Duration: 10/01/2013 to 09/30/2018

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Statement of Issues and Justification

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.

Related, Current and Previous Work

With the introduction and rapid spread of the causal fungus of chestnut blight in the early 20th century, a variety of eradication measures were deployed in a desperate measure to preserve forest chestnuts. When it became evident that eradication wasn't feasible, the search for blight resistance began and breeding programs were initiated; Asian chestnuts were crossed with American chestnuts to search for stable, blight-resistant hybrids. Success was elusive and these programs were abandoned (19). When the phenomenon of hypovirulence was discovered in the mid1960s there was a reawakening of the scientific community's interest in chestnut and the blight fungus, and a reexamination of previous breeding programs (92). Hope for chestnut restoration was rekindled. Since the inception of this multistate project considerable effort has been devoted to understanding the biology and spread of hypovirulence, with the intent of developing methods for manipulation in biological control efforts against the blight fungus. Hypovirus infection alters the reproductive capacity of the infected fungal strain, reducing asexual spore production and eliminating sexual reproduction (82.). CAES imported hypovirus-containing strains of C. parasitica from France in 1972, and began research on their use for biological control (7). Current efforts are focusing on hypovirus deployment. Studies are comparing aspects of recovering versus declining stands of chestnut with the goal of determining why hypoviruses spread in some situations and not others. The limitations to hypovirus spread imposed by a system of vegetative compatibility plays a prominent role in its effectiveness for biological control (24). Significant diversity in vegetative compatibility exists, restricting strain-to-strain hypovirus spread (102). Work is focusing on determining the extent to which recovering and non-recovering sites differ genetically for vegetative incompatibility (vic) loci and for microsatellite diversity. C. parasitica populations at recovering sites differ significantly from non-recovering sites in Michigan with regard to vegetative compatibility (128). Evaluating hypoviruses that have invaded C. parasitica populations provides a unique opportunity to understand their natural spread (38, 89, 90, 110). Tree growth and reproduction has improved each year as hypoviruses continue to spread (38). Based on these observations new hypotheses are generated addressing hypovirus spread, and new strategies have been developed for deployment (38). Related studies of disease progress and spread in a Wisconsin site with an artificially introduced hypovirus are ongoing (47, 101); this site represents the opportunity to document disease progress in mature chestnut along with the fate of artificially introduced hypoviruses (79). Both sites provide opportunities to manipulate ecological factors to enhance hypoviruses as biological control agents. Another avenue of research is investigating hypovirus spread within C. parasitica, determining how the genetic background of the pathogen and hypovirus affect the transmission of hypoviruses into fungal conidia (49, 109, 121). Understanding how hypoviruses perturb normal developmental processes of C. parasitica will provide a better understanding of the biology of hypovirulence, leading to a more effective deployment for biological control. The virus CHV-1 replicates on trans-Golgi vesicle membranes of C. parasitica (51, 76, 80) and integration of the p29 protein of CHV-1 into these vesicle membranes results in reduced fungal sporulation and pigment production, primary symptoms of virus infection, while not affecting fungal vegetative growth. Although p29 integration does not affect fungal virulence expression, one of the key enzymes contained within these vesicles, Kex2, is necessary for virulence expression (77). Results show that virus replication on the trans-Golgi vesicles of the fungus perturb secretion of proteins important in development (80), and that normal functioning of these vesicles and Kex2, a molecular marker of these vesicles, is necessary for fungal development. Characterization of these trans-Golgi vesicles and exploration of how the virus perturbs their function is on-going. Studies are also underway to determine whether genetically modified strains of C. parasitica can enhance biological control (120). In addition, fungi other then C. parasitica commonly isolated from cankers at locations in MI, WI and WV are being identified in an effort to understand what role bark saprophytes may play. Standing dead or cut chestnut logs may provide sources of hypovirulent inoculum (114), further contributing to biological control. Modeling and empirical work are being used to determine how interactions between the pathogen, hypovirus and non-blight competitors influence the fate of a canker. NE-1033 scientists have taken full advantage of emerging molecular technologies. Genome sequencing has redefined the ability to test hypotheses relating to genes required for virulence and susceptibility. The availability of the genome sequence for C. parasitica, and the collaborative nature of the annotation process, was made possible with the structure provided by NE-1033. Researchers were able to develop new research tracks in fungal pathogenicity, hypovirulence and vegetative incompatibility. Vegetative incompatibility (vic) nonself recognition systems in fungi restrict the spread fungal viruses (22). The vic system in C. parasitica has been the subject of considerable interest (68, 92, 105, 108) since reports that transmission of a reduced virulence phenotype from one strain to another through fusion of the hyphae was restricted when the two strains were vegetatively incompatible (5). The incompatible reaction triggered by allelic differences at the vic genetic loci results in localized programmed cell death (PCD) of the interacting strains and blockage of the transmission of hypoviruses that are now known to be the causative agent of the hypovirulence phenotype (23). While genetic analysis has revealed that the C. parasitica vic system is controlled by at least six genetic loci with only two alleles at each locus (3, 36, 71), the physical nature of the alleles has remained elusive. The comparative genomics approach to identify vic gene candidates has been enabled by several NE-1033 advancements, including a collection of 64 C. parasitica strains that represented all possible genotypes across the six vic loci (36), a genetic linkage map containing nucleotide markers linked to the vic genetic loci (85), and a reference C. parasitica genome sequence assembly generated by the Joint Genome Institute and the C. parasitica genome consortium. It was predicted that the C. parasitica vic alleles should exhibit heterogeneity at the nucleotide sequence level and would be identified as regions of sequence polymorphism near corresponding linkage markers (116). These regions of polymorphism would be expected to appear as gaps when the sequence reads from a re-sequenced C. parasitica strain with a different vic genotype were mapped by homology on the reference genome sequence. This hypothesis proved correct, resulting in identification of alleles at vic2, vic4, vic6 and vic7 (24). An extension of the comparative genomic approach is being used to identify genes associated with vic1 and vic3 loci to provide a clear picture of the complete complement of vic genes that restrict hypovirus transmission. 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 vegetative incompatibility diversity to hypovirus transmission. We anticipate that ongoing studies of the interactions of these genes with each other and the downstream PCD pathway will lead to strategies to enhance hypovirus spread. We have identified a putative orthologue of vib-1 in the C. parasitica genome sequence that shares 40% identity and 48% similarity with VIB-1 from the related fungus Neurospora crassa in which it is a transcription factor essential for the expression of genes required for programmed cell death (45). To assess a potential role for CpVIB-1 in vegetative incompatibility in C. parasitica, we created a gene knockout in the EP155 strain. This DCpvib-1 strain, however, did not manifest any deficiency in the incompatibility assay. We then created a second deletion of Cpvib-1, this time in the background of EU1, which has the vic profile 2212-22, which differs from EP155 at vic4. This EU1 DCpvib-1 strain behaved exactly as predicted demonstrating no change in compatibility with all other EU strains and EP155. Testing the two DCpvib-1 mutants together demonstrated that these previously incompatible strains were now compatible, and we conclude that CpVIB-1 does play a crucial role in regulating the incompatibility reaction triggered by mismatched alleles at vic4. The nature and function of effector molecules produced by a pathogen to enhance virulence is gaining attention. The effector appears dependent on host genes that trigger defense responses. We currently know little of the interaction between C. parasitica and chestnut at this level, but the family of LysM-domain proteins play important roles in pathogenic interactions (44, 104). Chitin is a major component in fungal cell walls results in production of chitinases by the plant host (18). Chitin can be recognized by LysM-containing receptor kinases or host chitin receptors (106, 135), which further act to initiate signaling pathways that lead to host defense responses to fungal pathogens (44, 104). With the completion of the C. parasitica genome sequence, we have identified five potential LysM effector genes and are analyzing their potential role in virulence. Virulence-attenuating viruses in strains of C. parasitica present intriguing possibilities for biological control. A contributing factor to the failure of the hypovirus-infected mycelium to transition to lipid metabolism might reflect a sequestration of these materials to support hypoviral replication (43), a process common to hypoviruses. If so, we would expect similar metabolic impairment associated with all hypovirus infections, but modulated by the degree of hypoviral genome accumulation. We established that a principle effect of hypoviruses on a laboratory fungal culture is a failure to properly transition into different metabolic pathways as the colony aged. Metabolomics measurements also showed that hypovirus-containing cultures accumulated different compounds associated with primary carbon metabolism, lipid metabolism and polyamines (43). An additional advance was development of a controlled expression system for C. parasitica that allows repression or induction of heterologous sequences depending on available copper ions (138). We are now developing a controlled expression tool that will allow us to initiate hypovirus infection from a genomic copy of the viral genome controlled by the regulatory sequence identified above. This provides a tool to examine changes that occur in the fungal host as the hypovirus begins to accumulate to the steady-state chronic infection normally observed. The linked processes of anastomosis and incompatibility likely provides for significant limitations to the spread of hypoviruses in natural populations (37, 112), and any method of biological control of the blight fungus using hypovirulence will benefit by understanding these mechanisms. NE-1033 also utilizes other fundamental approaches to chestnut improvement; selection and breeding is essential. Limited breeding programs were maintained prior to the discovery of hypovirulence, and CAES has trees of all species and most of the potential chestnut hybrids that are available to NE-1033 members (6). The back-cross breeding approach has been in place for nearly 20 years (66). Breeding efforts now are coordinated with numerous states through an expanding TACF chapter network and NE-1033. Breeders have sought to combine resistance to pathogens and pests with quality and stress tolerance through interspecific hybridization. However, many cultivars now being re-evaluated for nut production lack accurate pedigree records. Phenotypic characteristics cannot reliably reveal pedigree (66), and many investigators use genetic markers to examine diversity and differentiation in naturally occurring Castanea (70, 96, 113, 131). But genotyping cultivars routinely reveals multiple cases of synonymy and homonymy (26, 60, 96). The need for genetic fingerprinting is acute, as any given cultivar could have ancestors from any chestnut species, including American. In plants a set of 10-30k ESTs will typically contain several hundred or more EST-SSRs, most of which will be close to or embedded in functional genes (52). Indicative of variation in transcribed regions of the genome, they provide an estimate of functional diversity (53, 137) and a source of highly polymorphic SSR markers. As markers embedded in functional genes, EST-SSRs are more likely to be transferable across taxonomic boundaries than traditional gSSRs, significantly increasing their value for identification of interspecific hybrids, naturally occurring introgression and comparative studies of genome function, as recently demonstrated in oak and chestnut (48), ryegrass (129) and wheat (58). We evaluated the EST-SSRs detected in the ESTs developed for Chinese chestnut for amplifiability and informativeness in chestnut cultivars grown in the US and chose 11 EST-SSR. We have identified synonymies and homonymies among 214 entries representing 65 cultivars and 18 putative interspecific hybrids, and assessed the correspondence between the species or interspecific ancestry of record and the actual degree of genetic association. The goal is to develop a firm foundation for continued development of chestnut as a specialty crop in the US. The number of alleles per locus ranges from 8 to 30 (average 18.2). Megablast of the plant EST database at NCBI yielded hits to 10 of the 11 EST-SSR sequences. Nine of the 11 EST-SSR contigs contained sequence similar to genes of known function. Three aligned to annotated functional genes in multiple species, including transcription factors and protein kinases that regulate physiological responses in plants related to pathogen defense, senescence, meristem function and trichome development. We found extensive synonymies and homonymies among the cultivars and demonstrated that many presumed interspecific hybrids likely have ancestry different than that reported (99). Establishment of breeding programs focusing on sustainability and nut characteristics requires additional genetic fingerprinting of cultivars currently grown in the US. DNA analysis will clarify pedigrees, insure cultivar identity and permit rapid incorporation of insect and disease resistance. Our current focus is developing a simple test that will distinguish the plastid genotypes of the five Castanea species most likely in the cultivated gene pool and to identify a set of EST-SSR markers that will do the same for the nuclear genomes. The critical need to develop regionally adapted germplasm is progressing and includes collaborative efforts with Canadian scientists. Although the approach utilizes traditional breeding methods, molecular techniques are increasingly used to aid in selections. Advances in breeding 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. The breeding program provides an opportunity to blend traditional tree breeding with hypovirulence. Many hybrids express low-to-intermediate levels of resistance; they may not be killed as rapidly by blight, thereby supporting infections for longer periods and permitting greater opportunity for hypovirus acquisition. Combining the two approaches may improve chestnut survival and provide a greater chance for establishment of hypovirulence. An additional dimension to breeding is development of in vitro propagation to mass produce desirable clones or to regenerate somatic cells engineered with anti-fungal gene constructs (20, 30). Production efficiency has been enhanced (10, 21, 119) and the first transgenic chestnut trees have been planted. Repeatable transformation/regeneration systems are in place and research is focusing on screening candidate anti-fungal genes for their ability to confer resistance to C. parasitica and to further increase the efficiency of somatic seedling regeneration (88). With the success of traditional tree breeding and in vitro propagation, blight resistant material became available with a need for outplantings to evaluate growth, survivorship and reproduction (75). But knowledge of relevant contemporary silvicultural approaches was lacking. Historical documents and dendrochronological evaluations provided some insight into factors most relevant to restoration, but much remains unknown (98, 100, 127). Mortality of outplanted seedlings is high, with significant variation among nursery-grown individuals, suggesting a need to evaluate factors influencing survival. Understanding factors relating to seed quality, seedling genetics, seedling physiology, root architecture (84), planting sites, and seed and seedling predation (81, 118) are critical to future reforestation efforts (122). Appropriate nursery techniques for seedling production are a precursor to effective outplanting. Site selection is critical. In the central and southern Appalachians, knowledge of the epidemiology of P. cinnamomi and Ph. omnivore is essential (115, 139). Relationships between blight resistance and herbivore susceptibility are largely unknown (9, 81, 118). ACGW reduces tree vigor and nut production, and can cause tree mortality. After the initial discovery of the gall wasp in the USA, there were biological control releases of several parasitoids. Of these introduced parasites, only Torymus sinensis has been definitively recovered (31). However, eight additional native parasitoid species have been collected in association with the gall wasp; some may help regulate populations (117). Identification of the endemic species is progressing, but at least one endemic species, Ormyrus labotus, does help regulate gall wasp population but also has developed an antagonistic, hyperparastic relationship with the introduced biological control agent (34). Factors affecting gall wasp population dynamics (34), mechanisms associated with gall formation (32, 33, 35), natural enemy recruitment, and orchard management are under investigation. Some species of chestnut appear resistant, and preliminary results indicate that this resistance is simply inherited (9). The spread of ACGW in the eastern US provided an additional impetus for including C. ozarkensis in the CAES breeding program, both for production of blight resistant chinquapins and for use of the ACGW resistance. The granulate ambrosia beetle causes extensive chestnut mortality in young or small diameter trees, and the Asiatic oak weevil is causing significant concern. Management approaches in outplanted chestnut are being evaluated. Knowledge of how these pests might influence natural populations of chestnut sprouts, backcross trees from breeding programs, and trees grown for nut production is needed. This regional project has provided the background knowledge and impetus for active cooperation on major genomics projects. Large numbers of pedigree chestnut families are now available for genetic analysis (66, 86, 125). The relationships fostered by the project resulted in a funded NSF proposal entitled Genomic Tool Development for the Fagaceae (http://www.fagaceae.org). Similarly, a complementary proposal was funded by the Department of Energy's Joint Genome Institute (JGI) to sequence the genome of C. parasitica (www.jgi.doe.gov). A significant part of both projects involve NE-1033 members working together and with international collaborators. The intended outcome has been to identify genes responsible for resistance in chestnut and for virulence in the pathogen (133). There have been numerous other outcomes, including identification of genes responsible for the system of vic that restricts the transmission of hypoviruses between strains (90, 91). The project has developed and utilized molecular tools for highly efficient creation of strains in which specific genes are targeted for disruption to evaluate their functions in the biology of the fungus (25, 87, 130). Several hypoviruses have been sequenced and the function of a number of their genes identified (1, 46). Sophisticated molecular techniques have facilitated genetic engineering of hypoviruses with the potential of making them more effective modulators of fungal virulence. The fungus/hypovirus genomic data is allowing studies of how virus infection reduces sporulation and virulence and confirm the roles protease enzymes and vesicles play in expression of the hypovirulent phenotype (76, 77, 133). Several institutions have focused significant efforts on developing a US and Canadian chestnut industry. A portion of these efforts grew out of experiment station initiatives to consider minor high-value orchard crops that could be developed for the grower community. Test orchards to evaluate available cultivars and management approaches have been established. In Europe chestnut orchards have been treated with hypovirulent strains since before the 1980s (92). Similar to Europe, Michigan has naturally occurring hypovirulent strains on recovering chestnut trees where developing cankers do not kill the trees. Formation of the non-lethal cankers allows trees to survive blight (50, 103). Other types of hypovirulence have been found in Michigan, including a mitochondrial form (11, 12. 72, 93, 94, 95, 107). We have begun to use the same canker treatment protocols as the West Salem, Wisconsin chestnut stand on Michigan plot trees and farm orchard trees. This parallels the work in the American chestnut plots except we are targeting existing (natural) cankers and the trees consist mostly of hybrid European X Japanese hybrid germplasm. All of the trees will have been grafted, which means there are wounds created (although callused) and mixtures of germplasm (as grafts). This is an important study of using hypovirulence to manage blight in orchards. Some orchards already have a large number of vegetative compatibility types and these will be determined and dealt with based on past studies of vegetative compatibility in our laboratories (13, 41, 42, 54, 72). Many propagation and silvicultural problems exist including graft incompatibility, root stock varieties, efficient orchard designs and effective propagation systems (8, 124, 136). Many of these issues are common to breeding and reforestation goals. As grower interest in chestnut production has increased, so has the need for marketing strategies; most American consumers are unaware of the nutritional attributes of chestnut and the chestnut food products are available in other parts of the world (7, 61, 62, 63). Such efforts require coordination among NE-1033 researchers and with grower groups.

Objectives

1. Develop and evaluate blight resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome,
   
2. Evaluate biological approaches for controlling chestnut blight from the ecological to the molecular level by utilizing knowledge of the fungal and hypovirus genomes to investigate the mechanisms that regulate virulence and hypovirulence in C. parasitica, and
   
3. Investigate chestnut reestablishment in orchard and forest settings with special consideration of the current and historical knowledge of the species and its interaction with other pests and pathogens.
   

Methods

Objective 1. Develop and evaluate blight resistant chestnut trees for food and fiber through traditional and molecular techniques that incorporate knowledge of the chestnut genome. Traditional breeding programs that backcross Asian sources of blight resistance genes will continue. American chestnut trees that express some level of resistance also are being used in breeding programs in order to pyramid possible sources of resistance. To accomplish this, backcross breeding protocols will be utilized to incorporate resistance genes into genetic backgrounds that are adapted to specific regions in eastern North America. CAES breeding efforts are directed largely towards trees adapted to northern states and Canada, and TACF scientists are working with a network of regional coordinators and state chapters to maintain regional plantings that span from the northern to southern boundaries of the natural chestnut range. Current methods of screening for blight resistance involve laborious growing-season inoculations utilizing isolates of C. parasitica of known virulence after young trees have reached 5 years of age, or allowing natural infections to weed out susceptible trees. More rapid early screening methods will be sought including greenhouse inoculations. Progress with the genomic components of this project will result in rapid molecular screening that will be a useful tool for early identification of progeny that possess specific genes for resistance to blight and other pests and pathogens (CT, MI, MO, MS, ON, NC, PA, TN, VA). Molecular markers will be sought to identify species of Castanea and the percentage of each species in hybrids. These results will be available to those using genomics tools to identify critical regions of the genome associated with physical and physiological traits. Initial evaluation of nut cultivars will be for taste and nut size. Subsequent laboratory tests will establish nut nutritional characteristics for each cultivar and its paired pollinizer. Seed and pollen of C. ozarkensis are being provided to CAES by the Ozark Chinquapin Foundation (AR), the Cherokee Nation (OK), and the Nature Conservancy's Nickel Preserve (OK) to augment the CAES collection for breeding. Back-crossed hybrids of C. ozarkensis and biocontrol strains of C. parasitica will be sent to all cooperators in OK and AR (CT, AL, IN). To date, no strains of the blight fungus have been identified with levels of virulence that overcome the resistance expressed by the most blight resistant cultivars. However, strains differing in virulence have been reported. As an important ancillary component to the resistance breeding endeavor, strains of C. parasitica of known virulence will be crossed and the virulence of their offspring will be compared by inoculating them into moderately resistant trees. Further, as the fungal genomics component of this project advances, useful information on the genes that control virulence in C. parasitica will be generated, aiding in the evaluation of the risk new strains pose to the stability of resistance (MD, NY, VA). To further define the nature of resistance and as part of the tree genomics component of this project, thousands of cDNAs have been sequenced using RNAs generated from Chinese and American chestnut trees growing at research stations. A physical map of Chinese chestnut has been created using 10-20X coverage BAC libraries of genomic DNA. Genetic maps of Chinese and American chestnut will continue to be refined, with the goal of further understanding the genes for blight resistance. Genetic markers developed from both the BACs and cDNAs then will be used to align the genetic and physical maps (CT, NY, MS, NC, PA, SC, VA). Large numbers of desirable individuals will have to be propagated as part of an integrated approach to restore chestnut. Research teams will continue to initiate new embryogenic American chestnut cultures using protocols previously established under the umbrella of this project. Experiments will test variables including cold stratification, light quality and plant growth regulator treatments for their effects on somatic embryo and somatic seedling production. The feasibility of scaling up plant production using bioreactor-based approaches also will be explored. This research will facilitate transformations of the embryogenic cultures with vectors that carry anti-fungal candidate genes. Populations of somatic seedlings transformed with anti-fungal candidate genes, as well as empty-vector control and wild-type somatic seedlings, will be generated and screened for transgene expression and resistance to blight using small stem assays conducted in the greenhouse. This molecular approach to developing resistant individuals may provide trees that can be used to compliment the traditional breeding approach, thereby providing a more robust form of resistance to the chestnut blight fungus and perhaps to other organisms such as P. cinnamomi and ACGW. Lastly, this transformational system will be essential for confirming gene functions, such as those linked to resistance loci in Chinese chestnut (AL, GA, KY, NY, VA). Objective 2. Evaluate biological approaches for controlling chestnut blight from the ecological to the molecular level by utilizing knowledge of the fungal and hypovirus genomes to investigate the mechanisms that regulate virulence and hypovirulence in C. parasitica. Hypoviruses have naturally invaded populations of C. parasitica at numerous sites in Europe and Michigan resulting in remarkable levels of blight control. Unfortunately, attempts to duplicate this natural process in eastern North America have been less successful. Deployment studies will continue at sites within the native range of American chestnut and at sites in Michigan and Wisconsin, outside the natural range of the species. The goal is to understand the components of natural biological control that make it a successful phenomenon. To accomplish this, recovering and non-recovering populations will be censused annually after various treatments are applied. Hypovirulent inoculum production, the spread of inoculum, tree and stand response to hypovirus introduction and the contribution saprophytically-produced inoculum makes to the hypovirulent inoculum pool will be measured. Population growth projection matrices will be used to follow the course of disease and the spread of artificially introduced hypoviruses. Population level work is utilizing Lefkovitch size based transition matrices, evaluating stage-specific survival rates, to compare C. dentata populations in Michigan. These matrices are being used to project future size structure and growth rate of chestnut populations and evaluate the success of hypovirus introductions. In stands that are recovering from blight, certain trees recover and others do not. A genetic basis for this phenomenon is being investigated by comparing the genetic resistance of trees that support hypovirulent infections to those that do not. The structure of C. parasitica populations in Michigan are being determined for vic loci genotypes by utilizing the 64 vic genotype testers developed by Cortesi and Milgroom (36) and for overall genetic diversity using 10 microsatellite loci developed by Breuillin et al. (17). Transmission rates of hypoviruses used in field biocontrol studies into C. parasitica conidia is being evaluated for common vc types from Michigan and Wisconsin. NE-1033 collaborators at the University of Wisconsin La Crosse, Michigan State University and West Virginia University are initiating a new research effort investigating the dynamics of individual cankers. A cornerstone of this work will be the development of an individual based model (IBM) that will simulate the growth of the blight pathogen, reductions in virulence due to hypovirus invasion and competition with secondary invaders that may influence the growth of C. parasitica on the tree. Empirical work will be performed to parameterize the IBM and will also be used to test model predictions. Laboratory work will investigate competitive relationships among C. parasitica and various secondary invaders that are commonly isolated from blight cankers. Field studies will use experimentally initiated cankers to test the importance of spatio-temporal variables on the efficiency of hypovirus introductions and competition by secondary invaders. Fieldwork will be carried out in West Virginia (main range of C. dentata), West Salem, WI (site of a 20 yr experiment on hypovirus introductions) and Michigan (non-recovering and naturally recovering sites) to robustly evaluate the IBM. The potential for colonization and asexual inoculum production will be evaluated on dead and dying chestnut bark following artificial and natural infection by virulent and hypovirulent strains. Protocols used in European experimentation will serve as a guide for this experimentation. Replicate plots that contain American, Chinese, European and various Chinese X American backcross generations have been established in WV. They will be inoculated with virulent and hypovirulent strains of C. parasitica as well as subjected to natural infection. As cankers develop on the various species and hybrids they will be evaluated by measuring growth, sporulation and hypovirus infection, as well as survival and health of the trees (CT, MD, MI, ON, TN, WV). How the hypovirus CHV-1 replicates on trans-Golgi vesicle membranes of C. parasitica to perturb normal developmental processes is also under investigation. Purified vesicles will be analyzed for presence of individual proteins by standard proteomic methods (78), proteins identified and selected for further characterization and function analysis. Protein-protein interactions will also be identified that are important for virus-vesicle interactions and normal vesicle functioning. This information contributes to our understanding of the biology of virus-caused hypovirulence, and may lead to better deployment strategies of these viruses for biological control of C. parasitica. Molecular approaches are being utilized to more fully understand the difficulties of hypovirus transmission due to vegetative incompatibility and to characterize the genes involved. Specific objectives regarding the C. parasitica vic genes include 1) identifying the genes associated with the vic1 and vic3 loci to complete identification of genes associated with all six genetically defined C. parasitica vic loci; 2) testing for physical interactions between the two genes associated with the vic 6 locus (vic 6 and pix6); 3) providing vic allele specific PCR primers for population genetic studies related to hypovirulence to the chestnut research community; and 4) testing vic alleles for genetic linkage disequilibrium, balancing selection and trans-species polymorphism. The comparative genomics approach successfully used for identification of genes associated with vic2, vic4, vic6 and vic7 (24) will be employed to identify candidate genes associated with vic1 and vic3. Genome sequence data for C. parasitica vic geneotyping tester strains (36) EU-31 (1211-22), EU-60 (2221-22) and EU-55 (1221-22) will be generated at the University of Maryland Institute for Bioscience and Biotechnology Research DNA sequencing facility according to the Ilumina HiSeq 1000 protocols for generating 100-base paired-end reads (these strains differ from the reference strain EP155 (2211-22) at vic1, vic3 or both). The sequence reads will be mapped to the C. parasitica strain EP155 reference genome. Good linkage mapping data are available for vic1 to help the search for the sequence polymorphism expected at the vic1 locus. A general search for sequence polymorphic patterns observed for the other vic loci will be systematically conducted on the 10 scaffolds that contain 99% of the C. parasitica genome sequence to identify vic3 candidate genes. Candidate genes will be disrupted and tested for effects on the vegetative incompatibility reaction (barrage formation) and virus transmission as described in Choi et al. (24). Physical interactions between the vic6 and pix6 gene products, which were shown to interact genetically, will be performed by standard yeast two-hybrid and pull-down protocols using expressed protein. Allele-specific PCR primers will be prepared as described for the primers used to distinguish alleles for vic2, vic4, vic6 and vic7 in Choi et al. (24). Analysis for allelic linkage disequilibrium, balancing selection and trans-species polymorphism will require use of allele-specific primers for PCR genotyping of several hundred C. parasitica field isolates, sequence analysis of a sufficient number of the amplified alleles and identification and sequencing of vic gene homologs in C. radicalis, C. nitschkei, C. naterciae and C. macrospora. This will be accomplished by PCR amplification using degenerate primers or by re-sequencing of the other Cryphonectria species, if required. Gene knockouts will be made in the EP155 strain to evaluate fungal pathogenicity, hypovirulence, and vegetative compatibility. The deletion constructs prepared using the method of Colot et al. (29), in which the flanking regions were assembled with the Hygr marker using a yeast-based recombination system. Transformants will then be single-spored for nuclear homogeneity and verified by southern blot. Analysis of the secretion characteristics of the LysM proteins will be accomplished by fusing epitope tags to the gene sequences and verifying secretion by western blot. A conditionally-repressible hypovirus sequence will be cloned into the controlled expression vector available (138) and viral accumulation monitored by RNA visualization on an agarose gel. Epitope- and Green Fluorescent Protein-tagging of VIB-1 will be accomplished by standard molecular procedures. Transcriptome data will be prepared by using Illumina sequencing from RNA prepared across multiple samples to control for variation. Specific objectives addressing fungal pathogenicity include analyzing the potential role of LysM-containing proteins produced by C. parasitica as virulence factors, and identifying likely genes involved in pathogenicity from the available genome sequence. We intend to use a controlled expression system to manage the onset of hypoviral genome replication and explore the changes that occur in the fungal cells as the hypovirus accumulates, and explore correlations between physiological impact and the level of accumulation. We will identify which suite of determinants (vic genes) operate through the recently identified CpVIB-1 regulator by deleting this gene from different allelic backgrounds and determine the role of CpVIB-1 in initiating programmed cell death. Finally, we will identify CpVIB-1-interacting protein components using protein-interaction technology. Analysis of transcriptome data will also allow us to expand our activity model to include further downstream targets. The genomics component has added new dimensions to the project. Our understanding of fungal virulence mechanisms and utilization of hypoviruses as agents of biological control have increased considerably. NE-1033 project members, as well as international collaborators, are using sequence data for a variety of molecular studies, including protein analyses using two-dimensional electrophoresis to search for altered patterns between hypovirus-infected and uninfected mycelium, and using the genome information to identify the exact proteins involved. Further, engineered hypoviruses coupled with computer-predicted and experimentally-validated structural analyses will be used to determine which features of the viral genome are important for the maintenance of hypovirus infection (MD, CA, NJ, NM, NY). A further dimension to the study of the blight fungus involves a search for other effective viral or bacterial biological control agents. This requires detailed studies of the specific effects imparted by other organisms on C. parasitica and the mechanisms the fungus uses to defend itself. Particular attention is paid to genes identified through genomics methods such as microarrays and proteomic analyses that are responsive to biocontrol agent attack (NJ). Objective 3. Investigate chestnut reestablishment in orchard and forest settings with special consideration of the current and historical knowledge of the species and its interaction with other pests and pathogens. A first step in successful restoration is determining the historical context in which the restoration target initially thrived. Surveys of deeds and records are being examined to gain insights into site characteristics within the historical range of chestnut. Paleoethnobotany and dendrochronological techniques that examine high elevation bogs in the central Appalachians are being used to provide baseline data for selected restoration projects. Potential sites will be evaluated for competition, soil type, elevation and available light to each tree for field evaluation of experimental materials produced by TACF, ACCF and CAES. Analyses of plantings across various light environments will result in knowledge of minimum gap size and silvicultural techniques that favor chestnut establishment. Small-scale production of seedlings for research is done in state and forest nurseries. Standard nursery protocols will be used initially to learn when plants should be lifted and to evaluate their height, root collar diameter, stem taper and root architecture. Plantations have been established on southern National Forests and are evaluated annually through visual inspection, insect trapping, pathogen sampling, and measurements (27, 28). Impacts and management strategies targeting the Asiatic oak weevil, Cyrtepistomus castaneus, and granulate ambrosia beetle, Xylosandrus crassiusculus, are being evaluated. A variety of outplanting designs and spacings are used to allow for statistical analyses using mixed model methods and multiple regression or spatial analysis. Phytophthora cinnamomi, and more recently Phymatotrichopsis omnivore, cause root rots and are especially significant in the southern Appalachians. They are of particular concern to nurseries that produce chestnut seedlings. In anticipation of this issue, chestnut seed will be sown in nurseries of differing soil types that range from well-drained sandy loams to heavy, poorly drained clay soils. Nursery selections will be based on their fumigation histories, using nurseries that have not been fumigated as controls. The various treatment variables will be compared statistically with special emphasis placed on seedling survival and growth two or more years after outplanting (AL, MD, TN, VA). The spread of the exotic ACGW poses an additional threat to American chestnut restoration efforts and to nut production. Galling reduces tree vigor, prevents normal shoot development, flowering and nut production, and causes branch and tree mortality. Natural enemy recruitment is being assessed by evaluating the most frequently encountered adult parasitoids for their effectiveness as biological control agents against ACGW using the parameters of life cycle synchrony, natural enemy dispersal ability, and suppression of the target pest (65). Adult parasitoid collections are linked to larval parasitoids obtained through gall dissections by molecular characterization (34), since morphological characters have only limited use with respect to larval identification (64). PCR product from amplification of the ITS2 region is compared to known specimens (Cooper and Rieske, 2011) to link gall wasp mortality to specific natural enemies. Larvae of the introduced Torymus sinensis and the native Ormyrus labotus have been molecularly characterized; current efforts are focusing on Sycophila mellea (Fam. Eurytomidae) and Eupelmus vesicularis (Fam. Eupelmidae), both of which are encountered with some frequency. Once identified, characterizing the ecological associations of these species, and their effects as gall wasp population regulators, will be pursued (KY, OH, PA, TN, VA). To further develop the commercial nut industry, numerous chestnut cultivars being tested at different locations for their productivity and nut desirability. Resistance to C. parasitica and Phytophthora root rot, winter hardiness, graft compatibility, especially with dwarfing root stocks, and susceptibility to ACGW are traits being assessed for the most promising cultivars. Optimum orchard management practices, harvesting techniques and studies of the best conditions for nut storage will be investigated and the information generated will be shared with the grower community. Research will be conducted to foster the development of domestic chestnut markets including the creation of new chestnut products that capture high-value niche markets thereby contributing to the producers' economic viability (CT, MI, MO, TN, VA).

Measurement of Progress and Results

Outputs

• Development of regionally adapted, blight and ACGW resistant timber and orchard Castanea for timber, reforestation, and nuts.
• Evaluation of C. parasitica hypoviruses, both naturally occurring and introduced, including hypovirus selection, contribution of saprophytic growth and sporulation to inoculum production, effects, spread, and deployment for biological control of blight.
• Further evaluation of vegetative incompatibility in C. parasitica hypoviruses, including identification of genes associated with the six genetically defined vic loci and characterization of their physical interactions.
• Genome sequence reads for C. parasitica vic geneotyping tester strains EU-31, EU-60 and EU-55.
• Identification of LysM genes from C. parasitica, and an understanding of their role in virulence.
• A method for controlling the initiation of hypovirus replication.
• Characterization of the role of VIB-1 in triggering the programmed cell death response manifested during anastomosis of incompatible colonies.
• Development of guidelines and protocols for producing high quality seedlings, appropriate site selection and planting techniques, and management options for multiple diseases and insects of chestnut trees.
• Increased availability of improved chestnut cultivars for use as orchard trees and the development of chestnut as a US-produced product for the fresh market.

Outcomes or Projected Impacts

• Widespread use of regionally adapted Castanea in eastern N. America for reforestation, reclamation, conservation, and nuts using blight resistant germplasm.
• Improved tools for analyzing hypovirus accumulation and impact on the fungal host, and predicting its spread.
• Identification of genes associated with vic1 and vic3.
• Evidence for physical non-allelic interactions between vic genes to trigger PCD, opening opportunities to develop strategies for disruption of incompatible reactions.
• Use of allele-specific primers to obtain comprehensive vic genotypes for C. parasitica populations leading to more complete picture of the contribution of vic diversity to hypovirulence spread.
• Test of whether vic genes are subject to allelic linkage disequilibrium, balancing and positive selection and trans-species polymorphism leading to new insights into the generation, fixation and maintenance of fungal nonself recognition systems and the influence of hypoviruses on their origins and evolution.
• Characterization of population regulators of the ACGW, with opportunity for manipulation in biological control.
• Determination of ecological and silvicultural traits of timber and orchard chestnut trees based on experience with field plantings.
• Development of chestnut markets and marketable chestnut products.

Milestones

(2013): Screen F2 Canadian chestnut seedlings for blight resistance; Initiate development of dwarf chestnuts that may be of value to growers with limited space; Synthesize historical data to determine the most suitable sites for chestnut reestablishment; Identification of LysM genes from C. parasitica.

(2014): Evaluate preliminary results from field tests using embryogenic clones; Complete assessment of contribution of saprophytic growth of C. parasitica to hypovirulent inoculum production; Completion of the canker scale IBM that will investigate how hypoviruses and competing microorganisms cause recovery within a canker; Initial empirical tests of the IBM model; Completion of work characterizing the structure of Michigan C. parasitica populations for vic genotype and microsatellite diversity; Identify fungal genes responsible for differences in C. parasitica virulence levels through genetic and genomic analyses; Identify C. parasitica genes required for hypovirus replication and symptom expression through genetic and genomic analyses; Identification of genes associated with vic1 and vic3; Generation of knockout strains lacking each LysM gene; Establish guidelines for P. cinnamomi control in southern forest nurseries; Finalize assessment of ACGW population regulators.

(2015): Mutational characterization of the candidate genes associated with vic1 and vic3; Completion of two-hybrid and pull-down interaction studies for vic6 and pix6; Generation of a complete set of allele-specific PCR primers for all vic loci and distribution to interested researchers; Five-year evaluation of hypovirus introductions into C. parasitica populations at three non-recovering chestnut sites; Scaling up IBM to evaluate tree growth, survival and reproduction as well the population dynamics of recovering and non-recovering.

(2016): Completion of survey of vic-genotypes for C. parasitica field isolate populations; Construction of a controlled hypovirus expression system; Tagging of VIB-1 for studies of localization and interacting components; Generation of transcriptional data to determine downstream targets of VIB-1; Identification of VIB-1 interacting proteins; Empirical evaluation of the IBM in West Virginia, West Salem, WI and Michigan with regard to the IBM's predictions of conditions that promote chestnut recovery due to hypovirus invasion and competition by secondarily invading microorganisms.

(2017): Completion of field experiments in West Virginia, Wisconsin and Michigan that will provide parameter estimates of the IBM; Identification and sequence analysis of vic gene homologs in other Cryphonectria species; Sequence analysis of a sufficient number of vic alleles to be able to test for balancing and positive selection.

(2018): Initial deployment of hypovirus and other microorganisms based on predictions from the IBM.

Projected Participation

View Appendix E: Participation

Outreach Plan

The annual meeting of NE-1033 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-1033 web site. NE-1033 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. Reagents, including vic allele-specific PCR primers, are made fully available to the research community upon request and through collaborations especially with NE1033 participants. 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-1033 members will be updated annually and posted on the official NE-1033 website. The NE-1033 website has links to websites of some participating members (http://nimss.umd.edu/homepages/home.cfm?trackID= 3754). Additionally, the chestnut server at New Mexico State University collates information relating to the meeting and activities of NE-1033 research project (http://chestnut.nmsu.edu/index.html).

Organization/Governance

The organization of the regional research project 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 Chair-elect, whose duties begin the following year as Chair.

Literature Cited

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Attachments

Land Grant Participating States/Institutions

AL, CA, CT, KY, MI, MO, MS, NJ, NM, PA, SC, TN, WV

Non Land Grant Participating States/Institutions

American Chestnut Foundation, New York - Syracuse University, Univeristy of Notre Dame, University of Georgia, University of Wisconsin - La Crosse