NE1033: Biological Improvement of Chestnut through Technologies that Address Management of the Species, its Pathogens and Pests
Statement of Issues and JustificationThe 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.
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