Chestnut blight, incited by Cryphonectria parasitica (Murr.) Barr, devastated the American chestnut tree (Castanea dentata (Borkh.) Marsh) in the first half of the 20^thcentury, killing approximately 4 billion dominant and codominant trees in the hardwood forests of the eastern United States. Prior to blight, the tree had many uses, producing sawtimber, poles, posts, fence rails, cord wood for fuel, paper and tannin extraction, and nuts for humans, livestock and wildlife. It also can be characterized as a member of our charismatic megaflora; many people mourn its loss and participate in citizen-science projects to restore it. Restoration of the American chestnut would be a demonstration of an application of science for the public good in the face of continuing environmental degradation due to the advent of industrial and now postindustrial economies and the accompanying influx of exotic pests.

The United States Department of Agriculture (USDA), in cooperation with state and private agencies, began work in the 1910s to restore the chestnut tree after recognizing that it was on an inexorable path to destruction caused by the blight. As part of their work, exotic species of Castanea were introduced, which has resulted in a nascent orchard industry in numerous states from coast to coast in the US. Although the aggregate production of edible chestnuts is still too small to be tallied separately by the USDA, in 2015, the United States had 919 farms producing chestnuts on more than 3,700 acres. The states with the most chestnut acreage were Michigan, Florida, California, Oregon, Virginia, and Iowa. Most of those trees are not afflicted by blight, but are affected by other pests and diseases, which need management. Additionally, specialized cultivation techniques for the trees are required, and infrastructure to process and market chestnuts needs further development. NE-1833 members have performed research and obtained funding to address these needs and have formulated extension recommendations.

The NE-1833 project and its predecessors have been the central organization coordinating chestnut research since its inception as NE140 in 1982. For 40 years the project has exemplified what the original USDA model for Regional Research, now Multistate Research projects, was intended to accomplish; as detailed below, it has been and continues to be successful at every level. Members span numerous disciplines in forest pathology, plant sciences, microbiology, molecular genetics and biochemistry, and the annual meeting provides an opportunity for members to be exposed to this diversity. NE-1833 has provided a forum for new and established researchers to develop collaborative relationships and to share resources and expertise. NE-1833 meetings are well attended, and about 30 presentations are typically made by participants each year. Despite pandemic-related disruption in 2020 and 2021, the group still met virtually to share ideas and continue the work. International visitors and collaborators have often been included in these presentations, and two international symposia were organized and hosted by NE-1333, the immediate predecessor to NE-1833. As a result, numerous multi-state and international research efforts have been undertaken by NE-1833 members. The project was initiated to explore the diversity of hypoviruses and their efficacy for controlling blight on American chestnut at different locations in its natural range. That original goal persists, but range-wide studies additionally include breeding and evaluation of disease-resistant progeny as well as studies of orchard chestnuts for nut production. Additional activities requiring a multistate effort have been able to leverage rapid developments in genome sequencing technology and have led to the development of specific and useful genomic tools for Castanea.

The NE-1833 project comprises three objectives: 1) develop and evaluate disease-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 conservation and 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.

Objective 1: Develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome.

Restoration of American chestnut depends on producing a founder population that has adequate blight resistance, forest competitiveness, and genetic diversity to adapt to a large natural range and a changing climate. It is also important that the restoration population has resistance to phytophthora root rot (PRR) caused by the oomycete pathogen Phytophthora cinnamomi (Westbrook et al. 2019; Gustafson et al. 2022). This disease is most prevalent in the Southeastern U.S. but is expected to spread northward as the climate warms (Burgess et al. 2017).

For forty years, the American Chestnut Foundation (TACF) has pursued backcross breeding to introgress resistance to Cryphonectria parasitica and Phytophthora cinnamomi, from Chinese chestnut (C. mollissima), into a genetically diverse population of American chestnut. In parallel, scientists at the State University of New York college of Environmental Science and Forestry (SUNY-ESF) have worked since 1990 on transgenic approaches to enhance blight and PRR resistance in American chestnut (Steiner et al. 2017). 

To date, the most promising transgenic approach to enhance blight resistance has been the insertion of the oxalate oxidase (OxO) gene from wheat, which detoxifies oxalic acid produced by the chestnut blight fungus and significantly reduces the severity of chestnut blight stem cankers (Newhouse et al. 2014; Powell et al. 2019; Newhouse & Powell, 2020). In 2020 and 2021, the research team at SUNY-ESF submitted petitions to three federal regulatory agencies (USDA, EPA, and FDA) to obtain non-regulated status of the ‘Darling 58’ (D58) transgenic variety of the American chestnut containing the OxO gene (Newhouse et al. 2020). The experimental evidence presented in the petition indicates that D58 progeny pose no significant plant pest or food safety risks. Darling 58 progeny may be deregulated and distributed to the public as early as 2023. If and when D58 is deregulated, TACF and ESF plan to breed D58 progeny with a genetically diverse population of American chestnuts over three to five generations with the aim of representing 99% of the climate adaptive genetic diversity in the wild population of C. dentata (Westbrook et al. 2020; Sandercock et al. 2022). Furthermore, we plan to breed D58 progeny with backcross trees selected for resistance to P. cinnamomi to combine resistance to the two major diseases of American chestnut (Westbrook et al. 2019). The combination of biotechnology with breeding is a promising strategy to produce restoration-worthy trees. The initiatives outlined in the current and future work section describe our current best practices and future projects aimed at generating disease resistant and genetically diverse populations of American chestnut for restoration. 

NE-1833 participants maintain longstanding commitments to education, outreach, transparency, collaborations, and research toward safe and effective American chestnut restoration that date back to the conception of the original NE-140 project. Researchers at ESF have been working closely with federal regulators from all three agencies in the Coordinated Framework for Biotechnology: USDA-APHIS, EPA, and FDA. The D58 chestnut has provided a unique opportunity for both university researchers and federal regulators to understand how this process, historically applied to annual agricultural crops, can be extended to a wild tree intended for wild release and persistence in the environment. This series of rigorous reviews by all three agencies will help ensure safety and demonstrate to the public that the development and distribution of Darling 58 chestnuts is being done responsibly and transparently.

The project has always welcomed and incorporated public feedback while acting on scientifically sound advice. Indeed, the decision to pursue regulatory approval and public distribution has been driven and sustained by public feedback. As part of the deregulation petition for D58 there have been public comment periods (PCP) that have received supportive input from thousands of individuals and over 100 agencies (including The Sierra Club and The Nature Conservancy). In addition, dozens of commenters state that, while they are generally opposed to genetically-modified organisms as a general rule, they see the transgenic American chestnut as an amazing application of the technology and offer their support. In the first PCP (August 2020), the support from unique, individual commenters was strongly supportive (62%), a trend that continued in the second PCP (85% of unique comments are supportive as of 1/9/23, with over 5,000 unique comments contributed in total to date). This information is provided in more detail, with hyperlinks to comments, in the Outreach section, below.

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.

Chestnut blight appears to have been controlled by naturally occurring hypoviruses on C. sativa in Europe but not on C. dentata in North America, except in specialized settings. Research by NE-1833 members and their European colleagues contributed to the view that control in North America was hampered by the much larger number of strains of C. parasitica in different vegetative compatibility groups than occurred in Europe. Other factors hampering control in North America versus Europe may have included greater competition from other hardwood species, greater susceptibility to blight in C. dentata and differing forest management practices.

The RNA sequence of a hypovirus was first determined by members of NE-140 (the original precursor of NE-1833), and a number of species of virus were found based on sequence analysis. Viruses in families other than the Hypoviridae, including mitochondrial viruses, were found infecting C. parasitica, some associated with reduced virulence and biocontrol. Transformation of C. parasitica with cDNA of Cryphonectria hypovirus 1 resulted in transmission of the DNA in ascospores and regeneration of RNA viruses in progeny. This completed Koch's Postulates for the hypovirus. Unfortunately, while transformed fungus strains could produce progeny that infected adjacent chestnut trees with hypovirus-containing C. parasitica, disease remission did not occur in the general populations.

Regarding the molecular basis of fungal pathogenicity, NE-1833 researchers working in this and previous project cycles have found numerous fungal genes involved. Their protein products were components of complex signaling or response mechanisms critical for basic cellular functions, for example, G-protein signaling pathway components, some of which were also affected in expression by the presence of the hypovirus. Additionally, hypovirus-infected mycelium was found to fail to transition metabolism with colony age in the same manner as the uninfected mycelium. Thus, there are now known to be many genetic factors in the fungus that can influence pathogenicity, and several of these are now known to be affected by hypovirus infection.

To address the issue noted above concerning the variation in vegetative incompatibility groups in North American populations of C. parasitica and the potential negative impact on biological control potential, the strain Ep155 was crossed with a European strain and six vegetative compatibility loci, known as Vic genes, were genetically mapped in the progeny. The DNA of strain Ep155 of C. parasitica was sequenced and the European strain resequenced. The six Vic genes were identified, and a "super donor" strain prepared with five inactivated Vic genes (four Vic genes were knocked out). Demonstrating the efficacy of the superdonor strain should be able to transmit hypoviruses to strains with any combination of Vic genes. The strain is being tested in the forest for disease control and development of this potential tool is described in the current work section.

Strain Ep155 is regarded as highly pathogenic and has been used most effectively as the prototypic strain in the studies of fungal pathogenicity determinants. It is also used to screen chestnut trees for blight resistance in combination with a strain of low pathogenicity known as SG2-3. To investigate the genetic differences between these two strains that lead to the different phenotypes, the two strains were crossed and 96 progeny evaluated for pathogenicity. As part of NE-1833 all of these strains were sequenced to a high depth and preliminary work to identify quantitative trait loci (QTL) with the intent to test for pathogenicity effects by gene knockout. This should help lead to further understanding of mechanisms of pathogenicity in the fungus. The mechanisms of virulence reduction by hypoviruses in the fungus also remain an active area of investigation, as do other aspects of virus activity in the fungus. Reannotation of the fungal genome by NE-1833 researchers is facilitating transcriptomics and other detailed analyses in the above studies.

Blight cankers on chestnut are perennial and have been observed to persist more than 40 years. A rather complex community develops in cankers, especially as they age. NE-1833 members have documented numerous species of invertebrates and microorganisms in cankers. The blight fungus itself becomes a host for various viruses and similar entities, and multiple strains can be isolated from cankers. The development of these communities and association of their composition with canker longevity is a promising area for metagenomic investigation. Sadly, work at Shenandoah University in this area was significantly compromised by the COVID pandemic and resulting sample losses caused a significant setback to this part of the work.

Objective 3: Investigate chestnut conservation and 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.

In addition to the activities discussed under Objectives 1 and 2 above, research is ongoing on gall wasp, silvics, juvenile versus adult chestnut blight resistance, genetic variation in American chestnut, and integrating resistance with hypovirulence to control blight, inter alia.

It has been found that introduced and native parasites of the Asian chestnut gall wasp (Dryocosmus kuriphilus) control the pest after the first few years of infestation. Despite a plant quarantine, Michigan is now in the third year of gall-wasp infestation. While nut harvests are markedly decreased during those first few years of infestation, insecticidal treatments also would destroy the parasites, so the recommendation is to NOT spray insecticides to control gall wasp. Dispersal of parasites as a biological control is recommended, but none are being produced currently. There have been efforts to bring parasite-infested boughs to new areas of gall-wasp infestation, to introduce parasites earlier than occurs naturally. There is variation between cultivars in their susceptibility to gall wasp.

In general, silvicultural evaluations of American chestnut in several states have found that it is a very rapid grower, frequently much faster than planted oak and walnut, though naturally regenerated woody vegetation often grows faster than planted chestnuts in forest settings. Chestnut growth varies with site, like most hardwoods, and competitive dynamics will differ with site and light availability. Earlier research found that exotic chestnut species do not grow well in native forests, unlike American chestnut. This finding was part of the motivation leading to the proposal to backcross resistance from exotic into American chestnut.

The results of inoculating young seedlings of the three Chinese Castanea species do not match up with blight severity on mature specimens of the three species in China; this result needs more detailed experimental evaluation. Low levels of blight resistance occur in a few American chestnut trees. Intercrossing of these to enhance that resistance has been pursued for a long time. In combination with hypoviruses, impressive levels of blight control have been observed on some pure American chestnut trees with low levels of resistance. The hypothesis that hypoviruses coupled with resistance in backcross progenies will diminish blight severity is being evaluated.

Castanea species native to the U.S. (dentata, pumila var. pumila, and pumila var. ozarkensis), Castanea species imported from elsewhere (crenata, mollissima, henryi, seguinii, and sativa), and Castanea hybrids are maintained and studied by NE-1833 scientists and their citizen-scientist collaborators. These trees are in: Alabama, California, Connecticut, Delaware, Florida, Georgia, Indiana, Kansas, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, New Jersey, New Hampshire, New York, North Carolina, Ohio, Pennsylvania, Rhode Island, South Carolina, Tennessee, Vermont, Virginia, West Virginia, and Wisconsin. Strains of C. parasitica are shared by members of NE-1833 (according to APHIS PPQ permitting and restrictions), and important strains are deposited with the American Type Culture Collection. Strains with genetic markers are available, and information on the genetic determinants of vegetative incompatibility (vic genes) is available for use in population studies. Hypovirus types from France, Italy, MI, WV, KY, and China are studied and shared by NE-1833 members.

Members are renowned for their work on chestnut, Cryphonectria, and fungal viruses. In the duration of the NE-1833 project to present (2018 – end of 2022) NE-1833 members collectively published 49 peer-reviewed technical articles in scientific journals and participated in numerous articles in the popular press that have been distributed in both print and digital media through venues as diverse as the Washington Post and New York Times to the Sierra Club to modernfarmer.com and granitegeek.com. Student training also continued with 4 Ph.D. dissertations and 11 M.S. theses completed during the current project, and a large number of undergraduates contributing to the work at the participating institutions.

Venues for scientific presentations, although somewhat limited or sometimes virtual due to the COVID pandemic during the project period, nonetheless included the Plant and Animal Genome Conference, the American Phytopathological Society, the Mycological Society of America, the Fungal Genetics Conference (sponsored by the Genetics Society of America), the Ecological Society of America, the Society of American Foresters, various venues of the The American Chestnut Foundation (TACF), the Entomological Society of America, the American Society for Microbiology, and the American Society for Virology. The results of research have been extended to growers, especially in Pennsylvania, Michigan and Missouri, and to volunteer citizen scientists in 21 states guided by TACF and the American Chestnut Cooperators' Foundation (ACCF).

In recognition of these successes, researchers currently part of NE-1833 received the ESS Excellence in Multistate Research award in 2010 (NE-1833 was then known as NE-1033). The cohort of scientists from 12 years ago has undergone some natural turnover but remains strong with exceptional young scientists joining the project. Despite the significant impact of the COVID pandemic on research operations and the ability to meet in person, NE-1833 has largely met the milestones detailed in the project description and will continue to work on similar collaborative projects in the next five years. Data generated under the auspices of the NE-1833 project have been used by members to gain intramural and extramural funding for all aspects of chestnut biology and restoration.

In summary, the NE-1833 project remains a productive group of collaborators that has provided new and meaningful information to all clients interested in chestnut biology and restoration, from the bench scientist to the professional orchardist and to the individual volunteer grower of chestnut for restoration. In the next five years, we will continue to pursue collaborative projects under our three stated objectives leading to increased production of chestnuts in orchards and furthering the restoration efforts of the iconic American chestnut.