Duration: 10/01/1998 to 09/30/2003

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Within fifty years after its discovery, the pathogen Cryphonectria parasitica had caused considerable destruction that led to the elimination of chestnut trees as major components of the forests of Northeast America. Chestnuts once occupied 25% of our eastern hardwood forests. Restoration of this important species and the successful control of this devastating pathogen will increase forest productivity and profitability, and hence will positively impact local and regional economies.

Related, Current and Previous Work

Breeding work was begun with chestnuts at the turn of the last century, but the only program that has continued without interruption is that at The Connecticut Agricultural Experiment Station (4). Some studies of the disease and the fungus were done in the early 1900s, but interest waned with the lack of success in controlling the disease. The issue was reborn with the discovery of chestnuts in Italy that were surviving in spite of their chestnut blight cankers (11). The recovery phenomenon, termed "hypovirulence", usually involves viruses that cause a disease of the fungus, reducing its ability to kill chestnut trees (36). This discovery reawakened the scientific community to the long-standing issue of chestnut blight, a topic that had not undergone the scrutiny of modern scientific investigation. Plant and fungal physiology projects were begun to study the enzymes involved in the interactions of tree host, fungal pathogen, and viral parasite. Two additional institutions began breeding chestnut trees for blight resistance using , modern genetic principals; The University of Tennessee in Knoxville and The American Chestnut Foundation in Meadowview, Virginia (4). The large collection of species and hybrids of chestnut that had been maintained in Connecticut was made available to all who needed material. When it became clear that the oriental chestnut gall wasp (Dryocosmus kuriphilus), introduced into Georgia in 1974, was spreading northward, the breeding programs included screening for resistance to this pest in 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 discussion that ensued 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 group's activities, the following events have occurred:


Connecticut has improved the records of holdings and continues to maintain the finest collection of species and hybrids of chestnut in the world (6). New York (Cornell) confirmed, by making genetic tests of Asian and American populations, that the blight fungus came to the US from Japan (43). Connecticut imported hypovirulent strains from Dr. J. Grente in France (who first described the disease of the fungus) and showed that these strains could control chestnut blight cankers on American chestnut trees (8). Michigan described the spread of American hypovirulence viruses through the blight fungus population in a stand of trees planted in Michigan (35). Connecticut described a genetic system in the blight fungus that prevented strain fusion and the transmission of hypovirulence viruses (1, 2, 3). Michigan presented a physical and genetic map of the mitochondria) genome of a strain of the blight fungus (10). Michigan described a type of hypovirulence determined by genes in mitochondria (42). Michigan, New Jersey, and West Virginia described and compared three American hypovirulence viruses (19, 54) New Jersey, Maryland, Michigan, and Texas spearheaded the naming of hypovirulence viruses as a new genus, now recognized by the International Committee on Taxonomy of Viruses (37, 38) Maryland 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 (14, 15) Maryland, Connecticut, and West Virginia were granted permission from USDA/Plant Quarantine to test transgenic hypovirulent strains of the chestnut blight fungus in the forests of Connecticut and West Virginia. This was the first permit granted to test transgenic organisms for their ability to spread and effect biological control of a plant disease (7). Kentucky, Michigan, Texas, Maryland, Virginia, and Connecticut have made detailed studies of enzymes produced by the blight fungus that may be related to its ability to kill chestnut trees (5, 12, 13, 16, 17, 18, 20, 21, 22, 25, 27, 28, 31, 33, 34, 39, 40, 42, 46, 53, 55) Kentucky and Connecticut have studied enzyme systems in chestnut trees that may be related to their ability to resist the blight fungus (26, 29, 30, 45, 48, 49, 50, 51, 52) Virginia, Connecticut, and Tennessee have been breeding chestnut trees and selecting progeny with resistance to chestnut blight disease, and with the timber form of American chestnut trees (4). These will be ready for release within five years. Mississippi, Virginia, Massachusetts, and Connecticut prepared a genetic map of chestnuts and found three molecular markers associated with resistance to chestnut blight disease (41)

Objectives

1. To improve chestnut trees for timber and for nut production, and determine the cultural requirements of chestnut seedlings in nurseries and natural settings.
   
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.
   
3. 
   

Methods

Objective 1: To improve chestnut trees for timber and for nut production, and determine the cultural requirements of chestnut seedlings in nurseries and natural settings..

1. Traditional breeding programs are being carried out in Connecticut, Tennessee, and Virginia. 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 are 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 speed up selection using molecular markers. Assessment for form and nut quality may be done on 5-year-old trees.

2. Identification of molecular markers is being done in Mississippi with plant material supplied by all NE-140 members. Genetic input is provided by Virginia. 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. Seven 10-mer RAPD primers are used with extracted and purified tree DNA in PCR to identify three regions of DNA correlated with chestnut blight resistance, flanking regions, and "control" regions (used in Mississippi and Connecticut, so far). The next major step in this work will be to use bulk segregant analysis for fine-scale mapping of the three genetic regions associated with blight resistance.

3. Plant tissue culture laboratories in Georgia and New York (SUNY) will maintain and multiply important clones of chestnut hybrids produced by Virginia, Connecticut, and Tennessee. In addition, Georgia will develop cultures of trees in native populations of American chinquapins (C. pumila var. pumila and var. ozarkensis), threatened by chestnut blight and ink disease (caused by the root pathogen Phytophthora cinnamomi), to insure that important germplasm is not lost. Chinese chinquapins (C. Henrys) are available in Georgia, and will be included in the program. All three chinquapin types seem to have valuable resistance to the Oriental Chestnut Gall Wasp (Dryocosmus kuriphylus), which is currently found in Georgia, Alabama, Tennessee, and North Carolina. New York (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.

4. Cultivar trials (of commercially available chestnut cultivars for nut production) are underway in Connecticut and Tennessee, and commercial plantings in Michigan are being monitored. The most commonly planted cultivar in the U.S. (mostly on the west coast) is currently 'Colossal', which is a European X Japanese hybrid with some resistance to chestnut blight, but winter hardiness has not been documented and resistance to ink disease is unknown. We assume that it will be susceptible to infestation by gall wasp. The trials in Tennessee of this, and other commercial cultivars, will test for resistance to blight, ink disease, and gall wasp, and the Connecticut trial will test for blight resistance and winter hardiness. Plantings of 'Colossal' in Michigan will be checked for survival in that climate and for blight resistance.

5. Plantings of new hybrids for assessment of resistance to gall wasp, chestnut blight, and ink disease have been made and will continue to expand. This work is being done by Tennessee (plantings in Tennessee), and by Connecticut (plantings in Georgia in cooperation with Jerry A. Payne, U.S.D.A./ARS, Tree Nut Research Laboratory, Byron, and in North Carolina in cooperation with W. Henry McNab, U.S.D.A./Forest Service, Bent Creek Research Forest, Ashville). Tennessee is using Japanese stock plants with reported resistance to gall wasp, and Connecticut
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.

6. In addition, we must protect existing populations of American chestnut trees for future inclusion in the breeding programs and for studies of genetic diversity. This is being done by planting seed collected from natural populations, and by introducing hypovirulence viruses and transgenic hypovirulent strains of the blight fungus into natural populations and seed orchards. Native populations currently being protected are in Connecticut, New York, Massachusetts, Maine, New Jersey, Virginia, Pennsylvania, Maryland, West Virginia, North Carolina, Tennessee, and Georgia. Valuable plantings in Wisconsin are being maintained, as well. In Michigan and Ontario plantings of American chestnut trees are being protected from blight apparently by debilitation of the fungus with a cytoplasmically-transmissible, mitochondria) disease.

7. Trees grown from irradiated American chestnut seed that were planted at the National Colonial Farm in Virginia have been evaluated for blight resistance. Seedlings of these trees have been planted in Michigan for further testing (by Michigan, Virginia, and West Virginia)

8. Chestnuts imported by the U.S. D.A. and planted prior to 1960 are being located and evaluated for resistance to chestnut blight, ink disease, and gall wasp by Connecticut and Tennessee.

9. Natural populations and orchards in Connecticut and West Virginia will be examined for the effect of competing vegetation on tree survival (with the cooperation of Gary J. Griffin, VPI &SU, Blacksburg).

10. Forest and orchard planting techniques will be studied by Connecticut, Tennessee, Virginia, and Michigan.

Objective 2: To better understand the interactions and ecology of this host/pathogen/parasite system at the molecular, organismal, and environmental levels to develop effective biological controls for chestnut blight.

1. 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 Connecticut, New Jersey (cooperating with New York), New York, and West Virginia (with Michigan). These projects include work on the vegetative incompatibility system of the fungus, and specific vic genes used as markers.

2. The spread of hypovirulence viruses is being monitored in natural and planted populations in Connecticut (with Maryland), New Jersey (with New York), New York, West Virginia (with New Jersey, Michigan, and New York), and Tennessee (with Connecticut and West Virginia). American chestnut trees planted in West Salem, Wisconsin at the end of the last century now have chestnut blight disease. Hypovirulent strains of the fungus are being used to try and establish a biological control. The treatments are being supervised and monitored by Michigan, West Virginia, New York, and the Wisconsin Department of Natural Resources (Jane Cummings-Carlson). Trees planted in Michigan, where natural hypovirulence viruses developed, are being monitored by Michigan (with West Virginia).

3. The survival and spread of transgenic hypovirulent strains is being evaluated in Connecticut and West Virginia, both working with Maryland where the strains were developed.

4. Alternative controls for chestnut blight disease are being sought in Massachusetts, using naturally-occurring bark fungi, such as Trichoderma. This work will continue with studies of the nature of the control mechanisms.

5. 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. Sequencing and comparisons of the Hypoviruses will be done by New Jersey, Michigan, Maryland, West Virginia, all working closely with each other. Studies of the effect of specific fungal genes on Hypovirus replication and gene expression will be done in Maryland, New Jersey, and Texas. The function of the fungal genes as sex pheromones, and their involvement in virulence will be examined in Texas. 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 in Maryland.

6. Hypovirulence caused by mitochondria) mutations and mobile genetic elements will be studied in Michigan.

Measurement of Progress and Results

Outputs

• Molecular markers to speed up selection for tree form and nut quality on 5-year-old chestnut trees.
• Fine-scale marker maps of the genomic regions of chestnuts associated with chestnut blight resistance.
• Development of five chestnut breeding lines that are resistant to chestnut blight.
• Worldwide comparisons of genetic diversity of mycoviruses as biological control agents for chestnut blight disease.
• Identification of effective biological management practices to control or eradicate the virulent form of the chestnut blight pathogen in the U.S.
• Detection of genetic variability in young, open-pollinated chestnut trees in plantations.
• Identification of important chestnut seedling characteristics for successful artificial regeneration.
• Evaluation of early growth characteristics of chestnut genotypes planted on different sites.

Outcomes or Projected Impacts

• Increased populations of timber and nut-bearing chestnut trees in eastern North America.
• Improved stand diversity of eastern hardwood forests in North America.
• Improved economic opportunities in the local and international market for chestnut growers.
• Reintroduction of chestnuts onto xeric sites formerly populated by oak species that were decimated by gypsy moth infestations, in order to provide mast and wood products.

Milestones

(1999): Termination report summarizing 5-year accomplishments of previously completed project. Complete resistance mapping work in both 'Mahogany' and 'Clapper'-derived chestnut lines. Development of genetic markers for chestnut species and early hybrid identification. Complete evaluation of chestnut tissue culturing parameters including basal media, micronutrient levels, and continuous versus pulse 2,4-D embryo induction treatment. Development of a tissue culture bioassay to distinguish C. parasitica resistance levels among resistant Chinese, Chinese/American hybrid, and susceptible American chestnut callus tissue.

(2000): Chestnut seedling characteristics evaluated in nursery-run open-pollinated families. Open-pollinated chestnut progeny tests established on different forest sites in Tennessee, Pennsylvania, Connecticut, West Virginia, and Virginia. Third backcross chestnuts to be tested for chestnut blight resistance. Third backcross F3 from the 'Mahogany' and 'Clapper' chestnut lines are expected. Introduction of improved transgenic hypovirulent strains of C. parasitica for biological control of chestnut blight.

(2001): Additional forest site tests begun. Characterizations of putative genetically transformed American chestnut embryogenic cultures completed. Field evaluation of confirmed transgenic chestnut trees containing single-transgene constructs.

(2002): Sampling of sixty chestnut trees from each of 25 sites within a 135-mile grid completed, to assess the level of genetic diversity in wild American chestnut populations and to determine the geographic component of such diversity. Evaluation of transgenic hypovirulent strains of C. parasitica for biological control of chestnut blight.

(2003): Survival and growth of chestnut trees in genetic tests evaluated, information on the relationship of seedling characteristics to field survival and growth generated, and genotype by environmental interaction studied. Details of hypovirus genome structure/function relationships, and protein functions identified through comparative molecular studies of different Hypovirus isolates. Optimum strategies developed for the efficient repopulation of chestnut forests through combining biocontrol and resistance breeding.

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Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

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Literature Cited

Attachments

Land Grant Participating States/Institutions

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 State University -CESF, Southern Institute of Forest Genetics, USDA, University of Georgia, University of Tennessee at Chattanooga