the Report Covers: 10/01/1998 - 09/01/2003

Participants

(*NE-140 members);
*Kazmierczak, Pam, pjkkaz@ucdavis.edu, Univ. of CA Davis;
*Anagnostakis, Sandra, Sandra.Anagnostakis@po.state.ct.us, CT Agricultural Experiment Station; Anderson, J. F., John.F.Anderson@po.state.ct.us, CT Agricultural Experiment Station;
*Nuss, Donald, nuss@umbi,umd.edu, Univ. of MD; Dawe,Angus, Univ. of MD;
Geletka, Lynn, Univ. of MD; Root, Chris, Univ. of MD;
*Fulbright, Dennis, fulbrig1@msu.edu, MI State Univ.;
*Gold, Michael, NE-140 chair, goldm@missouri.edu, Univ. of MO; Hunt, Ken, HuntK@missouri.edu, Univ. of MO; Warmund, Michele, Univ. of MO; Rhoads, Julie, Univ. of MO; Bruhn, Johann, Univ. of MO;
*Hillman, Bradley, hillman@aesop.rutgers.edu, Rutgers Univ.;
Sisco, Paul, paul@acf.org, The American Chestnut Foundation;
*Hebard, Fred, fred@acf.org, The American Chestnut Foundation;
*Carlson, John, NE-140 chair-elect, jec16@psu.edu, PA State Univ.;
*Schlarbaum, Scott, tenntip@utk.edu, Univ of TN; Craddock, J. Hill, hill-craddock@utc.edu, Univ. of TN Chattanooga; Alexander, Mark, Univ. of TN Chattanooga; Alexander, Steven, Univ. of TN Chattanooga;
*MacDonald, William, macd@wvu.edu, WV Univ.; Double, Mark, mdouble@wvu.edu, WV Univ.; Bell, Brian, WV Univ.; Rittenour, William, WV Univ.;
Ellingboe, Albert, ahe@plantpath.wisc.edu, Univ. of WI;

Brief Summary of Minutes

The business meeting was conducted by Chair, Michael Gold.

1. Anderson, NE-140 Administrative Advisor, praised the group for their continued progress, and for successfully revising the project and having it approved.

2. Hillman nominated Carlson as Chair-elect. Anagnostakis seconded the motion. This is to fill the position vacated when Rhoades, formerly of the Univ. of KY left for another position. Kazmierczak nominated Churchill as secretary. Anagnostakis seconded the motion. Both nominations were approved unanimously.

3. Carlson will host the 2004 meeting in PA, either at State College or at the Mount Alto station at a date of his choosing in September 2004.

Accomplishments

1. To improve chestnut trees for timber and for nut production, and determine the cultural requirements of chestnut seedlings in nurseries and natural settings. a) Fulbright has been doing studies on improving pollination in chestnut orchards. Bad weather and lack of pollinizers may be the major problems in MI, but cross-compatibility is also being investigated. He is also working with a newly purchased peeling machine which removes shells and pellicles. It appears that successful peeling depends on cultivar, growing environment, maturity of nut, moisture and temperature after harvest, and temperature of nuts entering peeler. b) Hebard reported that B2F2 trees from open pollination were inoculated with C. parasitica, and several had good levels of resistance, supporting the hypothesis that some trees would have two copies of the 2 to 3 genes for resistance. c) Sisco recommended a joint effort to apply for a large grant for furthering the genetic work with chestnut. He also reported on the TACF efforts in the Eastern U.S., with most work being done in PA, VA, and MA. He hopes to extend European work with 12 linkage groups in European chestnut and European oak to American chestnut. d) Schlarbaum has about 2,500 nuts from 17 open-pollinated Ozark chinquapins in the Ouachita National Forest in AR and OK. The seed are being grown under contract by a Georgia nursery. He has found high mortality in American chestnut plantings in KY due to Phytophthora root rot (Ink Disease). e) Anagnostakis had new information on BC3 Japanese-American and BC2 Chinese-American trees planted on three sites in CT. The Japanese-American trees have grown the most over three years, and responded the most to site differences (which included soil pH from 3.9 to 5.5, and different soil calcium levels and soil nitrogen levels). The BC3 trees were all male fertile and the BC2 trees were all male sterile. Greenhouse and growth chamber studies with calcium levels in hydroponically grown American chestnuts showed high calcium inhibited tree growth and was present in leaf tissue. The high calcium level in the plants did not protect them from infection by C. parasitica after inoculation. f) Craddock has been working with the Chattanooga chestnut project to increase awareness of their breeding program, locate surviving American chestnut trees, and solicit support. A root rot, possibly caused by Phytophthora, has been a problem in many areas in TN. Hypovirulent inoculum is being used to protect trees infected with C. parasitica. Craddock's cultivar trial is being planted with an aim of replicating that made by MO. g) Alexander reported on screening B2F2 hybrids, Castanea sativa, C. dentata, C. pumila, and C. mollissima for resistance to C. parasitica, using virulent and hypovirulent strains. He found C. sativa, dentata, and pumila equally susceptible, C. mollissima highly resistant, and the B2F2 hybrids varying greatly in resistance. h) Hunt has established a replicated chestnut cultivar trial with at least 5 trees each of 12 cultivars in each location to determine which do best under MO growth conditions. A nut production orchard with three cultivars, 48 trees each, was planted in 2001 and two pruning systems are planned for evaluation. There are now 56 cultivars in the MO chestnut repository, being maintained and evaluated. i) Warmund is studying the effects of second-flush flowering on the energy balance of the trees and on nut production. j) Gold is working to establish a viable chestnut industry by considering market development and value added products, and working with Hunt and Warmund on tree improvement and cultivar selection. 2. To better understand the interactions and ecology of the host/pathogen/parasite systems at the molecular, organismal, and environmental levels in order to develop effective biological controls for chestnut blight disease. a) Hillman described his recent work with Reoviruses C-18 and 9-B-2-1 which were found in C. parasitica isolates from West Virginia. Segment-specific cDNA libraries were made, and studies of function have been begun. b) Double presented an update on the control of chestnut blight disease in the population of American chestnuts in West Salem, WI. This is a cooperative effort with several participating NE-1015 members. Two hypoviruses still persist and are spreading. c) Bell reported on experiments to determine whether canker treatment procedures can be improved. d) Kazmierczak is continuing to look at intracellular processing and secretion of hydrophobin and cryparin by C. parasitica. She concludes that Kex2 processing is not necessary for secretion of cryparin, the gene does not affect the ability of the fungus to break through the bark, and cryparin/GFP fusion products appear in discrete vesicles in the hyphae. e) Dawe described sequencing of C. parasitica using ESTs. The library produced and microarrays now available (4200 genes, 2200 clones) will allow more detailed studies of gene function. f) Nuss is using transfected hypovirulent strains on a population of American chestnut trees to test efficacy. g) Anagnostakis reported on the joint project with Nuss on transgenic hypovirulent strain release in CT. Six years after the first treatments, sprouts in the treated plot appear to be surviving longer than sprouts in the control plot. h) Carlson has been conducting silviculture studies for reforestation of American chestnuts, including direct-seeding studies in PA. Chestnut tree crosses are in progress for linkage mapping.

Publications

2003 Segers, G. C. and D. L. Nuss. 2003. Constitutively activated G alpha negatively regulates virulence, reproduction, and hydrophobin gene experession in the chestnut blight fungus Cryphonectria parasitica. Fungal Genetics and Biology 38:198-208 Parsley, T. B., G. C. Segers, D. L. Nuss, and A. L. Dawe. 2003. Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third G alpha homologue. Current Genetics 43:24-33 Dawe, A. L., V. C. McMains, M. Panglao, S. Kasahara, B. Chen, and D. L. Nuss. 2003. An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea. Microbiology 149:2373-2384 Craddock, J. H. 2002. Il Castagno negli Stati Uniti. p. 278-283 IN: Buonous, G. (ed.) Il castagno in Italia e nel Mondo: Colturo del passato per il futuro. Edagricole-Calderini, Bologna Bassi, G. and Craddock, J. H. 1999. Performance and description of the introduced chestnut cultivar 'Colossal' in Cuneo province, northwest Italy. In: Salesses, G. (ed.) Proc. 2nd. International Chestnut Symposium, Bordeau, France. Acta Horticulturae 494:207-212 Craddock, J. H. and Bassi, G. 1999. Effect of clonally propagated interspecific hybrid chestnut rootstocks on short-term graft incompatibility with four cultivars of Italian "Marrone." In: Salesses, G. (ed.) Proc. 2nd. International Chestnut Symposium, Bordeaux, France. Acta Horticulturae 494:207-212 Craddock, J. H. and Bassi, G. 1999. Introduction into Italy of eight Castanea mollissima cultivars from China. In: Salesses, G. (ed.) Proc 2nd. International Chestrnut Symposium, Bordeaux, France. Acta Horticulturae 494:319-321

Impact Statements

<li>We have compared the genetic diversity of mycoviruses worldwide, for their efficiency as biological control agents for chestnut blight disease.<li>We have identified effective delivery methods of hypoviruses to manage the chestnut blight pathogen in the U.S.<li>We have evaluated early growth characteristics of chestnuts of different genotypes planted on different sites.<li>We have developed an understanding of chestnut seedling/site requirements for successful artificial forest regeneratio
n.<li>We have planted chestnut cultivar trials for nut production in different climatic and geographic regions.<li>We are continuing with comparative studies to strengthen and expand Hypovirus taxonomy.