NE-1033 began in 1982 as NE-140, a collaborative project that included five experiment stations. The impetus for the regional project was the discovery of hypovirulent strains of the chestnut blight fungus, Cryphonectria parasitica, that afforded some level of resistance to the disease that had decimated chestnut resources in many parts of the world. The introduction of the exotic chestnut blight fungus to North America had unparalleled ecological and economic impacts, and functionally eliminated American chestnut from North American forests.

The discovery of hypovirulence rekindled interest in the blight fungus and brought renewed hope for chestnut restoration. But the phenomenon of hypovirulence is complex. Understanding its biology, ecology, and spread, as well as developing methods of manipulation, has been challenging. In areas of Michigan and Italy, hypovirulence appears to be the only explanation for the recovery from blight of significant stands of American and European chestnuts (16, 55, 67). But successful manipulation of hypovirus-infected strains in the eastern U.S. for biological control of the blight fungus has been problematic. Ongoing studies are comparing aspects of recovering versus declining stands of chestnut in Michigan with the goal of determining why hypoviruses spread in some situations and not others. Hypoviruses are being deployed in declining chestnut populations to test hypotheses of its spread, investigate within canker dynamics, and evaluate contributions of dying chestnut to hypovirulent inoculum. Overcoming the challenges of utilizing hypovirulence as a tool for blight suppression, and ultimately for restoration of chestnut resources, is a driving force behind this project.

Technological advances have added new dimensions. Genome sequencing has redefined the ability to test hypotheses relating to genes required in pathogens (for virulence) and hosts (for susceptibility). An important outcome of the work by genomics researchers in NE-1033 is the investigation of the fungus genome to learn what factors allow C. parasitica to be so virulent. For example, pathways for synthesis of secondary metabolites, which may serve as toxins and virulence factors, can be investigated more efficiently with access to the genome sequence. Molecular approaches also provide mechanisms to develop genetically altered strains of C. parasitica with enhanced production of hypovirus-laden spores, thereby increasing the probability of hypovirus spread. Understanding the roles different hypoviruses play in altering the virulence of C. parasitica is a critical component of the genomics research. Such efforts are expected to lead to development of molecular strategies that will enhance the effects that hypoviruses have on the C. parasitica strains they infect, thereby reducing pathogen virulence. This is an important step for biological control. Another consideration is the enhanced resistance of chestnut provided as part of the breeding initiative. Incremental increases in blight resistance expressed by backcross generations of trees, coupled with diminished virulence provided by hypovirulent strains, may provide a viable integrated approach to blight control. Hypovirus infection also lends itself to the study of virulence factors, as comparative studies of isogenic strains that are or are not hypovirus infected may unravel the mechanisms by which virulence genes in the fungus are suppressed, ultimately leading to successful forest restoration.

Genomics also provides tools to investigate thoroughly a system of vegetative incompatibility (vic) that regulates hypovirus transmission among strains of C. parasitica, thereby reducing their effectiveness as biological control agents. Identification of the physical genes involved in restricting hypovirus transmission are providing powerful tools for population genetic studies to more precisely determine the contribution of vic gene diversity to hypovirus transmission. Additionally, a wider survey of the vic allele sequences in C. parasitica field isolates and in related fungal species is providing new insights into the generation, fixation and maintenance of fungal nonself recognition systems and the influence that mycoviruses may have on their origins and evolution. It is anticipated that ongoing studies of the interactions of these genes will lead to enhanced hypovirus spread. The goal of those working with the genomics of chestnut, the blight fungus and its viral pathogens is to understand the interaction of the three at the molecular level, including delineating genetic defense mechanisms necessary to resist infection.

Today, NE-1033 utilizes several fundamental approaches to chestnut improvement. The first is selection and breeding of blight resistant trees for forest and orchard settings. Although the approach utilizes traditional breeding methods, molecular techniques are increasingly used to aid in the selections. Advances in breeding efforts have provided genetic material needed to accomplish much of the genomic work that is instrumental in identifying genes that impart resistance to blight and other organisms. Identification of genes that confer blight resistance will require molecular comparisons of Chinese, Japanese, and European chestnuts, American and Chinese chinquapins, and the hybrids developed from the breeding program. Validation of the original two lineages of hybrid chestnuts is a priority. Breeders have sought to combine resistance to pathogens and pests with nut quality, cold hardiness, and stress tolerance through interspecific hybridization. The vigor and female fertility of the interspecific hybrids resulted in the introduction of many cultivars with hybrid ancestry (57, 97), but interest in chestnut breeding and nut production in the US waned in the 1950s and '60s, and most programs were abandoned. Although many of the original crossing records still exist at the Connecticut Agricultural Experiment Station (CAES) (3), many of the cultivars being re-evaluated for nut production have no pedigree records. Success for the grower depends on reliable yields and high quality nuts which in turn depend on development of stress-tolerant cultivars suited to climatic variability. Verification of identity and interspecific ancestry will increase the efficiency of chestnut breeding programs, encourage the growth of a wholesale nursery industry based on true-to type nursery stock, and enable growers to minimize risk by choosing cultivars based on predicted performance for a given location. Breeding efforts have expanded and include numerous state programs, as well as Ontario, Canada.

A critical need that has emerged as a result of chestnut breeding success is generation of large numbers of the most desirable blight resistant genotypes, which are required for performance testing and general research. Project members are investigating somatic embryogenesis and an embryo germination/ micropropagation system for chestnut propagation. Both systems have the potential to be scaled-up to supply hundreds of seedlings. Even though the principle breeding program is designed to incorporate resistance genes from Asian species, there also are alternative molecular technologies that can exploit an array of anti-fungal genes that, if successfully incorporated into somatic cells, may impart blight resistance to plants that are regenerated from those cells. If successful transformation systems can be developed, the plants that result can be incorporated into the genomics efforts that are designed to identify how genes function to create resistant individuals.

As breeding efforts progress, several additional pathogens and pests have emerged as concerns. In the central and southern Appalachians, root rots cause by Phytophthora cinnamomi and Phymatotrichopsis omnivore pose significant threats to both nursery grown seedlings and to outplanted stock. Galling by the Asian chestnut gall wasp (ACGW), Dryocosmus kuriphilus, reduces tree vigor, flowering and nut production, and causes branch and tree mortality. Although some species of chestnut appear resistant, knowledge of how this pest might influence natural populations and backcross trees is needed. The granulate ambrosia beetle, Xylosandrus crassiusculus, causes extensive chestnut mortality, linked to the presence of symbiotic fungal associates, in young or small diameter trees. The Asiatic oak weevil, Cyrtepistomus castaneus, is causing significant concern in restoration plantings. Knowledge of how these pests might influence natural populations of chestnut sprouts and backcross trees generated from The American Chestnut Foundation's (TACF) breeding program is essential. These pests must be considered as the breeding program advances.

When NE-140 was initially formed in 1982, there was no edible sweet chestnut industry in the eastern US. Since then, several project participants have made significant progress in creating a horticultural chestnut industry and consumer marketplace in their respective states. Chestnut is a temperate tree nut that more closely resembles a fresh fruit than a nut, as it shows rapid respiration after harvest and can mold during storage. The nut is low in fat but high in nutritional benefits. Because chestnut is novel to most Americans, marketing must be emphasized. Various new chestnut food products are appearing in high-value niche markets which further encourage grower interest. This expanding horticultural industry requires regional testing of old and new cultivars for productivity, food quality, pathogen and pest resistance, and regional adaptability. In Michigan the highest yielding chestnut cultivars planted are the European X Japanese hybrids (57). Many of these European X Japanese hybrids claim blight resistance, but this has not been demonstrated conclusively. As blight becomes more common in Michigan chestnut orchards, hypovirulence treatments using hypoviruses isolated from recovering American chestnut stands in Michigan have been implemented (54, 55, 56, 59, 111, 126). The Michigan group has worked to better understand the dynamics in chestnut stands when hypoviruses are present versus when they are absent (39, 40). Specific knowledge of root stocks, graft compatibility and propagation systems, developed as part of the micropropagation efforts, may prove to be an invaluable synergism.

As the project progressed we have also investigated basic silvicultural aspects of chestnut restoration. The restoration of American chestnut into eastern North American forests by the introduction of blight-resistant chestnuts is greatly anticipated by the general public. As the actual release of resistant seed and seedlings approaches, attention must be directed to the ecological and silvicultural considerations that will affect the success of the reintroduction efforts. Clearly understanding specific aspects of how to plant, protect, and grow chestnut in our forest ecosystems is paramount to the success of restoration efforts. This begins with sound nursery practices to produce large numbers of healthy seedlings, followed by a solid knowledge of site selection and outplanting techniques.

The knowledge of chestnut gained and shared among scientists who participate in NE-1033 has brought renewed hope for chestnut restoration in eastern North American forests and created a promising chestnut industry. Current and emerging molecular approaches have opened a floodgate of opportunities that were unimaginable in the project's formative years. An understanding of the mechanisms that regulate resistance to blight, as well as to emerging pathogens and insects, and a working knowledge of how best to grow chestnut trees, is critical to the improvement of chestnuts for nut production and ultimately for deployment as forest trees.

Importance of the Work: The history and productivity of this project are testament to its value. When NE-140 first began there was limited hope for chestnut as a component of North American forests or as a nut producer. Significant progress, both basic and applied, has been made since then. Issues associated with blight host interactions are complex, and emerging pathogens and pests continue to pose new challenges. Nevertheless this multi-state project must be considered a huge success, as remarkable progress has been made toward a detailed understanding of the issues and approaches that are necessary to affect solutions. As findings and technologies continue to unfold, they aid in identification of critical issues and further research progress.

Technical Feasibility of the Research: Research participants in NE-1033 contribute significantly to our understanding of the chestnut Cryphonectria pathosystem. Initial studies largely utilized traditional plant pathological techniques, but as the complexity of this host/pathogen/virus interaction began to unfold and molecular approaches became feasible, many of the fundamental questions posed by the chestnut blight dilemma were solved. This regional project has expanded in concert with rapid advances in technology. The ability to examine the actual genetic make-up of the host, pathogen and pathogen-infecting viruses brought a new dimension to the project. The progress by collaborators on the NE-1033 project cannot be overstated: this is the only plant system world-wide for which the interactions of the plant host, its major fungal pathogen, and a suite of natural biological control agents of that pathogen have been characterized at the level of primary sequence. Numerous complete sequences of biocontrol-associated viruses have been determined and their role in suppression of the chestnut blight fungus examined; the genome sequence of the fungus is complete, and genetic mapping of American chestnut and its blight-resistant Chinese chestnut counterpart are complete. None of these efforts would have been possible by independent research groups alone. The spin-off potential of these analyses is already evident. The identification of genes involved in expression of disease resistance will be a remarkably powerful tool for developing blight resistant trees. Knowledge of the genetic make-up of C. parasitica provides insights into the genetic mechanisms the fungus utilizes to cause disease in chestnut, as well as the fungal defenses that restrict the movement of biological control agents among strains. Further, combining knowledge of all three systems is providing an understanding of the biochemical alterations that result when the blight fungus is infected by cytoplasmic agents or the host is challenged by a variety of pathogens and pests. We are at the cusp of finding answers to many long-standing questions relative to a variety of threats to chestnut and impediments to its improvement. This regional project continues to exploit new technologies and provide the impetus for what has evolved into a model system for the study of the interactions among a woody plant host and the many pests and parasites that threaten it.

Value of a multi-state approach: The components of chestnut blight, chestnut improvement, and accompanying restoration issues are far more complex than initially thought when NE-140 was initiated. In the ensuing years NE-1015, followed by NE-1033, has been highly successful in fostering collaborative work to examine multiple facets necessary to address this complexity. Increased research effort and improved technologies, coupled with insights and efforts of scientists from numerous disciplines, have led to significant accomplishments. This multi-state project involves scientists across multiple disciplines from the land-grant system as well as numerous other academic institutions, government agencies, and private organizations. These collaborations are truly interdependent; many of the individual projects would not have been possible had it not been for the resources and interactions fostered under the CSREES multi-state model. In the case of C. parasitica, the availability of the genome sequence and collaborative nature of the annotation process was made possible by the structure provided by NE-1033. The availability of this information has made it possible for research groups to develop new research tracks in areas related to fungal pathogenicity, hypovirulence and vegetative incompatibility. The formation of the regional project can be credited with renewing interest in restoration of American chestnut, and in part is responsible for the emergence of TACF, a non-profit organization that invests its resources in breeding efforts to develop blight resistant trees.

Projected Impacts: The overall impacts of the NE-1033 project is further progress toward restoration of American chestnut as a functional tree in North American forests, and support for utilization of chestnut as a nut tree for the American marketplace. The notable stature of chestnut in the history of the US is evident by the existence of member-funded organizations such as TACF, the Canadian Chestnut Council and the American Chestnut Cooperator's Foundation. These organizations focus solely on chestnut and can trace their roots to the resurgence of interest in the species, in part generated by the NE-140/NE-1015/NE-1033 project. Since initiation of this project, the US Office of Surface Mining began using chestnut as a mine site reclamation species, and the National Wild Turkey Federation has embraced chestnut restoration through an official partnership with TACF. These and other stakeholders, including private landowners, are intensely interested in efforts to restore this once important forest species.

While the complexities of the host/pathogen/virus interactions, and the issues associated with chestnut restoration will not be solved by the end of this of this project, our progress has been significant and encouraging, and the future steps we've outlined will bring us closer to our goals. One of the most significant undertakings is development of blight resistant chestnuts that are regionally adapted to a variety of forest environments from Canada to the Gulf States. Progress with traditional breeding efforts has been substantial, but there are many obstacles. Development of regionally adapted chestnut has been advanced by the addition of the genomic component to the project, resulting in a genetic map for chestnut. The genomics approach is leading to identification of resistance genes and technologies to facilitate rapid screening of chestnut progeny that possess genes imparting resistance to blight and other pests and pathogens. This approach might be useful in evaluating chestnut resistance to P. cinnamomi, the ACGW, and other pests of concern.

The need to produce large numbers of chestnuts requires establishment of seed orchards and also exploitation of technologies that utilize novel regeneration systems to produce large numbers of individual clones. Regeneration systems also can allow the incorporation of antifungal genes from a variety of sources that may impart resistant or tolerance to C. parasitica, a novel approach to addressing the disease problem. Both avenues to generate offspring have their place as part of the project and are complimentary.

Analysis of the fungal genome is clarifying the genetic basis for pathogenesis by C. parasitica, and will help determine why the fungus is such an efficient pathogen of American chestnut but not of Asian chestnuts. Studies of the metabolites produced by the fungus and how these products are linked to specific synthesis and regulatory pathways will aid in understanding the process of pathogen invasion. Likewise, the system of vegetative compatibility is tied closely to particular genes that regulate anastomosis between strains. Mapping specific vic genes is providing an understanding of how compatibility restricts the transfer of debilitating hypoviruses from strain-to-strain. The biological implications of hypovirus infection provide fundamental and applied research opportunities. The fungal and hypovirus genome projects provide a more global view of the influences different hypoviruses and their encoded gene products have on gene expression. Understanding the mechanisms by which hypoviruses regulate fungal pathogenesis is fundamental to manipulating them as biocontrol agents, and also raises the possibility of genetically altering specific processes in the fungus tied to hypovirus infection, thereby making hypoviruses more effective fungal mortality agents. Despite numerous forest settings where hypoviruses have naturally contributed to biological control, successful manipulation of artificial hypoviruses has been elusive.

Understanding the components of natural hypovirus spread is essential; transgenic strains that transmit their hypoviruses more efficiently are being utilized to evaluate this. Another dimension of the hypovirus research is their use in conjunction with the breeding program. Trees produced by the breeding program that are only moderately resistant to blight may be able to support hypovirulence infections, allowing them to grow competitively in forest settings.

Numerous stakeholder groups are poised to undertake large-scale plantings of blight resistant chestnuts, but deployment requires extensive knowledge of silvicultural approaches. Chestnut has never been the focus of contemporary silvicultural research. Even if systems to produce large numbers of trees were in place, optimal site characteristics, soil parameters, and planting approaches have not been clearly defined. Historical records are being examined to determine where chestnut once thrived; however sites that support chestnut today may just be isolated areas where the species survived, and not where it thrived, and thus not the best choice for reestablishment.

An important continuing dimension of NE-1033 is nut production. Developing a chestnut industry in the US requires that suitable cultivars be regionally tested, and that systems of orchard culture and management, including diseases and insects, are evaluated. Market development is essential. While many problems associated with successful nut production are unique, many are common to both forests and orchards.

The overall impact of this project is to further the progress that has been made toward restoration of chestnut as a tree in North American forests and as a nut in the American marketplace. Some specific impacts include:

• Establishment of breeding orchards to generate large numbers of backcross generations for forest and orchard testing for pest resistance and regional adaptability;
  
• Evaluation of Castanea genomic data to identify genes that confer desirable traits and enable rapid screening for those traits;
  
• Development of in vitro mass propagation systems for Castanea spp. so that elite genotypes can be clonally propagated for reforestation;
  
• Evaluation of the blight fungus genome to further our understanding of the genetic basis for pathogenesis and hypovirus regulation;
  
• Development and deployment of genetically engineered virus for enhanced biocontrol;
  
• Utilization of biological control agents to reduce the impacts of blight and other pests and pathogens; and,
  
• In the longer-term, the project will lead to the return of an important timber species, mast species for wildlife, and commercial nut crop.