Duration: 10/01/2008 to 09/30/2013

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The NE-1015 project had its beginning in 1982 as NE-140, a regional project with five collaborating experiment stations. The initial impetus for the regional project came with the discovery of the phenomenon of hypovirulence and the level of blight control that it brought to areas of the world once decimated by the disease. This discovery rekindled interest in chestnut blight, a disease that had unparalleled ecological and economic impact to eastern North American forests during the early part of the 20th century. Understanding the biology of hypovirulence and how to exploit it successfully, however, has been a complex issue, and initiating biological control artificially using hypovirus-infected strains has not been widely successful in the eastern U.S. Yet, in areas of Michigan and Italy, hypovirulence remains the only plausible explanation for the recovery of the significant stands of American and European chestnuts from blight. The challenge of utilizing hypovirulence still drives much of the research associated with this project. While research on the deployment of hypoviruses continues, two new dimensions have emerged that require research attention. Molecular technologies have provided the mechanisms to develop genetically engineered strains of Cryphonectria parasitica that increase the probability of hypovirus spread by enhancing the production of hypovirus-laden spores. Field tests are required to compare these seemingly improved strains to those that are infected naturally by hypoviruses. Another consideration relates to the enhanced resistance of chestnut provided as part of the breeding initiative. Incremental increases in resistance expressed by backcross generations of trees, combined with diminished virulence provided by hypovirulent strains may result in a successful integrated approach to blight control.

Today, NE-1015 embraces research in several fundamental areas. The first is the selection and breeding of blight resistant trees for forest and orchard settings. Although the approach utilizes traditional breeding methods, one goal is to incorporate molecular techniques to aid in the selection of desirable trees. Advances in the breeding effort over the past decade have provided genetic material needed to accomplish much of the genomic work that should prove instrumental in identifying the genes that impart resistance to blight and other organisms that threaten chestnut. Fortunately, the breeding efforts have expanded and now are collaborative with numerous state programs and efforts in the province of Ontario. Another noteworthy breeding-related issue has been the emergence of the Asian chestnut gall wasp (ACGW) in the early 1970s. Since the first reports of this exotic pest, the insect has been identified in a significant portion of the natural range of the American chestnut in the southern Appalachians. Although some varieties of Chinese and Japanese chestnut appear resistant, knowledge of how this pest might influence natural populations of chestnut sprouts and backcross trees from The American Chestnut Foundation's (TACF) breeding program is required. For the central and southern Appalachians, root rot cause by Phytophthora cinnamomi also poses a significant threat, if not to nursery grown seedlings then to outplanted stock. This disease, therefore, also must be considered as the breeding program advances.

A critical need that has emerged as a result of the progress with breeding is the generation of large numbers of the most desirable blight resistant chestnut genotypes, which will be required for performance testing and general research needs. Several project members are investigating two propagation systems, somatic embryogenesis and an embryo germination/ micropropagation system. 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 oriental 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.

The powerful molecular tools currently available to biologists have opened a floodgate of opportunities to this project that did not exist in its formative years. The ultimate 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. This involves delineating the genetic defense mechanisms necessary for the American chestnut to resist infection. Understanding and exploiting plant resistance is a powerful tool for controlling insects and pathogens. Therefore, knowledge of genes that regulate resistance to blight, as well as pathogens and insects such as P. cinnamomi and the ACGW, is critical to the development of chestnuts if they are to be successful forest trees. Identification of genes that confer resistance will require molecular comparisons of Chinese, Japanese and European chestnuts and the hybrids between them that have been developed from the breeding program. An equally important outcome of the genomics work is the investigation of the fungus genome to learn what factors allow C. parasitica to be such a virulent pathogen of chestnut. 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. Genomics research also provides the tools to investigate thoroughly a system of vegetative incompatibility (vic) that regulates hypovirus transmission among strains of C. parasitica thereby restricting their effectiveness as biological control agents. Likewise, 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 the development of molecular strategies that will enhance the effects hypoviruses have on the strains they infect, thereby reducing their virulence, a step potentially beneficial to biological 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.

As this project has evolved, it would be shortsighted not to focus on the silvicultural aspects of chestnut restoration. The reintroduction of blight-resistant American chestnuts into eastern North American forests is one of the most anticipated events in natural science by the general public. This groundswell of interest, in part been generated by TACF, is unique for a forest species and confirms the public's interest in restoration. 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 eastern forest ecosystems is paramount to the success of any restoration effort. Much of this effort will begin with sound nursery practices designed to produce large numbers of healthy seedlings.

When NE-140 was first conceived, an edible sweet chestnut industry was almost non existent. Since then, several project participants have made significant progress in creating a fledgling horticultural chestnut industry and consumer marketplace in their respective states. Chestnut is a temperate tree nut more closely resembling a fresh fruit than a nut as it does not have a hard shell, shows rapid respiration after harvest, and can mold during storage. The nut is low in fat but high in nutritional benefits. Because chestnut is new to most Americans palates, marketing must be emphasized. Various new chestnut food products are beginning to occupy high-value niche markets, a movement that further encourages grower interest and involvement. This expanding horticultural industry requires regional testing of old and new cultivars for their productivity, food quality and regional adaptability. Specific knowledge of root stocks, graft compatibility and propagation systems, being developed as part of the micropropagation portion of this project, may prove to be an invaluable synergism to our silvicultural efforts. The knowledge of chestnut gained and shared among scientists who have participated in this project has created a nascent North American chestnut industry.

Importance of the Work: The history of this project is testament to its value. When it was initiated, there was limited hope for the American chestnut. Since then, great progress has been made on applied fronts directed at restoration of the species, and on basic fronts that are applicable to this and other pathosystems. Issues associated with blight and other pests of chestnut are complex and certainly cannot be solved in the short period this project has been in existence. Yet, this multi-state project must be considered a huge technical success, as remarkable progress has been made toward a detailed understanding of the issues and approaches that are necessary to effect solutions. As findings and technologies continue to unfold, they will aid in the identification of critical issues and enable the research to move forward.

Technical Feasibility of the Research: Researchers who have participated in NE-1015 projects have made significant contributions 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, it became evident that the rapidly expanding field of molecular biology would contribute technologies to answer many of the fundamental questions posed by the chestnut blight dilemma. Essentially, the 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 has brought a new dimension to the multi-state project. The progress by research collaborators on the NE-1015 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 panel of natural biocontrol agents of that pathogen have been or are close to being characterized at the level of primary sequence. To date, more than 10 complete sequences of biocontrol-associated viruses have been determined and used to examine their role in suppression of the chestnut blight fungus; the genome sequencing of the fungus is close to completion and sequencing of the American chestnut and its blight-resistant Chinese chestnut counterpart are well underway. None of these efforts would have been possible by independent research groups alone. The spin-off potential of these analyses is already beginning to be appreciated. Certainly the identification of genes involved in the expression of disease resistance by chestnut will be a remarkably powerful tool in the development of blight resistant trees. Knowledge of the genetic make-up of C. parasitica will provide insight into the genetic mechanisms the fungus uses 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 will aid in our 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. This regional project continues to 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: With the increase in research in recent years came the realization that the components of chestnut blight were complex and required the concerted efforts of scientists from numerous disciplines. NE-1015 has been highly successful in fostering collaborative work to examine the many facets of research necessary to address this complex biological issue. Not only has it involved scientists associated with the land-grant system but also has attracted scientists from numerous other academic institutions. 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. The formation of the regional project can be credited with renewing interest in the American chestnut and in part is responsible for the emergence of TACF, a non-profit organization that now spearheads the breeding efforts to develop blight resistant trees.

Projected Impacts: The overall impact of the NE-1015 project will be to further progress toward restoration of American chestnut as a tree in North American forests and to support the utilization of chestnut as a nut tree for the American marketplace. The notable stature of chestnut in the history of this nation is made ever more evident by the existence of member-funded organizations like TACF, the Canadian Chestnut Council and the American Chestnut Cooperator's Foundation. These are organizations that 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 project. Since the last iteration of this project, the US Office of Surface Mining has shown significant interest in utilizing chestnut as a species for mine site reclamation. Likewise, the National Wild Turkey Federation, with a membership of over 500,000, has embraced the restoration issue by their official partnership with TACF. These and other stakeholder groups are interested in any means to restore this once important species.

While victory over the blight certainly will not be declared by the end of this or future revisions of this project, the progress that has been made is significant. Steps outlined in this project will bring us closer to the restoration goal. One of the most significant undertakings is the development of blight resistant chestnuts that are well adapted to a variety of eastern forest environments extending from Canada to the Gulf States. While progress with the traditional breeding efforts has been remarkable, the obstacles are many. This undertaking will be advanced by the addition of the genomic component to the project which will result in a genetic map for chestnut (www.fagaceae.org). This genomic approach should lead to the identification of resistance genes and technology facilitating the rapid screening of chestnut progeny that possess genes imparting resistance to blight and other pests and pathogens. In this regard, two invasive organisms, the ACGW and P. cinnamomi, will receive special project attention.

The need to produce large numbers of chestnuts will not only require the establishment of seed orchards but also the exploitation of technologies that utilize novel regeneration systems to produce large populations 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 to one another.

Analysis of the fungal genome will add significantly to the project. Fundamental studies that clarify the genetic basis for pathogenesis by C. parasitica will help determine why the species is such an efficient pathogen of American chestnut but not of the Oriental species. 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 invasion by this pathogen. Likewise, the system of vegetative compatibility will be tied closely, for the first time, to particular genes that regulate anastomosis between strains. The mapping of specific vic genes is necessary to understand how compatibility restricts the transfer of debilitating hypoviruses from strain-to-strain.

The biological implications of hypovirus infection provide a variety of continuing fundamental and applied research opportunities as part of this project. The fungal and hypovius genome projects will 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 exploiting them as biocontrol agents. Such knowledge also raises the possibility of genetically altering specific processes in the fungus tied to hypovirus infection thereby making hypoviruses more effective biocontrol agents. Despite numerous forest settings where hypoviruses have naturally contributed to biological control, the success of artificial hypovirus release has not been immediately apparent. Understanding the components of natural hypovirus spread is the intent of studies that are underway or planned in an effort to develop protocols to better establish hypovirulence as a biological control. Utilizing strains that are transgenic and transmit their hypoviruses with greater efficiency to both sexual and asexual spores is one method that will be compared to the spread of strains that are not genetically altered but are hypovirus infected. Another dimension of the research with hypoviruses that will continue is their use in conjunction with the breeding program. The test is whether trees produced by the breeding program that are only moderately resistant to blight will support infections by hypovirulent strains allowing them grow competitively in forest settings.

While many groups are poised to undertake large-scale plantings of blight resistant chestnuts, any release requires that numerous issues be evaluated given the species has never been the focus of contemporary silviculture research. Even if systems to produce large numbers of trees were in place today, locations of ideal sites where plantings will be most successful are not clearly defined. A new dimension of the project is to determine from historical records where chestnut once grew. Sites that support chestnut today may only be sites where the species has survived and not where it once thrived and thus not the best choice for reestablishment.

An important continuing dimension of the NE-1015 project is with nut production. The US chestnut industry requires that suitable cultivars be regionally tested. Systems of orchard culture and management including disease and insect controls also require evaluation. Assistance with the development of marketable chestnut products also is essential. While many problems associated with successful nut production are unique, many are common to forests and orchards alike.

The overall impact of this project will be to further the progress being made toward the 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 for generating larger number of backcross generations for forest and orchard testing of pest resistance and regional adaptability;
  
• Evaluation of genomic data of Castanea 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 from breeding programs and genotypes engineered with anti-fungal genes can be clonally propagated for reforestation;
  
• Evaluation of the chestnut blight fungus genome to further our understanding of the genetic basis for pathogenesis and hypovirus regulation;
  
• Development and deployment of the first genetically engineered virus for enhanced biocontrol of a plant pathogen;
  
• Utilization of biological control agents to reduce the impact of chestnut blight and other pests and pathogens; and,
  
• In the longer-term, the project will lead to the return of an important timber species, major mast species for wildlife, a new cellulosic biomass energy crop, a new commercial nut crop; and, a new 'green' alternative to pressure-treated lumber for durable wood and outdoor uses.

Related, Current and Previous Work

The early 20th century failure to control chestnut blight by a variety of eradication treatment methods led to the search for resistance and the initiation of several breeding programs in the early 1920s. By the early 1960s, most breeding programs had been abandoned as these early efforts were fraught with problems and it appeared most efforts would be unsuccessful (5). The discovery of the phenomenon of hypovirulence resulted in a reawakening of the scientific community's interest in chestnut and the reexamination of the previous breeding programs (31). Fortunately, limited programs had been maintained at The Connecticut Agricultural Experiment Station and the University of Tennessee. However, it wasn't until TACF was formed that the breeding approach was reassessed and the principles of contemporary genetics considered. Research utilizing the back-cross breeding approach was instituted by TACF and has now been in place for over 15 years with promising results (17). Breeding efforts now are coordinated with numerous states through an expanding TACF chapter network. Thus, the critical need to develop regionally adapted germplasm is underway and includes collaborative efforts with scientists in Ontario.

The development of blight resistant material brings with it the need to establish outplantings to test growth, survivorship and reproduction of this material (18). The challenge is to develop the techniques needed for restoration of a species for which there is almost no contemporary silvicultural information. Some historical documents and dendrochronological evaluations provide some insight into factors most relevant to the restoration effort, but much is unknown (32, 33, 45). Recent experimentation has shown that mortality of outplanted seedlings can be as high as 60%. Significant variation among nursery-grown individuals has been demonstrated, suggesting the need to better understand how root architecture relates to survival (23). Understanding factors related to seed quality, seedling genetics, appropriate planting sites, and the problems associated with animal predation are all critical to establishing test plantings or for future reforestation efforts (41). Development of appropriate nursery techniques for growing seedlings is a precursor to effective outplanting strategies. Certainly the selection of suitable forest sites will require carefully executed field experimentation. Additionally, P. cinnamomi is a documented threat on certain sites in the central and southern Appalachians. Knowledge of the epidemiology of this root pathogen in both nursery and field settings is required for its successful management (36, 50).

Although initial research with the ACGW has detected no differences in foliar chemistry or herbivore performance, herbivory differed among parents and backcross groups but relationships of these traits to blight resistance is largely unknown (38). Likewise, investigation of the community of parasites and predators with biological control potential for ACGW have been surveyed but their role now must be defined (10, 21, 37).

Another ancillary dimension to the breeding research is the development of in vitro propagation technologies to mass produce clones of desirable trees or to regenerate somatic cells that have been engineered with anti-fungal gene constructs (7, 9). Production efficiency has been enhanced only recently to the point where hundreds of somatic seedlings can be produced (4, 6, 39). Likewise, the first transgenic chestnut tree now has been produced. Now that repeatable transformation/regeneration systems are in place, research will focus on screening candidate anti-fungal genes for their ability to confer resistance to C. parasitica and to further increase the efficiency of the system of somatic seedling regeneration (27).

Studies of hypoviruses that have invaded C. parasitica populations at some Michigan sites provides a unique opportunity to understand their natural spread in a North American setting (28, 30, 35). On sites that have been studied, tree growth and reproduction has improved with each successive year as hypoviruses continue to spread in the fungal population (11). Related studies of disease progress and the spread of an artificially introduced hypovirus are ongoing in a Wisconsin stand of American chestnut growing outside the native range of the species (34). Settings in both states provide opportunities to study how ecological factors can be manipulated to enhance the effects of hypoviruses as biological control agents. At other locations, study of the spread of introduced transgenic hypovirulent strains adds a further dimension to understanding hypovirulence and whether using genetically modified strains of C. parasitica can enhance this system of biological control (40).

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 (17, 24, 44). The relationships fostered by the project have resulted in a funded NSF proposal entitled "Genomic Tool Development for the Fagaceae" (http://www.fagaceae.org/ outreach). This 4-year project will advance the understanding of the genes in chestnut that regulate many host functions including resistance to blight and to other insects and pathogens that threaten the species. 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/sequencing/why/CSP2007/chestnutblight.html). A significant part of both projects involve NE-1015 members working together and with international collaborators. The intended outcome is to identify genes responsible for resistance in chestnut and genes that regulate virulence in the pathogen (48). There are, however, numerous other potential results that are equally important. They include identification of the genes responsible for the system of vic that restricts the transmission of hypoviruses between strains thereby reducing their effectiveness as biological control agents (25, 29, 30). Preliminary analyses of vic genes have laid the groundwork that will be advanced by knowledge of the fungal genome. Insights into the mechanisms by which C. parasitica causes disease in chestnut also are now possible. The project has the molecular tools for the highly efficient creation of strains in which specific genes are targeted for disruption to evaluate their functions in the biology of the fungus (8, 26, 46). Similarly, several hypoviruses have been sequenced and the function of a number of their genes identified (1, 12). As a result, sophisticated molecular techniques have facilitated the genetic engineering of hypoviruses with the potential of making them more effective modulators of fungal virulence. As sequence data of the fungal genome become available, a more complete understanding of the processes by which viral genes alter the phenotype of their fungal host should follow (13, 42). The fungus/hypovirus genomic data will allow studies of how virus infection reduces sporulation and virulence and confirm the roles protease enzymes and vesicles play in expression of the hypovirulent phenotype (19, 20, 47).

Several institutions have focused a portion of their efforts on the development of a US and Canadian chestnut industry. In some cases, these efforts grew out of experiment station initiatives to consider minor high-value orchard crops that could be developed for the grower community. As a result, test orchards to evaluate available cultivars and management practices have been established. Many silvicultural problems exist including issues of graft compatibility, root stock varieties, efficient orchard designs and effective propagation systems (3, 43, 49). Many of these same issues are common to the breeding and reforestation goals of TACF. 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 many chestnut food products that are available in other parts of the world (2, 14, 15, 16). Such efforts require coordination among NE-1015 researchers as well as with grower groups such as the Chestnut Growers of North America, the Northern Nut Growers and the Chestnut Growers' Association of America.

Objectives

1. To develop and evaluate blight resistant chestnut trees for food and fiber through traditional and molecular techniques that incorporate knowledge of the chestnut genome
   
2. To 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 <i>C. parasitica</i>
   
3. To 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- To 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 oriental sources of resistance genes will continue in the US and Canada. Additionally, 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. TACF scientists 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 will coordinate much of the regional adaptability research. 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 3-4 years of age. More rapid early screening methods will be sought including greenhouse inoculations. Progress with the genomic components of this project will result in a rapid molecular screen 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).

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 that have been developed. 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 will be sequenced using RNAs generated from Chinese and American chestnut trees growing at research stations. A physical map of Chinese chestnut will be created using 10-20X coverage BAC libraries of genomic DNA. Genetic map of Chinese and American chestnut will be generated using the progeny of crosses made as part of the breeding programs with the goal of locating the genes for blight resistance in Chinese. 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).

As part of an integrated approach to restore chestnut, large numbers of desirable individuals will have to be propagated. Research teams, therefore, 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 like P. cinnamomi and ACGW. Lastly, as the chestnut genome is mapped, 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- To 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.

The fungal genomics component of this project brings a new dimension to this research which will aid our understanding of fungal virulence mechanisms and utilization of hypoviruses as agents of biological control. To date, genomic DNA and total RNA from C. parasitica have been provided to the JGI. The JGI team has generated approximately 400Mb of raw sequence reads of the fungal genomic DNA through a shotgun sequencing approach yielding 8-10X coverage and provided 20,000 express tag sequences of C. parasitica mRNAs. They have assembled the genome DNA sequence and are constructing a machine-annotated draft copy to aid in identifying potential genes. Members of this project, as well as international collaborators, will then use a JGI-provided web-browser to manually annotate the draft assembly before its public release six months later. This sequence data opens the doors to a wide variety of molecular studies including protein analyses using two-dimensional electrophoresis to search for altered patterns between hypovirus-infected and uninfected mycelium 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 (CA, NJ, NM, NY).

The availability of the genome sequence of the fungus will allow project participants to progress on several other fronts. The role of secondary metabolites produced by C. parasitica during the invasion process can be investigated by searching for genes predicted to be involved in their synthesis. C. parasitica homologues of pathways with known virulence functions in other fungi can be identified as candidates for immediate functional analysis studies utilizing targeted gene disruption. The ultimate goal is to disrupt the key biosynthetic genes for all secondary metabolite pathways identified in the genome so that their function can be identified. Secondary metabolite gene clusters usually contain genes for toxin efflux to the host, as well as genes to protect the fungus from its own toxins and those from the host and other organisms. This information could be a valuable component to devising novel approaches to protect American chestnut from fungal-produced toxins. Another important component is to understand which host factors interact specifically with proteins produced by the infecting hypovirus. Recent evidence suggests that the interaction of hypovirus with membranes of trans-Golgi vesicles in the fungus is important in causing key aspects of the hypovirulence symptoms associated with infection. The reductions in sporulation and virulence that accompany infection are of particular importance. Published methods of vesicle fractionation will be used along with purification, cloning and antibody production to evaluate the interaction of hypovirus and fungal proteins. A key component that appears to be correlated with the success of hypovirulence in forest settings is a vic system that regulates hypovirus transmission via hyphal fusion between strains. This recognition system is controlled by multiple genes that now can be examined via genomic analyses. This research will require cloning and characterizing vic genes, a process that will be dramatically enhanced by using the genome sequence data generated for C. parasitica. Once this information is in hand, the role vic genes play in programmed cell death during incompatible reactions can be studied. Acquiring this information is key to knowing how hypoviruses suppress or regulate vic relationships and whether the system can be altered to favor hypovirus transmission (CA, MD, MS, NC, 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 will be paid to genes identified through genomics methods such as microarrays and proteomic analyses that are responsive to biocontrol agent attack (NJ).

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. In stands that are recovering from blight, certain trees recover and others do not. A genetic basis for this phenomenon will be sought by comparing the genetic resistance of trees that support hypovirulent infections to those that do not. The long-term goal of the field studies is to identify the best strains to use, as well as the optimal time and method to deploy hypoviruses to maximize their effectiveness as biological control agents. A further dimension of this research will be to continue the evaluation of transgenic strains as agents of biological control. These strains are capable of transmitting their hypoviruses in high numbers to both sexual and asexual spores. By treating infections and evaluating the hypovirus infection status of the treated cankers and the amount of hypovirulent inoculum produced, the effectiveness of transgenic strains will be determined (CT, MD, MI, ON, TN, WV).

Objective 3- To 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.

One of the most important aspects of any restoration project, orchard or forest plantings, is to determine the historical conditions of the site. Surveys of deeds and records will be used to gain insight to the historical range of chestnut especially on sites where sprouting no longer occurs. Paleoethnobotany and dendrochronological techniques that examine high elevation bogs in the central Appalachians will be 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. 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 will be 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. A variety of outplanting designs and spacings will be used that allow for statistical analyses using mixed model methods and multiple regression or spatial analysis. Phytophthora root rot infections is an especially significant problem in the southern Appalachians and will be 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 introduction and establishment of populations of the ACGW poses an additional threat to American chestnut restoration efforts. This pest is being investigated on several fronts including the mechanisms responsible for resistance to ACGW with specific consideration of the role jasmonic acid plays in stimulating tannin production which may be associated with resistance. Additionally, community associations with other insect parasites, particularly parasitoid larvae will be assessed with special attention to those species and ecological conditions that result in mortality of ACGW (KY, TN, VA).

To further development of the commercial nut industry, numerous chestnut cultivars will be tested at different locations for their productivity and the desirability of the nuts they produce. Additionally, resistance to C. parasitica and Phytophthora root rot, winter hardiness, graft compatibility, especially with dwarfing root stocks and susceptibility to ACGW are traits that will be 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 blight-resistant trees that are adapted to multiple regions of eastern North America
• Preparation and integration of detailed genetic maps of Chinese and American chestnut
• Selection of hypoviruses and deployment methods best suited for biological control
• An integrated global view of the influence of different mycoviruses and their encoded gene products on <i>C. parasitica</i> gene expression, pathogenicity and fitness
• Completion of the DNA sequence of <i>C. parasitica</i> and an annotated web browser-based genome assembly made available to the research community
• Annotation of the <i>C. parasitica</i> genome, with attention to genes identified by microarrays and proteomic analyses as being responsive to hypovirus attack
• Evaluation of how <i>C. parasitica</i> defends itself against viral and bacterial pathogens
• Increased knowledge of the genetic potential for toxin syntheses by <i>C. parasitica</i> and the roles secondary metabolites play in disease, fungal development and chemical self-defense
• Identification of fungal factors that interact specifically with viral proteins resulting in symptoms associated with the expression of hypovirulence
• Guidelines for chestnut seedling quality, planting environment and genetic superiority
• Determination of ecological and silvicultural traits of chestnuts based on surveys of historical records, pollen analyses and dendrochronology experience with field plantings
• Increased options for controlling multiple pests and diseases of chestnut trees
• Delineation of the range and impact of ACGW to American chestnut in eastern North America
• Assessment of the impact of ACGW and characterization of the North American insect community associated with its biological control
• Identification of protocols to produce chestnut seedlings in forest nurseries to minimize the effects of <i>Phytophthora</i> root rot or other pests or pathogens
• Increase the availability of improved chestnut cultivars for use in agricultural production as orchard trees for nuts and the development of chestnut as a new, US-produced product for the fresh market to replace imported nuts
• Development of dwarf chestnut trees that may be of value to growers with limited space
• Improvement in grafting efficiency between newly registered trees and known cultivars
• Evaluation of combined action of increased blight resistance and hypovirulence
• Assessment of outplanting sites most conducive to chestnut restoration
• Evaluation of the contribution saprophytic growth and sporulation provide to hypovirulent inoculum production
• Continued assessment of hypovirus deployment of cytoplasmic and transgenic strains on disease remission and biologicall control
• Assessment of the community of organisms responsible for ACGW mortality
• Tests to evaluate the cultural and chemical controls for <i>P. cinnamomi</i>-free nursery stock

Outcomes or Projected Impacts

• Utilization of American chestnut by the US Office of Surface Mining for mine site reclamation
• Reforestation of eastern N. American forests with blight resistant American chesntut, in conjunction with the National Wild Turkey Federation
• A genetic map for chestnut, a result of the NSF Fagaceae project
• Identification of resistance genes in <i>C. parasitica</i>, a result of the JGI project
• Regeration systems that will allow the incorporation of antifungal genes that may impart resistance or tolerance to <i>C. parasitica</i>
• New chestnut cultivars that may increase regional production
• Development of marketable chestnut products.

Milestones

(2009): <ul><li>Complete the genetic map of Chinese and American chestnut <li>Complete the genetic analysis of regions near chromosome-specific markers <li>Collect tissue from chestnut families segregating for resistance to the blight <li>Refine the genetic linkage and genome sequence maps for map-based cloning of fungal vic and pathogenicity genes <li>Complete assembly and community manual annotation of the <i>C. parasitica</i> genome sequence <li>Use the <i>C. parasitica</i> genome sequence to develop new microarray chip and proteomics platforms for analysis of global gene expression in the blight fungus when challenged by viral pathogens <li>Identify secondary structural motifs within the hypovirus genome <li>Complete anatomical studies of Chinese chestnut stems and determine the relationship between protophloem fibers and grafting failure <li>Identify the major components of chestnut volatiles <li>Determine specific genetic fingerprints of tested cultivars through the use of microsatellite markers <li>Develop direct root cutting methodologies <li>Establish cultures of new embryogenic clones of known parentage <li>Regenerate multiple transclones with 3 to 6 first-generation anti-fungal gene constructs <li>Complete delineation of range and impact of ACGW</ul>

(2010): <ul><li>Delineate the region of the Chinese chestnut genome that is responsible for resistance to the blight pathogen <li>Identify common secondary structural motifs shared with other hypoviruses <li>Identify proteins altered in the fungus in response to infection with hypovirus <li>Complete generation of robust genetic linkage map for <i>C. parasitica</i> based on genomic sequence <li>Complete characterization of <i>C. parasitica</i> antiviral RNA silencing pathways <li>Complete five-year summary of early performance of 'Eaton' and 'Auburn Super' scions grafted onto 'Little Giant' interstem and 'Cropper' seedling rootstock <li>Elucidate the genetic and physical causes of graft union failure in newly budded trees <li>Finalize nitrogen recommendations for chestnut orchards <li>Determine the major components of chestnut volatiles as potential attractants for adult chestnut weevils <li>Develop DNA marker technology <li>Engineer altered secondary structures and assess impact on hypovirus viability and transmission <li>Test resistance of embryogenic clones in greenhouse <li>Establish field tests using embryogenic clones <li>Complete assessment of contribution of saprophytic growth of <i>C. parasitica</i> to hypovirulent inoculum production</ul>

(2011): <ul><li>Commplete assessment of gains provided by release of transgenic strains of <i>C. parasitica</i> in the field <li>Complete five-year summary of early performance of 'Eaton' and 'Auburn Super' scions grafted onto three dwarfing rootstocks <li>Determine the genetic and physical causes of delayed incompatibility on bearing trees <li>Determine the effects of different pruning methods on nut yield and quality <li>Determine the attractiveness of chestnut volatiles to adult chestnut weevil <li>Make F2 crosses with Canadian chestnut and collect and store fruit <li>Sequence regions of the Chinese chestnut genome responsible for resistance to the blight pathogen and identify candidate resistance genes <li>Identify, clone and disrupt <i>C. parasitica</i> vic genes <li>Complete identification of all <i>C. parasitica</i> secreted proteins and plant cell degrading enzymes and hypovirus-mediated modulations</ul>

(2012): <ul><li>Plant F2 Canadian chestnut seedlings <li>Initiate functional tests by genetic transformation of candidate genes for resistance to the blight pathogen identified in the Chinese chestnut genome <li>Evaluate preliminary results from field tests using embryogenic clones <li>Identify fungal genes responsible for differences in <i>C. parasitica</i> virulence levels through genetic and genomic analyses <li)Complete assessment of stand health at lake state locations wher

Projected Participation

View Appendix E: Participation

Outreach Plan

The annual meeting of NE-1015 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-1015 web site. NE-1015 members will continue to make research results available through scientific journals, both refereed and non-refereed, extension bulletins, and national and international conferences and workshops. Information to the general public will be disseminated via publications in the popular press, magazines, oral and written presentations at workshops and at producer field days. A listing of all publications developed by NE-1015 members will be updated annually and posted on the official NE-1015 website. The NE-1015 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 was recently set up to collate information relating to the meeting and activities of NE-1015 research project (http://chestnut.nmsu.edu/index.html).

Organization/Governance

The organization of regional research project NE-1015 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.

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Attachments

Land Grant Participating States/Institutions

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

Non Land Grant Participating States/Institutions

American Chestnut Foundation, Beltsville Area, New York - Syracuse University, Ontario - Canada - Hort Research Institute, University of Georgia, University of Wisconsin - La Crosse, USDA Forest Service