Duration: 10/01/2003 to 09/30/2008

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Before the introduction of chestnut blight, American chestnut was one of the most valuable trees in eastern North American forests. When Europeans first settled eastern North America, chestnut was a major and eminently notable forest tree everywhere from Ontario to Georgia. It was the one eastern forest tree that approached the dimensions of the gigantic Pacific Coast rain forest conifers; thus, it was referred to as the 'Redwood of the East'. Chestnut occupied a wide range of site types, ranging from mesic to xeric. Quality chestnut on mesic sites attained a clear trunk length of up to 72 feet with an overall height of 120 feet and a diameter of 5 feet. Dry, thin, acidic ridge top soils provided the poorest growing conditions, but chestnut still managed to grow to a height of 70 feet. The tree was prized for its high quality wood, tannin extracts, and nuts. Chestnut blight disease eliminated the American chestnut as a canopy species and elevated oaks as the most dominant tree group in the southern Appalachians. However, widespread oak decline has occurred in the southeastern U.S. over the past 20 years, leading to declining forests. It is believed that southern Appalachian forest ecosystems will not be considered healthy until chestnut can be reintroduced as a functioning component of that ecosystem. Loss of the American chestnut resulted in significant biological and economic changes. Replacement species generally include red oak, black oak, and chestnut oak, all much slower growing than chestnut. Return of chestnut to eastern forests could vastly improve existing forest stands, and positively impact wildlife, since chestnuts produce seed every year as opposed to oaks and hickories.

The NE-140 Technical Committee was organized in 1982, and since then, members have worked cooperatively to investigate the complex issues involved with the biological improvement of chestnut, along with habitat restoration and horticultural development of American chestnut and chestnut cultivars. Environmental and land stewardship issues that are concerns to stakeholders are addressed by NE-140 through efforts to understand the ecology and biology of eastern North American forests following the loss of American chestnut. Control of chestnut blight disease through the use of hypoviruses and resistant chestnut trees may vastly improve forest stands in eastern North America, answering many concerns regarding forest ecology. Other important issues of this continued work are improved forest management, including biological-based controls of invasive insect and disease organisms threatening chestnut. This will serve as a model for natural resource managers. Food safety issues are addressed via the creation and use of IPM-based controls of both forest and orchard trees, reducing the use of pesticides. Increased use of chestnuts in the American diet will improve human health, since chestnuts are unique among nuts with fat content ranges from 1-10%, compared to 70% in most other nuts. The viability of rural communities will be enhanced through creation of a new horticultural chestnut market.

Restoration of chestnut as a timber tree and improving orchard chestnuts in the United States continue to be the long-term goals of the members of this regional project. To succeed in our efforts to achieve these long-term goals, continued activity of the award-winning NE-140 group is critical. NE-140 is working to successfully develop and deploy biological systems that protect natural resources and the environment. NE-140 must sustain its rapid, productive trajectory in order to develop effective, biological-based solutions to address the ecosystem disruption caused by invasion of the chestnut blight fungus (Cryphonectria parasitica) and death of billions of American chestnut trees (Castanea dentata).

In the shorter term, we continue to build on our understanding of two biological approaches to the control of chestnut blight disease: use of biocontrol agents to control the fungus populations, and tree breeding to improve the fitness of the trees. Efforts to understand the molecular, organismal, and ecological basis of C. parasitica infection, and the critical role of hypoviruses and defective mitochondria, are essential for successful development and deployment of IPM programs. NE-140 is further developing hypovirulent strains of C. parasitica for use in IPM programs to protect timber and orchard chestnut trees, and seeking environmentally safe techniques to control pathogens such as Phytophthora root rot and pests such as the oriental chestnut gall wasp.

Currently NE-140 is:
1. incorporating, by standard breeding and transgenic approaches, chestnut blight resistance and gall wasp tolerance into chestnut for planting in forests and orchards
2. working to conserve native Castanea population diversity for future use in forests and orchards, including working with the National Plant Germplasm System of the USDA to establish germplasm repositories in California and Missouri
3. studying the ecology and cultural requirements of chestnut in nurseries and natural settings, leading to the reintroduction of chestnut into the forest
4. testing new chestnut cultivars, systematically documenting information on existing cultivars, establishing germplasm repositories for cultivar material, and testing new orchard management methods to enable U.S. growers to compete against U.S. fresh market imports

NE 140 has broadened its membership over the past five years to reflect the increasing breadth of chestnut research activity and bring additional research expertise to bear. The members of this group are strongly dependent on each other for the success of their individual projects. Working collaboratively, they seek to reverse the effects of devastating pathogens, find ways of dealing with new imported pests, and conduct research in support of the nascent horticultural chestnut industry.

The technical feasibility of the research.
NE-140 is one of the nation's most productive research groups. Over the last five years (1998-2002), publications included 86 research papers, book chapters, reviews, and popular articles (plus an additional 169 publications from 1993-1997). Due to the growing interest in chestnut throughout the U.S., NE-140 also has expanded the breadth of institutions, researchers, and research sites beyond the Northeast region to more effectively address the multiple issues and foci related to chestnut.

The advantages for doing the work as a multistate effort.
The scope, breadth, and magnitude of the effort required to combat chestnut blight disease, address other critical pathogen and pest problems, understand ecosystem processes involved in reestablishment of chestnut in the natural forest, and develop chestnut as a new orchard crop alternative for growers are far beyond the capability of any individual researcher or institution. Combined talents in molecular biology, genomics, virology, entomology, pathology, ecology, and horticulture, available across many U.S. institutions and use of numerous test sites throughout the Northeast and into the Midwest (plus a site in California) are required to effectively address the issues facing NE-140.

What the likely impacts will be from successfully completing the work.
Successful completion of NE 140 goals will lead to a number of positive impacts including: reestablishment of timber chestnut trees in eastern N. America; improved stand diversity of eastern hardwood forests; successful reintroduction of chestnuts into xeric sites formerly populated by oak species decimated by gypsy moth infestations, in order to provide mast and wood products; establishment of productive nut-bearing chestnut trees in both eastern and western N. A. where a new industry is rapidly developing; and, improved economic opportunities for chestnut growers in local and international markets.

Related, Current and Previous Work

The total decimation of the American chestnut by chestnut blight disease had significant biological and economic impacts. During the first two decades of the 20th Century, exhaustive attempts were made to control the disease. When it was apparent that all control attempts were unsuccessful, several breeding programs were initiated. The goal of these breeding programs was to develop blight resistant American chestnut. The USDA initiated one of the first resistance breeding programs in the 1920's. Unfortunately, this program was discontinued in 1960, as their efforts in breeding a blight-resistant American chestnut were unsuccessful. Hope was renewed when C. parasitica isolates with reduced virulence (hypovirulent) were discovered in Europe.

The phenomenon of hypovirulence results in a lowered pathogenicity of the fungal pathogen, allowing the tree to utilize its natural defense mechanisms and maintain vigor. Cankers are not eliminated, but the tree can produce adequate callus tissue and wall off the infection. This discovery reawakened the scientific community to the long-standing issue of chestnut blight disease. Modern scientific investigative techniques could be applied to both the fungal pathogen and the tree host. Two additional breeding institutions began breeding chestnut trees for blight resistance using modern genetic principals: The University of Tennessee-Knoxville and The American Chestnut Foundation in Meadowview, VA. These two programs complimented the one at The Connecticut Agricultural Experiment Station (CAES), the only program that has continued uninterrupted since its initiation by Arthur Graves in 1930. The large collection of species and chestnut hybrids, maintained over the years by CAES, are available to all who need breeding material. When it became clear that the oriental chestnut gall wasp (Dryocosmus kuriphilus), introduced into GA in 1974, was spreading northward, the breeding programs discussed ways to incorporate screening for gall wasp resistance into their plans.

NE-140 had it beginnings in 1978, when approximately 200 people attended a symposium held in Morgantown, WV at West Virginia University. There were 34 papers given, and the ensuing discussion convinced the group that a Regional Research Committee was needed. In 1982, five experiment station scientists agreed to participate. Within a few short years, the committee grew to include 13 experiment stations and other participating academic institutions and governmental units. As a result of this groups activities, the following events have occurred:

1. CT has improved the records of holdings and continues to maintain the finest collection of species and hybrids of chestnut in the world (6). CT imported hypovirulent strains from Dr. J. Grente in France (who first described the cytoplasmically-transmissible disease of the fungus) and demonstrated that these hypovirus-infected strains could control chestnut blight cankers on American chestnut trees (8). CT described a genetic system of vegetative compatibility in the blight fungus that prevented strain fusion and the transmission of hypovirulence viruses (1, 2, 3).
   
2. NY (Cornell) confirmed, by making genetic tests of Asian and American populations, that the blight fungus entered the US from Japan (57).
   
3. MI described the spread of American hypovirulence viruses through the blight fungus population in a stand of trees planted in MI (45). MI described a type of hypovirulence determined by genes in mitochondria (56). MI presented a physical and genetic map of the mitochondrial genome of a strain of the blight fungus (13). MI, NJ, and WV described and compared three American hypovirulence viruses (29, 71).
   
4. NJ, MD, MI, and TX spearheaded the naming of Cryphonectria hypoviruses as a new family (Hypoviridae), now recognized by the International Committee on Taxonomy of Viruses (49,50).
   
5. MD transformed virulent strains of the blight fungus with cDNA copies of hypovirulence virus RNA genes, and produced stable, transgenic hypovirulent strains with virus genes and fungal genes together in the nucleus (21,22).
   
6. MD, CT, and WV were granted permission from USDA/Plant Quarantine to test transgenic hypovirulent strains of the chestnut blight fungus in the forests of CT and WV. This was the first permit granted to test transgenic organisms for their ability to spread and effect biological control of a plant disease (7).
   
7. KY, MI, TX, MD, VA, and CT have made detailed studies of enzymes produced by the blight fungus that may be related to its ability to kill chestnut trees (5, 15, 20, 23, 24, 25, 30, 31, 32, 35, 37, 38, 41, 43, 44, 52, 53, 56, 62, 70, 76).
   
8. KY and CT have studied enzyme systems in chestnut trees that may be related to their ability to resist the blight fungus (36, 39, 40, 61, 65, 66, 67,68,69).
   
9. VA, CT, and TN have been breeding chestnut trees and selecting progeny for resistance to chestnut blight disease and with the timber-form of American chestnut trees (4). These will be ready for release within five years.
   
10. MS, VA, MA, and CT prepared a genetic map of chestnuts and found three molecular markers associated with resistance to chestnut blight disease (54).
    
11. MO, TN, MI, KS, CT, and CA have initiated cultivar trials of exotic chestnuts (Chinese, Japanese, and European in origin), with the goal of developing chestnut research orchards, thereby creating a U.S. chestnut industry.
    

More recent findings, 1998-2002, include:

• Studies conducted by MI, MN, NY, WV, and WI on the effectiveness of two hypoviruses to affect biological control, conducted at an American chestnut stand in West Salem, WI, have shown that hypovirus spread and biological control has been most significant on trees where hypoviruses were introduced (27,51). The best surviving hypovirus is the one less debilitating to C. parasitica. Also, new vegetative compatibility groups of C. parasitica were discovered and named. A mitochondrial plasmid was found in some West Salem isolates, but its role in disease expression is as yet unknown (11).
  
• A MI laboratory study on the effects of specific C. parasitica vic genes on the restriction of the hypovirus transmission was confirmed by WV in a forest setting (12). Also, a study conducted by WV and NY, compared vegetative compatibility types in the US with those in Europe (26,58). NJ and WV examined and characterized a cryptic dsRNA (CHV4-SR2) found in 30% of the isolates recovered from Appalachia.
  
• MD reported that "transgenic hypovirulent" strains are able to transmit virus to sexual (ascospore) progeny. This novel virus transmission property is predicted to provide increased biological control potential by circumventing barriers imposed by the fungal vegetative incompatibility system (9).
  
• WV and VA, involved in an ongoing long-term study of hypovirus dissemination in cleared and non-cleared, cut-over areas, demonstrated that chestnut regeneration was perpetuated by continuous clearing, while hypovirus remained after repeated hypovirus introductions. MD, CT, and WV have shown that the sexual recombining ability of transgenic with virulent isolates was high when tested in a forest setting but evidence for hypovirus dissemination was lacking (9,28).
  
• WV and NY have collaborated on several projects related to C. parasitica population structure. C. parasitica hypoviruses that survived in WV forest settings 15 years after their release, typically were avirulent (16,17,55).
  
• WV conducted two experiments to examine some of the parameters associated with hypovirus transmission in the field. The influence of mycelium age on hypovirus transmission within cankers was examined using two hypoviruses, CHV1-Euro 7 and CHV3-COLI 11-1 (data not published). Canker age and time of hypovirus introduction on the development of hypoviruses in cankers was examined using the above mentioned hypoviruses (data not published).
  
• Work conducted in MD has resulted in the preparation of Expressed Sequence Tagged (EST) libraries with mixed mRNA populations isolated from both hypovirus-infected and uninfected cultures. cDNAs were cloned into a lZipLox system to facilitate easy recovery of insert fragments contained in the pZL1 vector background. Recovered ampicillin resistant colonies were picked into microtiter plates, catalogued, and stored at -80oC. Characterization of the libraries are in progress.
  
• MD has identified hypovirus encoded genes that contribute to alterations in C. parasitica colony morphology, growth characteristics, sporulation levels and the size and morphology of canker formation on chestnut tissue (18,19,59,72,73).
  
• Work in CA has progressed to understand the molecular interaction between C. parasitica and the hypovirus CHV-1, specifically the attenuation of virulence. This includes isolation of C. parasitica genes down-regulated by the virus at the level of transcription, and characterization of cryparin, one of the down-regulated genes shown to play a critical role in the formation of fruiting bodies on wood. The viral polymerase and its products have been characterized. Cloning, sequencing and deletion of Mf1-1 and Mf2-1, mating type specific pheromones has been accomplished. Identification of 2 extracellular and 1 intracellular laccase has occurred. Deletion of one of the extracellular laccases was shown to have no phenotypic effect. Cryparin secretion has been characterized.
  
• MA has found that microorganisms antagonistic to C. parasitica, such as the fungus Trichoderma atroviride and the bacterium Bacillus megaterium, show biocontrol potential (47,48,74). MA research has demonstrated that both B. megaterium and T. atroviride contain genes for antagonism against C. parasitica . Similar genes for antagonism to C. parastica, however, may also exist in other fungi and bacteria on the bark of the American chestnut, which may play a key role in the microbial ecology of the chestnut blight disease.
  
• A symposium on hypovirus deployment was presented at the annual American Phytopathological Society meeting in Milwaukee, WI (July, 2002). MI, NY (Cornell), TN, and WV were involved in the symposium.
  

Objectives

1. To improve chestnut trees for reestablishment in forest ecosystems, and chestnut cultivars for nut production by selection, breeding, and marketing, and determine the cultural criteria of all chestnuts for successful production in nurseries, orchards, and/or natural settings.
   
2. To evaluate and integrate multiple approaches for the biological control of the chestnut blight fungus and other pathogens and pests that threaten chestnut, by investigating host/pathogen/parasite relationships from the molecular to the ecological level.
   
3. 
   

Methods

Objective 1: To improve chestnut trees for reestablishment in forest ecosystems, and chestnut cultivars for nut production by selection, breeding, and marketing, and determine the cultural criteria of all chestnuts for successful production in nurseries, orchards, and/or natural settings. Traditional breeding programs are being carried out in CT, TN, and VA. All three programs share chestnut pollen and seed as needed. Crosses are made in the spring (early July), seeds harvested in the fall (October), and planted the following spring. Tests for blight resistance are made when the seedlings are 3 to 5 years old by inoculating branches with pure cultures of the blight fungus and noting canker enlargement rate. All three programs will soon be able to enhance selection using molecular markers. Assessment for form and nut quality may be done on 5-year-old trees. Identification of molecular markers is being done in MS and MA with plant material supplied by all NE-140 members. Genetic input is provided by TACF staff in VA and NC. The work to date has concentrated on identifying Chinese chestnut-specific markers for resistance to chestnut blight disease and is now being expanded to examine Japanese chestnut markers as well. A collaboration with Forest Service personnel in Delaware, OH, is being initiated to use RAPD markers to select among backcross progeny for American traits other than susceptibility to blight. There has been difficulty identifying all 12 linkage groups in crosses of Chinese and American chestnut, due to segregation distortion and underlying anomalies. Blight resistance is associated with the linkage groups where segregation distortion has been observed. An attempt is being made to cross-reference the American x Chinese chestnut genetic map with a European chestnut genetic map, where all 12 linkage groups have been identified, and to associate linkage groups with chromosomes by fluorescent in situ hybridization (FISH). Mapping populations that are not based on recombination between species also are being assembled, where segregation distortion is not expected to occur. More than 500 markers have been mapped, including about 250 RAPD markers (MS), 250 AFLPs (NC), 20 SSRs (MS), 19 cDNA RFLPs (MA), two cloned functional genes (cystatin and the 5-S Ribosomal RNA gene) (MS), and 6 isozymes (AL). Plant tissue culture laboratories in GA and NY (SUNY) will maintain and multiply important clones of chestnut hybrids produced by VA, CT, and TN. To enhance somatic seedling production from embryogenic cultures of American chestnut for mass clonal propagation and gene transfer applications, work will continue to establish new embryogenic cultures, develop protocols for high-frequency somatic embryo production and test cultural and environmental variables for enhancement of somatic seedling production. GA will also develop cultures of trees in native populations of American chinquapins (C. pumila var. pumila and var. ozarkensis ), threatened by chestnut blight disease and Phytophthora root rot, to ensure that important germplasm is not lost. Chinese chinquapins (C. henryi) are available in GA, and will be included in the program. All three chinquapin types seem to have valuable resistance to the oriental chestnut gall wasp (D. kuriphylus), which is currently found in GA, AL, TN, and NC. NY (SUNY) efforts include the development of five constructed genes for resistance to blight that will be put into somatic embryos of chestnut using Agrobacterium-mediated transformation. Plantings of new hybrids for assessment of resistance to gall wasp, chestnut blight, and Phytophthora root rot have been made and will continue to expand. This work is being done by TN (plantings in TN), and by CT (plantings in cooperation with U.S.D.A./ARS, Tree Nut Research Laboratory, Byron, GA and with U.S.D.A./Forest Service, Bent Creek Research Forest, Asheville, NC). TN is using Japanese stock plants with reported resistance to gall wasp, and CT is using American chinquapins and Chinese chinquapins with reported resistance. The breeding programs will seek to identify the two genetic bases of resistance and then combine their lines if that seems useful. Existing populations of American chestnut trees must be protected for future inclusion in breeding programs and for inclusion in genetic diversity studies. Chestnut biomass is being maintained by planting seed collected from natural populations. Cytoplasmic and transgenic hypoviruses are then introduced into maintained populations and seed orchards. Native populations currently being protected are located in CT, NY, MA, ME, NJ, VA, PA, MD, WV, NC, TN, and GA. Valuable plantings in WI are being maintained, as well. In MI plantings of American chestnut trees are apparently being protected from blight by debilitation of the fungus by a DNA plasmid in the mitochondria of C. parasitica. Trees grown from cobalt-irradiated American chestnut seed in the 1950s, which were planted at the National Colonial Farm in VA, have been evaluated for blight resistance. These trees have large, healing cankers that produce significant amounts of callus tissue. Seedlings of these trees have been planted in MI to determine whether the callus production is a function of the trees or the area in which they are planted. Testing is being conducted by MI, VA, and WV. Chestnuts imported by the U.S.D.A. and planted prior to 1960 are being evaluated for resistance to chestnut blight disease, Phytophthora root rot, and gall wasp by CT and TN. A $10,000 request has been made from the fruit and nut tree improvement group at UC Davis to help establish newly imported chestnuts from other areas in the U.S. This initiation is intended to relocate the germplasm repository for chestnut currently located at College Station, TX to the National Clonal Germplasm Repository at UC-Davis. The University of Missouri-Columbia has offered to serve as the eastern U.S. chestnut germplasm repository in concert with UC Davis. To help reestablish chestnut in its native range, edaphic, silvicultural, and landscape factors influencing forest planting techniques will be studied by CT, TN, VA, and MI. Further, effects of chestnut restoration on forest ecosystem dynamics and community composition will be evaluated in CT, MI, WI, WV, VA, and TN. Natural populations and orchards in CT and WV will be examined for the effect of competing vegetation on tree survival. Nut production trials of commercially available chestnut cultivars are underway in CT, TN, MO, and KS. Commercial plantings in MI are being monitored in a pollination study to determine whether pollination is a problem in a two-cultivar orchard. Resistance to chestnut blight disease is documented for many cultivars from China and Japan, but winter hardiness is unknown. To date, the most commonly planted cultivar in the U.S. (mostly on the west coast) is 'Colossal', a European X Japanese hybrid with some resistance to chestnut blight. Trials in TN are underway to test 'Colossal' and other cultivars for resistance to chestnut blight disease, Phytophthora root rot, and oriental chestnut gall wasp. MI and CT will conduct blight resistance and Phytophthora root rot trials, and assess 'Colossal' for winter hardiness and blight resistance. To help foster a U.S. chestnut industry, chestnut production and management techniques for orchard production will be tested in MI, MO, KS, TN, CT, and CA. Optimum growing practices will be developed and resulting information shared with producers. Marketing research will be conducted in MI and MO focused on development of domestic markets for chestnuts produced in the U.S. Work will be undertaken to better understand underlying market dynamics and identify niche market and value added opportunities. Chestnuts from orchard selections are being evaluated for nutrient and lipid content. Objective 2: To evaluate and integrate multiple approaches for the biological control of the chestnut blight fungus and other pathogens and pests that threaten chestnut, by investigating and linking host/pathogen/parasite relationships from the molecular to the ecological level. Spread of the chestnut blight fungus is being studied in natural populations of American chestnuts and orchards of species and hybrids. This work is in progress in CT, NJ (cooperating with NY), NY, and WV (with MI). These projects include work on the vegetative incompatibility system of the fungus, and specific vic genes used as markers. The spread of hypoviruses is being monitored in natural and planted populations in CT (with MD), NJ (with NY), NY, WV (with NJ, MI, and NY), and TN (with CT and WV). Also, trees planted in MI, where natural hypoviruses developed, are being monitored by MI (with WV). American chestnut seeds planted in the 1880s, 600 km beyond the natural range in West Salem, WI, have produced a population of more than 3,000 American chestnut trees. These trees grew blight-free for more than 100 years, but in 1986 the stand become infected with chestnut blight disease. Hypovirulent strains of the fungus were introduced between 1992-1997 in an attempt to establish biological control. The treatment is supervised and monitored by MI, WV, NY, and the WI Department of Natural Resources (Jane Cummings-Carlson). Assessment of disease progress, spread of two introduced hypoviruses, and canker evaluation, continues at the West Salem stand. Demographics of the West Salem stand will be quantified via a multi-institutional collaboration between NY, MI, MN, WI, and WV. An annual assessment of the crown health and disease status of the stand will be conducted, as will survey and evaluation of the vegetative compatibility, mating type, and virulence/hypovirulence status of individual C. parasitica infections. GIS will be employed to map all chestnut stems and the coordinates combined with the above data to quantify disease and hypovirus spread. Ecological effects of disease development on the population dynamics of the stand will be measured by relating changes in stand composition to the developing epidemic. The contribution of host genotype to biological control will be measured by taking scion wood from trees displaying the most recovery and trees displaying the least. Grafts will be made onto rootstock in MI, and after a period of growth, the grafts will be inoculated with C. parasitica to see if trees respond differently. The ability of transgenic hypovirulent C. parasitica strains to produce hypovirulent sexual and asexual inoculum when introduced into natural and artificially-established blight cankers in a forest setting will be evaluated by researchers in WV and CT, both working with MD where the strains were developed. Forest sites with high numbers of 6-12 cm dbh American chestnut sprouts have been selected for release of transgenic hypovirulent C. parasitica strains. Naturally-occurring and artificially-established infections will be exposed to sexually compatible, transgenic inoculum containing nuclear cDNA copies of either the highly debilitating hypovirus CHV1-EP713 or the moderately debilitating hypovirus CHV1-Euro 7. The hypovirus infection status of sexual and asexual inoculum produced by the treated cankers will be measured, as will new cankers that arise, to further define the importance of balancing hypovirulence and ecological fitness for establishment of effective biological control. Studies conducted in CT and WV (in conjunction with MD) will help elucidate whether C. parasitica population replacement by hypoviruses-infected strains is possible. Breeding studies conducted by TACF (VA) will be linked to hypovirulence studies by comparing the ability of various generations of Chinese X American hybrids and American chestnut to harbor virulent and hypovirulent C. parasitica infections. This work will be cooperative between MD, VA (TACF), and WV. Test orchards of American and American X Chinese chestnut hybrids of known lineage will be established at the WV University Experimental Farm. After a 3-5 year growth period, artificial inoculations will be made using C. parasitica isolates that previously have been characterized for their level of virulence/hypovirulence. As infections develop, they will be evaluated for their growth and production of sexual and asexual inoculum. Comparisons will then be made to estimate how the resistance of the back-crossed-chestnuts combines with the debilitating effects of hypovirus infection to regulate chestnut blight disease. Alternative controls for chestnut blight disease are being sought in MA, using naturally-occurring bark microorganisms, such as Trichoderma sp. and Bacillus sp. Work will continue to determine the microbial ecology of the interaction between C. parasitica and the fungi and bacteria that exist on the bark of American chestnut trees. Through work done in MD, the molecular biology of the host/pathogen/parasite system of Castanea/C. parasitica/Hypovirus is beginning to yield valuable information about the basis of the interactions. Mapping of the viral determinants responsible for changes in fungal phenotype has been accomplished with the aid of infectious cDNA clones of the severe hypovirus strain CHV1-EP713 and the related mild strain CHV1-Euro7 (16). Mapping approaches include construction of functional chimeric viruses from the CHV1-EP713 and CHV1-Euro7 infectious cDNA clones, deletion mutagenesis of infectious hypovirus cDNA clones, systematic repair of deletion mutants, site specific mutagenesis and construction of hypovirus based gene vectors (2,10,15,16). These studies will be expanded during the next funding period to map additional viral determinants. Identified determinants and the polyprotein processing of a large portion of the second viral-encoded open reading frame, ORF B, will be characterized in detail. NY has determined that C. parasitica isolates have a mixed mating system in which both selfing and outcrossing occurs in the same population. Selfing isolates of C. parasitica will be examined to determine whether they have both mating types in the same thallus. Results from this study will help answer the following questions: Under what conditions does selfing occur? If it is genetically determined is the genetic structure of populations affected? Additionally, the diversity and evolution of Cryphonectria hypoviruses will be examined to determine how these viruses have evolved. In particular, how they have migrated among different geographic regions? Phylogenetic analyses of CHV-1 among continents will be conducted. The mechanism in which viruses are transmitted horizontally between vegetative compatibility (vc) types is being pursued by studying sequence variation in CHV-1 from different vc types. Sequencing and comparisons of the hypoviruses will be done by NJ, MI, MD, WV, and CA working cooperatively. Studies of the effect of specific fungal genes on hypovirus replication and gene expression will be done in MD, NJ, and CA. The function of the fungal genes as sex pheromones, and their involvement in virulence will be examined in CA. The use of hypoviruses as gene expression vectors, and the introduction of hypoviruses into other fungal pathogens (for biological control of other plant diseases) will be studied (MD). To further our understanding of the molecular interaction between C. parasitica and the hypovirus CHV-1, the attenuation of virulence will be characterized and the role of a kex2 specific secretion pathway in C. parasitica development and virulence will be analyzed (CA). Hypovirulence caused by mitochondrial mutations and mobile genetic elements will continue to be studied (MI). Studies will be initiated to create a better understanding of the function of the anthraquinone pigments and other secondary metabolites in the biology of the chestnut blight fungus. Isolation and characterization of genes of the pigment biosynthetic pathway are being studied to further understand the viral/fungal interaction. Molecular genetics approaches are being used in combination with analytical chemistry analyses to identify genes for polyketide biosynthesis in C. parasitica (NY).

Measurement of Progress and Results

Outputs

• New, blight resistant selections of chestnut to better utilize hypovirulence for management of chestnut blight disease.
• More efficient use of natural resources to meet diverse social needs, by restoring chestnut as a timber crop for lumber and poles.
• Strategies for planting chestnuts in harvested and disturbed ecosystems, providing a wood with natural enhanced wood decay inhibitors to help replace pressure treated lumber.
• Increased options for controlling pests and diseases of chestnut trees.
• Greater availability of improved chestnut cultivars for use in agricultural production as orchard trees for nuts.
• Development of chestnuts as a new, US-produced product for the fresh market to replace imported nuts. New chestnut food products to be developed now that U.S. chestnuts are being peeled for processing. Development of an endowed chestnut research station at Michigan State University (built in 2003-4) to develop chestnut trees for food production. Elucidation of the Castanea/Cryphonectria/Hypovirus interaction to help understand other pathosystems.

Outcomes or Projected Impacts

• NE-140 provides continuous outreach and leadership to organizations that will provide products to the end users. Since NE-140's inception (1982), successful nonprofit chestnut groups have been established (forest restoration or orchard establishment). These include The American Chestnut Foundation, The Midwest Nut Producers Council, and The Western Chestnut Growers. NE-140 hosted an international meeting of chestnut scientists. Through these active groups, existing in large part due to NE-140 member participation, sharing research and information, end users will receive products from chestnut forests and orchards.
• Understanding the roles of secondary metabolites in the biology and ecology of C. parasitica could lead to the identification of alternative control methods for chestnut blight disease (NY). Infectious C. parasitica hypovirus cDNA clones have also been used to infect and reduce virulence of fungal pathogens of apple, pear, peach, and eucalyptus (MD).
• Results derived from studies of hypovirus-encoded genes will allow a more rational approach for engineering hypoviruses for enhanced biological control and lead to a better understanding of the cellular regulatory pathways underlying fungal pathogenesis (MD, NJ).
• Expressed Sequence Tagged (EST) libraries have been prepared with mixed mRNA populations isolated from both hypovirus-infected and noninfected cultures. A web-searchable database of more than 2500 C. parasitica genes to be used by NE-140 researchers and the fungal researchers in general will be established. The availability of this EST library/database will allow researchers to monitor the activity of thousands of genes simultaneously following hypovirus infection, disruption of specific genes, under different growth conditions and during infection of chestnut tissue (MD).
• C. parasitica strains have been genetically engineered to contain a nuclear copy of hypovirus CHV1-EP713. These transgenic hypovirulent strains are able to transmit hypovirus to sexual (ascospore) progeny, giving them a distinct advantage over traditional cytoplasmic hypoviruses that are not transmitted to sexual spores. By crossing the sexual cycle, hypoviruses can be transmitted to additional vegetative compatibility groups. This novel transmission property may provide increased biological control potential by circumventing barriers imposed by the fungal vegetative compatibility system (MD, WV, CT).
• Blight-resistant, timber chestnut is the goal of TACF (VA). However, simply creating blight resistant trees does not ensure reforestation. Natural plantings in KY and TN will address silvicultural obstacles that must be overcome for successful reforestation (PA, TN, VA, CT). A US chestnut industry requires suitable cultivars and market development. Two import collection sites (CA, MO) and five cultivar trial sites (MO, MI, TN, CT, CA) will allow cultivars to be imported, established, and evaluated. Region specific recommendations to growers on cultivar selection, orchard culture and management, disease and insect resistance, and control options and market opportunities will be provided. Successful cultivars can then be made available to the public (MO, TN, CA). MI purchased a chestnut peeler from Italy to produce a high quality, fresh, pre-peeled chestnut for the consumer market. New products can now be developed since the shell is easily removed (MI, TN, MO, VA). Non-traditional control of chestnut blight may be an important component in a multifaceted approach to biological control. Anthraquinone pigments in C. parasitica and other non-related microorganisms (i.e. Trichoderma sp. and Bacillus sp.) may be important antagonistic factors in the control and management of chestnut blight (NY, MA).

Milestones

(2004): <u1> <li>Breeding program begun for selecting improved protein content, peelability, and blight resistance in high yielding orchard-based chestnut cultivars (Endowed chestnut experiment station, Rogers Reserve Farm, MI). <li>Nuts collected for nutritional analyses and size comparison from orchard selections and cultivars of chestnut. <li>Hypovirulent strains of C. parasitica developed and deployed for blight control on native chestnut trees at each of three clear-cut forest areas and one nursery area planted with hybrid chestnut trees. <li>Scion wood collected from WI American chestnut trees surviving well with hypovirulence in the C. parasitica population, grafted onto root stocks in MI. <li>Site selected for release of new transgenic-hypovirulent strains of C. parasitica in WV. </ul>

(2005): <ul> <li>Evaluation and comparison of improved methods of hypovirus introduction into cankers under field conditions. <li>Sequencing and genetic structure analyses of a gene cluster for anthraquinone pigment biosynthesis in C. parasitica completed, and targeted gene knockout strains constructed for the PKS gene and other genes to determine gene function, and studies conducted of pigment gene knockout mutants for possible phenotypic changes. <li>Characterization of the role of hypovirus p29 in virus RNA accumulation in C. parasitica, and virus transmission through conidia of the fungus. <li>Generation of polyclonal antibodies against 5 overlapping regions of hypovirus ORF B, and construction of a C. parasitica EST database. <li>More American chestnut scions collected in WI (as in 2004) and grafted on trees in MI. <li>Orchard established in WV with advanced, back-cross chestnut trees from VA for assessment of host resistance with hypovirulence in the C. parasitica population. </ul>

(2006): <ul> <li>Chestnut market analyses completed and findings reported. <li>New processed chestnut products introduced. <li>New chestnut cultivars established in several cooperating locations.. <li>Role of canker age and vegetative compatibility on the perpetuation of hypoviruses determined, following their introduction into forest chestnut trees. <li>Publication of a C. parasitica EST database containing approximately 2500 ESTs. </ul>

(2007): <ul> <li>ORF B polyprotein processing pathway in C. parasitica confirmed, ORF B mature proteins responsible for altering fungal cell signaling pathways mapped and DNA microarray analysis of hypovirus-mediated alteration of fungal gene expression initiated. <li>Complete assessment of field performance of transgenic hypovirulent strains of C. parasitica with CHV1-Euro 7. <li>Pigment knockout mutants of C. parasitica used to assess role(s) of pigments in fitness and competitive abilities in chestnut, and gene knockout strains for other PKS genes cloned. <li>Hypoviruses found in native C. parasitica strains analyzed for evidence for spontaneous infection or long-distance transmission based on molecular sequence. <li>MI grafted (5-year-old) American chestnut selections from WI inoculated with C. parasitica to determine reaction to various strains. <li>Experiments initiated to assess roles of other C. parasitica PKS genes in virulence, fungal development, and chemical defense. <li>Naturally occurring chestnut bark microorganisms analyzed for their antagonism toward C. parasitica. </ul>

(2008): <ul> <li>Analysis of blight resistance of cobalt-irradiated American chestnut trees completed. <li>Trials of commercially available chestnut cultivars analyzed and reported. <li>Polyprotein processing maps completed for hypoviruses CHV1-EP713 and CHV1-Euro 7, and a detailed view compiled of the changes in cellular transcriptional profiles caused by infection of C. parasitica with mild and severe hypoviruses. <li>Optimal site conditions determined for chestnut establishment in forest ecosystems. <li>Quantification of the progress of the C. parasitica and hypovirus epidemic and measureme

Projected Participation

View Appendix E: Participation

Outreach Plan

The annual meeting of NE-140 serves as the mechanism to keep members and other interested parties abreast of current research and related chestnut activities (i.e., ancillary symposia, annual meetings, international exchanges). Information on this meeting and shared projects is available on the NE-140 web site. NE-140 members will continue to make research results available through scientific journals, both refereed and non-refereed, extension bulletins, and national and international conferences and workshops. Information to the general public will be disseminated via publications in the popular press, magazines, oral and written presentations at workshops and at producer field days. A listing of all publications developed by NE-140 members will be updated annually and posted on the NE-140 website in NIMSS at www.lgu.umd.edu.

Organization/Governance

The organization of regional research project NE-140 was established in accordance with the format suggested in the "Manual for Cooperative Regional Research". One person at each participating agency is designated, with approval of the agency director, as the voting member of the Technical Committee. Other agency individuals and interested parties are encouraged to participate as non-voting members of the committee. Each year, members elect a Secretary. The Secretary, whose duties begin the following year, then becomes Chair-elect, followed by Chair the third year.

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Attachments

Land Grant Participating States/Institutions

AL, CA, CT, KY, MA, MI, MO, NJ, NY, PA, TN, WV

Non Land Grant Participating States/Institutions

Alabama, Tennessee valley Authority, American Chestnut Foundation, New York - Syracuse University, University of Georgia, University of Tennessee at Chattanooga, USDA Forest Service