Duration: 10/01/2023 to 09/30/2028

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

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.

Related, Current and Previous Work

Objective 1

A: Diversify transgenic blight tolerant populations and incorporate phytophthora root rot resistance via breeding transgenic with backcross trees

We have begun introgressing the OxO gene into a diverse population of American chestnut by “speed breeding” D58 progeny with diverse wild type and backcrossed American chestnuts from across the species range. Simulations suggest that outcrossing a single transgenic founder over five generations to 700 diverse wild type parents will increase the effective population size to >1000 and minimize inbreeding coefficients to less than 1% (Westbrook et al. 2020a). Genome-environment analyses based on whole genome sequencing on a sample of 356 American chestnut individuals suggests that the C. dentata native range is composed of 3 to 4 locally adapted subpopulations (Sandercock et al. 2022; Sandercock et al., unpublished). Preliminary analyses suggest that breeding with 209 total wild type individuals or 92 individuals from the northeastern zone, 87 from the central zone, and 30 from the southwestern zone is sufficient to match climate adaptive allele frequencies in each zone at R² = 0.99 (Sandercock et al., unpublished). In 2022, Virginia Tech researchers performed whole genome sequencing on 350 backcross progeny representing most of the ~250 wild type parents used in TACF’s breeding program to determine how much of the adaptive diversity is represented. Under permits from the USDA, the multistate cooperators on this project have taken Darling 58 progeny to the third generation of outcrossing to over 100 wild type and backcross American chestnut parents.

Researchers at the University of New England, SUNF-ESF, TACF’s Meadowview Research Farms, and Penn State have established systems to grow seedlings under long day and high fertilizing conditions to induce transgenic Darling 58 seedlings to produce catkins and pollen within the first growing season. Between these locations, we expect to produce and cryostore over 2000 vials of pollen before the start of the 2023 growing season. This pollen supply is enough to pollinate more than 200,000 female flowers.

Scientists at TACF and Virginia Tech have collaborated on an extensive genotyping and phenotyping effort encompassing over 5,500 American chestnut backcross hybrids planted in 88 orchard locations in 20 states. A total of 421 backcross trees that descended from a range wide sample of unique wild type American chestnuts have been prioritized for breeding with D58. These trees inherited between 85% and 100% of their genome from C. dentata. TACF has also collaborated with RAPiD Genomics to develop a genotyping method that will enable us to select against residual inheritance of genes from Chinese chestnut and select progeny that inherited all of their genome from C. dentata. Genotyping also enables us to select progeny that inherited less than 10% of their genome from the D58 founder tree. Selecting against the D58 genome minimizes the potential for deleterious inbreeding in the restoration population and ensures that the trees that we deploy for restoration have locally adapted genetics. Upon deregulation of D58, we plan to plant 6 + seed orchards collectively composed of 5000 + D58 progeny derived from crosses with a range wide sample of 200 + American chestnuts. These seed orchards collectively will have the capacity to produce >1 M open pollinated seed by age 10 years.    

We have begun breeding D58 progeny with the PRR resistant selections to generate ‘dual resistance’ progeny with resistance to both chestnut blight and PRR. Through a combination of progeny testing and genomic prediction, we’ve also identified 146 backcross trees with elevated resistance to P. cinnamomi ranging from 25 to 77 on the 0 American chestnut to 100 Chinese chestnut scale. In 2021 and 2022, U.S. Forest Service, Clemson University, and TACF collaborators inoculated 220 OxO positive progeny from ten crosses between Darling 58 progeny and PRR resistant backcross trees with P. cinnamomi at the U.S. Forest Service Resistance Screening in Asheville, NC. To date, a total of 65 seedlings have survived the greenhouse inoculations with intact root systems and were planted at a permitted TACF orchard site in Georgia. Our plan is to eventually intercross the partially PRR resistant survivors and select for higher levels of PRR resistance and OxO homozygosity among the progeny. 

Finally, Breeding D58 with Ozark, Allegheny, and Alabama chinquapins holds promise as a method to enhance blight resistance in these blight susceptible and sexually compatible species. To introgress OxO into chinquapin populations, we plan to outcross D58 with chinquapin varieties over 3 + generations and use genetic markers to select for maximal chinquapin ancestry in each generation.

B: Compare traditional breeding as a standalone strategy to the combined biotech/breeding approaches to enhance blight and phytophthora resistance

Even if D58 is approved, we anticipate that some landowners will prefer non-transgenic trees for species restoration. The traditional breeding program to introgress blight resistance into American chestnut from Chinese chestnut has been challenging due to the latter’s complex genetic architecture of blight resistance. Estimates of trees’ genetic resistance are negatively correlated with the proportion of genome inheritance from C. dentata (Westbrook et al. 2020b). Simulations suggest that controlled crosses between backcross trees selected for blight resistance (hereafter referred to as best x best crosses) will produce progeny with average blight resistance index values of 50 and approximately 85% American chestnut ancestry. Genomic selection of the most resistant 10% of the progeny from best x best crosses is expected to further improve average blight resistance indices to ~ 65 while also potentially reducing C. dentata inheritance to 75%.

Another traditional breeding option for improving blight resistance in American chestnut is to perform controlled pollinations between partially blight resistant wild type American chestnuts and select for enhanced resistance in the progeny through repeated cycles of breeding and selection. In the late 1990’s and early 2000’s TACF performed controlled pollinations between large surviving American chestnuts and planted the progeny at Meadowview Research Farms. Current TACF staff recently assessed the long-term blight resistance of 48 progeny of intercrosses among 12 large surviving wild trees. All the progeny were inoculated with virulent strains of the blight over a decade ago and the trees ranged in age from 16 to 25 years old. We visually assessed these trees for survival of the main inoculated stem, percent of the tree canopy that was healthy, presence/absence of large cankers, exposed wood, stump sprouts, and blight fungal sporulation from cankers. The blight resistance data from the individual trees and their relatives to estimate the genetic resistance of the progeny of large surviving trees relative to typical blight-susceptible American chestnuts, resistant Chinese chestnuts, and partially resistant American chestnut backcross hybrids. A blight resistance index was created from the sum of the individual traits and scaled this index from 0 = average of typical blight susceptible wild type American chestnuts to 100 = average of blight resistant Chinese chestnuts. The blight resistance indices of the 10 most resistant progeny of the large surviving trees varied from 43 to 25. Nine  out of 10 of these progeny and all trees had 100% American chestnut ancestry (Westbrook et al. unpublished). For context, the blight resistance indices of the top 5% most resistant backcross selections (137 selected trees) varied from 40 to 80 (average 50) while American chestnut ancestry varied from 60% to 100% (average 88%). These results confirm that blight resistance in American chestnut is heritable.

TACF is planning to compare the resistance of progeny D58, best x best backcross trees, and large surviving American chestnuts using small stem assays for blight resistance replicated at two greenhouse locations in 2023. If we find that the blight resistance index values of best x best progeny exceeds our minimum standard 40, then it would be worthwhile to pursue both transgenic and non-transgenic approaches in parallel to be able to offer different types of trees depending on landowner preference.

C: Develop a basic understanding of the biology and genes underlying blight and phytophthora root rot resistance in Castanea

Understanding of the biology of resistance and susceptibility to chestnut blight and phytophthora root rot in Castanea is a prerequisite to additional biotechnology aimed at enhancing resistance to these diseases in American chestnut. To discover candidate genes underlying resistance to chestnut blight and PRR, researchers from Virginia Tech, University of Kentucky, TACF, and Oak Ridge National Laboratory are currently pursuing a combination of quantitative trait locus mapping in American chestnut backcross populations, RNAseq and metabolomic timecourses, genome scans for signatures of natural selection, and comparative genomics. This work has been aided by the recent completion of a chromosome scale annotated reference genome for Castanea dentata and two haplotype resolved genome assemblies for C. mollissima by colleagues at Hudson Alpha Institute of Biotechnology. These genomes are available on Phytozome (https://phytozome-next.jgi.doe.gov/).

The quantitative trait locus mapping completed to date indicates that blight resistance is controlled by tens to hundreds of small effect loci on all twelve chromosomes, while phytophthora resistance is controlled by fewer larger effect loci concentrated on chromosome five. We found regions of overlap between QTLs for blight and PRR resistance and signatures of natural selection in resistant C. mollissima and C. crenata. In the RNAseq timecourse, we identified over 3,000 differentially expressed genes (DEGs) in response to blight inoculation in either C. mollissima or C. dentata. A subset of 700 DEGs were specifically differentially expressed in C. mollissima and an additional 800 DEGs were specific to C. dentata. The species specific DEGs may contain genes important for blight resistance and susceptibility in Castanea. We identified a smaller subset of 58 hub genes whose expression was strongly correlated with the expression of genes specific to the C. mollissima response. As this work continues, our aim is to narrow the list to dozens of well supported candidate genes to target for gene knockouts with CRISPR or transgenic insertion to test for enhanced resistance or susceptibility in C. mollissima and C. dentata.  

D: Develop additional founder lines containing the OxO gene and a higher throughput system to do genetic transformations in American chestnut.

The Darling 58 variety was developed by transforming a single founder wild type American chestnut from New York state. Researchers at University of Georgia and SUNY-ESF are collaborating to transform up to three additional American chestnut genotypes from Georgia, Virginia, and Pennsylvania with the OxO gene to reduce the potential for deleterious inbreeding and founder effects. Some of the new OxO events in these founder lines will be expressed with a wound and pathogen inducible promoter (win3.12) from Populus (Carlson et al. 2022). Inducible OxO expression has the potential to reduce growth penalties and other deleterious effects that might result from constitutive OxO expression. Next steps for the development of these new transgenic founder lines include verifying transgene expression and copy number, developing whole plants from transformed somatic embryos, long read sequencing to determine the transgene location, high light pollen production, initial breeding with wild type trees, and conducting blight resistance assays on the progeny. Once these steps are complete, we plan to petition for non-regulated status to use these new founder lines in our larger breeding effort to introgress OxO into wild populations of C. dentata.

Any further biotechnology aimed at enhancing disease resistance in American chestnut will be aided by developing a more efficient transformation system. Recently, it was demonstrated that gene editing could be performed on live plants by simultaneously inducing and transforming meristems by injecting stems with developmental regulators and gene editing constructs (Mahar et al. 2020). This method bypasses the time-consuming development of somatic embryos and potentially could be adapted to multiple genotypes. In 2023 and 2024, scientists at Virginia Tech and University of California Davis are planning pilot studies to test if meristem induction and transformation could work in American chestnut. 

Objective 2

Hypovirus infection of C. parasitica often reduces canker expansion rates (Anagnostakis & Waggoner 1981). Heiniger and Rigling (1994) postulated that hypovirus infection led to recovery of many stands of C. sativa. Widespread recovery has not occurred in the U.S. on C. dentata despite 45 years of effort. Several factors have been proposed to explain this failure, including higher diversity of vegetative compatibility groups in the US (Anagnostakis et al 1986), the extreme susceptibility of C. dentata to blight, strong competition from other tree species in the US (MacDonald & Fulbright 1991; Heiniger & Rigling 1994), and different silvicultural practices (Mittempergher 1978; Griffin et al 2005).

Michigan State University (MSU; Jarosz) found that hypovirus infection of C. parasitica led to recovery of some C. dentata stands (Davelos & Jarosz 2004). However, further modeling suggested that equilibrium has not been attained (Davelos-Baines et al, 2014). Further, the frequency of hypovirus infection can change over time, leading to the decline of large trees (Springer et al , 2013). Chestnut blight cankers are complex communities of interacting cohabitants (vertebrates, arthropods, microbes) that may modulate canker severity. Interactions can be further complicated by infection of C. parasitica by hypoviruses, other viruses and microbes, and by host resistance. We can predict the fate of a canker only at the extremes of these interactions. Recent data suggest that microbial invaders of cankers may play an important role in canker severity. Invader frequency increases over time in cankers that do not kill the distal stem. In addition, the prevalence of virulent C. parasitica declines steadily over time while hypovirulent C. parasitica remain at a moderate level (~25%) (Double et al, 2018). We hypothesize that these invading microbes play an important role in canker severity and longevity.

MSU (Sakalidis) and CAES (Kërio) have also considered population genomics of C. parasitica to understand pathogenicity and host responses. As a tripartite model system, interactions among the pathogen (C. parasitica), the host (Castanea species) and the virus (Hypoviridae family) of chestnut blight disease have been intensively studied in Europe and North America. Despite substantial research of C. parasitica populations in Europe, there is very limited recent research of C. parasitica populations in North America. Additionally, none of these studies have used genome resequencing. The most recent study in North America focused on four locations using microsatellite analysis (Dutech et al. 2012). The high diversity of VC groups in the US has led to failure of biocontrol through hypovirulence. To overcome restrictions caused by vegetative incompatibility, a super mycovirus donor (SD82+SD382/CHV1-EP713) that can donate the mycovirus to any VC group has been engineered (Zhang and Nuss 2016). However, there is limited data as to how this super donor strain will success in the field, and in the presence of other mycoviruses. In addition, we do not know how the presence of established populations of C. parastica on both domesticized chestnut and wild chestnut will affect the reestablishment of American chestnut in US forests.

A critical aspect impacting the success of the American chestnut restoration is whether the achieved resistance in the best backcross hybrids is efficient against multiple strains of the pathogen present in domesticized and wild chestnuts. Currently, majority of the inoculation tests used to screen chestnut progenies for resistance utilize only the EP155 and SG2-3 strains of C. parasitica. The highly virulent EP155 strain was initially isolated from Bethany, Connecticut (Anagnostakis and Day 1979), and was used to sequence the C. parasitica genome (Crouch et al. 2020). However, using these two isolates may not sufficiently capture the host resistance to naturalized and potentially recombining strains of the pathogen. Genetic analysis of 230 isolates of C. parasitica strains mainly from Europe indicate that a highly invasive European lineage of the pathogen arose through recombination of European isolates (Stauber et al. 2021). In this context, the combination of inoculation experiments and population genomic analyses can prove as a successful strategy to identify virulence genes (Tabima et al. 2019). It is also of importance to test the resistance of the trees carrying the wheat oxalate oxidase (OxO) transgene (Newhouse et al. 2014) to a wider selection of pathogen isolates. Knowledge of the genetic diversity of C. parasitica in North America will facilitate the selection of American chestnut with resistance to a broad selection of C. parasitica genotypes. Additionally, this creates the opportunity to identify candidates to be used as superdonors for more effective hypovirulence transmission.

Rutgers University. Like other regions of the Northeast, New Jersey has stands of C. dentata trees that are stable with large mature trees recovering from blight. Hypovirulent strains of C. parasitica have been isolated from these blighted trees over the course of this project. A wide array of viruses have been isolated from these strains and have been shown to have a measurable effect on fungal growth and virulence. (Hillman and Suzuki, 2004; Eusebio-Cope et al., 2015; Hillman et al., 2018 for reviews). The Hillman lab at Rutgers continues to characterize new viruses from C. parasitica isolates from northeast forests, particularly local New Jersey forests, to examine their roles in control of blight in natural settings, and to examine the interplay among viruses in mixed infections of C. parasitica and virus/transposon interactions in the fungus. Recently, a hypovirulence-associated reovirus characterized in C. parasitica was shown to require the presence of a virus from a different family, the Hypoviridae family, for stable infection (Aulia et al., 2019; Aulia et al., 2021). Interplay between two closely related members of the Hypoviridae, CHV1 and CHV2, in coinfected fungal isolates has shown the opposite effect, with one of the viruses, with CHV2 being lost in coinfection (Hillman et al., unpublished). The role of such coinfections in other in natural C. parasitica infections is unknown.

West Virginia University. With few exceptions (Yu et al, 2013), mycoviruses have evolved exclusive intracellular lifestyles (Buck, 1986), limiting their transmission to intracellular mechanisms via conidia or anastomosis. Vegetative incompatibility (Vic) systems restrict mycovirus transmission (Boland, 2004; Caten, 1972; Hall et al, 2011; Biella et al, 2002) due to apoptosis triggered when vic incompatible individuals anastomose (Saupe, 2000; Jacobson et al, 1989; Glass et al , 2000). Through a combination of systematic gene disruption and classical genetics, Zhang and Nuss (2016) developed the SD328/82 super donor (SD) strain, containing gene disruptions at vic1, vic3, vic6 and vic7, and both alleles of vic2. Under laboratory conditions, SD328/82 was able to transmit hypoviruses to uninfected strains heteroallelic at any vic locus. Preliminary testing for their efficacy in controlling blight on C. dentata in forest environments has demonstrated the feasibility of the approach with increased natural dissemination of hypovirus (Stauder et al., 2019), although improvement in limiting disease spread may be enhanced with use of a less debilitating hypovirus isolate.

In 2022, the University of Maryland (UMD) demonstrated that CRISPR/Cas9-mediated genetic transformation of C. parasitica is more efficient than the prevailing homologous gene replacement (HGR) method.    UMD developed a strain of C. parasitica strain, DC9, with the Streptococcus pyogenes Cas-9 gene inserted into it and driven by a fungal promoter, and then compared the results of transformations using HGR and HGR plus guide RNA targeting the gene of interest.  Guide RNA is necessary to direct the Cas-9 endonuclease to make double stranded breaks in the DNA at the intended locus.   The experiment was carried out twice targeting the CpSec66 gene, the deletion of which produces a visible change in phenotype as well as reduced virulence against chestnut.   In both cases HGR plus guide RNA resulted in an equal or greater number of transformed colonies.  More importantly, about half of the transformed colonies produced with HGR plus guide RNA contained only the mutant locus.  With HGR alone, transformed colonies always contain both the wild-type and transformed versions of the target locus, requiring an additional step to separate transformed and untransformed spores or hyphae.   Attempts to transform DC9 spheroplasts with guide RNA alone did not produce visibly recognizable mutants, but whether there were invisible mutations remains to be determined by amplicon sequencing of pooled DNA from treated colonies.

Mississippi State University (MissSU) has prepared a new, experimentally validated, annotation of the C. parasitica genome sequence (Ren and Dawe, in preparation). This significantly improves both gene identification and putative functional assignment. ARV-1 is a predicted gene in C. parasitica that shares similarity with genes that code for proteins with important roles in sterol homeostasis in other organisms. The knockout of ARV-1, serendipitously made when investigating LysM proteins during the NE-1333 project period, is avirulent and has a heavily impaired vegetative growth phenotype. MissSU has developed and verified a quantitative assay for ergosterol production in C. parasitica by modifying published protocols and using a GC/MS system in collaboration with the lab of Todd Mlsna in the Department of Chemistry at Mississippi State. Using derivatization techniques to tag the appropriate class of compounds show that ergosterol accumulation is much reduced in the ARV-1 mutant, as expected. When tested, however, the hypovirus infected strain EP713 reproducibly shows a reduction of ergosterol accumulation similar to that of the mutant, suggestion that a component of the membrane alterations induced by the hypovirus may be due to altered ergosterol presence.

A putative orthologue of Neurospora crassa vib-1 was found in C. parasitica. vib-1 is part of the transcription cascade leading to apoptosis (Dementhon et al., 2006). We knocked out Cpvib-1 to assess its role. The knockout did not change vegetative incompatibility in strain Ep155 but did in strain EU1. EU1 differs from Ep155 at vic4, yet the strains with a Cpvib-1 knockout were vegetatively compatible. Thus, we concluded that Cpvib-1plays a crucial role in the incompatibility reaction modulated by vic4. By tagging the VIB-1 protein with an epitope it was possible to demonstrate DNA binding and complete a ChIP-Seq analysis to identify a recognition sequence. Intriguingly, when tested from cultures stimulated by using rapamycin to mimic the vegetative incompatibility response, the recognition sequence was altered indicating that VIB-1 protein is a transcription factor that is responsive to cues from the vegetative incompatibility signaling pathways.

TACF uses two strains of C. parasitica, Ep155 and SG2-3, to screen for blight resistance. They are near the top and bottom, respectively, of pathogenicity for virulent strains. The two strains were crossed, and progeny tested, revealing significant variation in pathogenicity. DNA was extracted from 92 progeny and sequenced. Subsequent genetic mapping has shown that there are deficiencies in the some of the data leading to parts of the map that do not resolve properly. There are indications of potential QTL locations on chromosomes 1 and 2 but the size of the regions indicated means that identifying individual genes is not feasible at this stage. MissSU will address this with long-read (MinION) sequencing to improve the genetic map and increase resolution. Once identified those genes of interest can be subjected to knockout to assess the role of their protein products. Knockout approaches will be able to leverage advances in this technology by UMD (above).

Objective 3

USDA Forest Service, Delaware, OH, in collaboration with the University of Tennessee and other partners, is evaluating silvicultural methods for reintroducing improved C. dentata to the northern parts of its range.  Several studies established in Pennsylvania (2015-2017), track backcross chestnuts planted across a gradient of soil moisture availability, (study 1), with and without protection from deer herbivory (study 2), and across several silvicultural treatments that create varying levels of light availability and competition from woody species (study 3). Early results demonstrate the plasticity of American chestnut; planted chestnuts can survive across site quality and light availability gradients (Pinchot et al., 2017. Pinchot et al., 2020a). Sites with higher resource availability; soil moisture or light; encourage rapid growth of competing woody species and may require competition control to ensure the chestnuts attain dominant or co-dominant canopy positions (Pinchot et al., 2020a, Pinchot et al. 2020b). Fencing or other means of protection from herbivory will be necessary to reintroduce chestnut to forests with moderate to high deer densities (Pinchot et al., 2022).  The Forest Service and partners will continue to monitor these plantings to study the competitive dynamics of the planted chestnuts and naturally occurring woody vegetation. 

The USDA Forest Service, Southern Research Station is also cooperating with The University of Tennessee and other partners like TACF, Mississippi State University, and Clemson University to evaluate silvicultural test plantings in NC, TN, and VA. Ten plantings are still being monitored that were established between 2009 and 2015. Chestnut blight disease has led to large-scale mortality across plantings more than 10 years of age, resulting in a 34% survival rate for the most advanced hybrid seedlings (BC3F3). Disease resistance rankings were consistent with orchard inoculation tests from TACF. Nursery studies were also established that determined nut size/weight and genetics affected seedling quality.  Soil fungi was not a reliable predictor of seedling planting performance in chestnut and in co-planted white oak, even though chestnut species were associated with different fungi before being planting as bareroot seedlings. These multi-disciplinary projects have yielded a number of publications reporting on novel research findings (Brown et al., 2022; Case et al., 2016; Clark et al., 2010, 2012, 2014, 2016, 2019a, 2019b, 2020; Coughlin et al., 2020; Knapp et al., 2014; Reazin et al., 2019

In collaboration with TACF, Virginia Tech is experimenting with the use of unmanned aerial and ground-based remote sensing to quantitatively characterize tree growth and architecture, stress, and apparent blight resistance. These data collected with Lidar (Light Detection and Ranging) and multispectral sensors are to be analyzed in relation to ancestry genotype and phenotype data via machine learning techniques to evaluate potential for high-throughput tree phenotyping. Virginia Department of Forestry will be joining the collaboration by contributing phenotype/genotype data and access for remote sensing to backcross and single-ancestry orchards at Lesesne State Forest.

TACF and Virginia Tech will also be exploring several questions related to plant-soil interactions in chestnut orchards and restoration plots. Chief among these will be soil mesocosm trials at Meadowview Research Farms to evaluate the impacts of varying soil textures on chestnut rhizosphere, biomass, and blight resistance while controlling for climate and other spatially variable effects.

TACF is embarking on the establishment of permanent common garden infrastructure spanning the historic range of the American chestnut, as well as point north potentially encompassing areas due to become suitable with continuing climate change. Apart from evaluating all current and future breeding projects across climates, this will allow researchers to evaluate apparent local adaptation and phenotypic plasticity of populations from across the range through reciprocal transplant. This, in turn, will drive decision=making on provenance selection for geographically diverse reintroduction and restoration plantings.

With support and input from SUNY ESF, TACF is beginning a side-by-side evaluation of backcross bred, transgenic American, and backcross X transgenic ‘Stacked Resistance’ trees in nursery, orchard, and forest settings. This will provide highly valuable and actionable data that will – by the end of this decade – help researchers and conservationists make decisions about relative returns on investment in pursuing traditional backcross breeding, OxO-based transgenic, both, or a hybrid of these approaches toward restoration into forest systems. Trees will be challenged with C. parasitica at the seedling stage (small stem assays) and sapling stage (orchard and forest settings), while monitoring blight resistance, overall growth rate and form, as well as response to competition. Ultimately, fecundity will be estimated as trees enter reproductive age.

Penn State University has a suite of silvicultural trials, installed using wild-type American chestnuts, which will be used to ascertain and improve best management practices for reintroduction of disease-resistant American chestnuts. In addition, and in collaboration with William and Mary and Wilkes University, ongoing research will continue investigating the metrics which will determine American chestnut forest regeneration. Several stands of naturally regenerating American chestnuts found in Maine, Vermont, Indiana, and Wisconsin have been studies for 15+ years. Some data have been published regarding the founder effects (Rogstad and Pelikan 2014) and dispersal mechanisms at these sites (Elwood et al 2018). Genomic data collected on these sites within the past year will be evaluated to determine whether calculations established decades ago are currently applicable to reforestation initiatives. These data will then be applied as recommendations for reintroduction plots of disease-resistant American chestnuts.

TACF, Penn State University, and Villanova University are collaborating on the creation of broad and fine-scale habitat suitability models which can be made publicly available and in a usable format for landowner use. Multiple models have been developed on various local and regional bases (Noah et al 2021). The organizations above aim to collate and merge them into a single on-line tool which can be update over time to reflect changing climate and analogous seed zone recommendations.

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.
   
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.
   

Methods

Objective 1

Genotyping by sequencing was performed on over 5,500 trees in TACF’s breeding program. A description of the genotyping and bioinfomatic pipeline can be found in Westbrook et al. (2020).

Blight resistance phenotype data were taken on ~5000 backcross and large surviving American chestnut trees from 88 orchard locations in 20 states. All phenotyped trees had been inoculated at least two years beforehand. Data included whether the inoculated stem was dead or alive, whether the canker exceeded 15 cm vertically, presence/absence of sporulation and exposed wood in the canker, presence/absence of basal sprouting, percentage of the original canopy that was alive, and diameter-at-breast height (1.4 m above ground) of the largest trunk. Phenotype data were scaled such that high values were representative of blight resistance.

A genetic-based blight resistance index was created from the sum of estimated additive genetic values for this suite of traits.  Additive genetic values for blight resistance traits were estimated with the following generalized linear mixed model in ASReml-R v. 4.1 (Butler et al., 2018). Genetic liabilities for presence/absence traits were estimated with a binomial model and percent canopy survival was modeled as a continuously distributed gaussian trait. The effect of tree age was treated as a fixed covariate. Genotype effects were assumed to be normally distributed and were estimated with a blend of pedigree and genomic relationships (VanRaden 2008; Aguilar et al., 2010) estimated with the R package ‘AGHmatrix’ (Amadeu et al. 2016). To construct the genetic-based blight resistance index, genetic values were scaled 0 to 1 and these scaled values were multiplied by trait heritability (h² = s²_genotype / s²_{genotype +} s²_error). Blight resistance index values were then scaled from 0 = mean for susceptible AM chestnut and 100 = mean for resistant CH chestnut controls.

Reference genome assembly methods for one C. dentata individual and two C. mollissima individuals are described on the Phytozome website (https://phytozome-next.jgi.doe.gov/).

A time-course experiment was performed to quantify differences in gene expression and metabolic responses of Chinese chestnut, American chestnut, and F1 hybrids of these species in response to chestnut blight infection at an early stage of infection (3 days) and a later stage (10 days). Comparison of allele-specific expression in F1s to differentially expressed genes in Chinese and American will provide insights into whether gene expression differences between resistant and susceptible species are primarily regulated in cis or trans. The experiment included seven genotypes: (2 blight resistant Chinese trees, 2 F1s, 1 Large surviving American, and 2 susceptible American chestnuts), 3 time points: (just before inoculation, 3 days and 10 days post inoculation), three treatments (unwounded/uninoculated, wounded only, wounded and inoculated), and three biological replicates (grafted clones) per genotype, treatment and timepoint.

Small stem assays will be used to compare the blight resistance of progeny of D58 x wild type American chestnut, best x best backcross trees, and intercrosses between large surviving American chestnuts. The cut stem tips of 500+ progeny from each of these cross types and their combinations (e.g., D58 x backcross, D58 x LSA, LSA x backcross) will be inoculated with a virulent strain of C. parasitica following methods developed by Cipollini et al. 2021. Canker length, rating, and presence/absence of sporulation will be assessed 60 d and 90 d post inoculation. Mean canker severity for each cross type will be compared with ANOVA followed by multiple test correction using Tukey tests.

A high throughput genotyping platform will be developed to assess 1) species ancestry, 2) provenance from the wild C. dentata population, and 3) genome inheritance from the D58 founder tree. Whole genome sequencing on 20+ individuals of each Castanea species has been performed to discover SNP markers that are informative for species ancestry (Sandercock et al., 2022). A subset of markers spaced at approximately one centimorgan intervals with fixed differences between C. dentata and C. mollissima, C. dentata and C. sativa, and C. dentata and C. pumila varieties will be selected. Markers associated with climatic variation in C. dentata and with Fst values > 0.25 between subpopulations will be selected to determine provenance.

Markers that are homozygous in the D58 founder but variable in the wild-type American chestnut population (minor allele frequency > 0.25) will be selected to detect and select against the D58 founder’s genome in progeny.

The Connecticut Agricultural Experiment Station (CAES) will conduct work that aims to identify mechanisms underlying the high resistance of Chinese chestnuts to C. parasitica. A unique resource available at the CAES Lockwood experimental farm is a 20-year-old full-sib progeny of two C. mollissima trees, ‘Mahogany’ and ‘Nanking’, which both have high blight resistance (Steiner et al. 2017). The progeny has approximately 150 trees which have observable differences in the occurrence of natural blight infections. Susanna Keriö at CAES will utilize this resource to identify chemical, molecular, and genetic components associated with the high blight resistance in Chinese chestnuts and the hybrids. Potential avenues to explore this include pathogen plate assays to identify antifungal molecules, studying the expression of candidate resistance genes (Westbrook et al. 2020; Barakat et al. 2009) in naturally infected trees, and cell death assays (Kim et al. 2008; Vijayaraghavareddy et al. 2017; Majtnerová and Roušar 2018).

Objective 2

The CAES will contribute to the population genomic analysis of C. parasitica by collecting pathogen isolates from different chestnut species and hybrids available on CAES farms and established field plots. First stage of the population genomic analysis is to collect the pathogen isolates. The CAES has a diverse collection of pure chestnut species and chestnut hybrids with varying degrees of blight infection. The CAES has also established several field plots with hybrid trees which have natural blight infection. Additionally, the CT chapter of TACF maintains several germplasm conservation orchards where some of the trees have natural blight infection. This offers the opportunity to study the diversity and virulence of the pathogen isolates colonizing hosts with varying genetic backgrounds and varying resistance. The second stage is to characterize the vegetative incompatibility of these isolates by agar plate tests and PCR assays (Short et al., 2015). In the third stage, the CAES will pursue funding to conduct resequencing of these isolates in collaboration with the participants of the NE1833 Chestnut Multistate Project consortium. Finally, in collaboration with TACF and the participants of the NE1833 Chestnut Multistate Project consortium, CAES will facilitate controlled inoculation experiments (small stem assays) on selected American chestnut genotypes, other pure species or hybrids, or on American chestnut seedlings carrying the OxO gene.

Michigan State University will focus on the population genomic analysis of C. parasitica aiming to 1. characterize the NA populations of C. parasitica using a genome resequencing approach and VC typing; 2. Fine level characterization of C. parasitica in MI orchards and remnant American chestnut stands including population genomics and VC typing; 3. epidemiological study focused on spore release timing; 4. comparison of the efficacy of the natural CHV3-GH2 hypovirus with the super hypovirus donor (SD82+SD382/CHV1-EP713) to control the spread of C. parasitica. The CHV3-GH2 hypovirus is naturally found in Michigan and data is already available on the diversity of VC groups in Michigan C. parasitica populations (Springer et al., 2013). The Sakalidis lab maintains a collection of Michigan C. parasitica isolates and the CHV3-GH2 hypovirus. With the presence of domesticated chestnut varieties in an active and expanding chestnut fruit production system, naturalized American chestnut, populations of C. parasitica and a native hypovirus used in chestnut blight control Michigan is uniquely suited to studies focused in this tripartite model system of the host-pathogen-virus interaction in the environment. Michigan is unique in that it contains populations of C. parasitica in forest settings and a large commercial orchard industry (the largest in the US). This setting provides a unique environment to characterize gene flow between C. parasitica populations in domesticized chestnut and wild chestnut.

Rutgers will continue to identify and characterize new viruses found in isolates of C. parasitica from New Jersey forests. Our focus is especially on complex virus/fungus interactions that may result from multiple virus infections in a single fungal isolate or complex, multi-segmented viruses. Virus-transposon interactions may later be examined. For that project, single ascospore progeny have been selected from genetic crosses of a strain bearing complete, active copies of a hAT-like element (Linder-Basso et al., 2001) and a strain bearing a helitron copy that is predicted to be complete and possibly autonomous (Du et al., unpublished).

Biological characterization of several viruses identified from C. parasitica strains from northern and eastern New Jersey are being performed and will continue. Molecular characterization of viruses from isolates showing hypovirulent phenotype is proceeding following double-stranded RNA analysis, small RNA isolation and sequencing. Small RNA libraries from hypovirulent, virus-infected isolates and from their isogenic, virus-free counterparts are being sequenced commercially. Data returned to us are run through a pipeline that subtracts C. parasitica genomic sequence and identifies putative viral sequences. Through this methodology, we have recently identified 10 RNA viruses from the turfgrass dollar spot pathogen Clarireedia (Cohen et al., in prep).

A fruitful collaboration with Dr. Nobuhiro Suzuki continues. Hypovirulent C. parasitica isolate GH2 from Michigan contains a complex virus, CHV3-GH2, which comprises a genomic RNA segment and two accessory RNAs: a defective RNA segment, and two satellite RNA segments (Smart et al., 1999; Hillman et al., 2000; Yuan and Hillman, 2001). The roles of satellite and defective RNAs are well-studied in plant and animal virus systems, where they may have profound effects on virus pathogenesis, but are poorly understood in fungal viruses. We are now investigating the roles of the accessory segments in CHV3 biology.  During a study examining the behavior of several C. parasitica isolates in chestnut trees, Suzuki et al. (2021) noted that the CHV3-GH2 isolate used in the study was more virulent than had been previously reported. Subsequent work showed that the isolate contained only CHV3 genomic RNA and had lost the accessory satellite and defective RNAs. Through thorough examination of single conidial isolates and transmission experiments, we are initiating studies to define the specific roles of the defective and satellite RNAs in CHV3-GH2. A full-length cDNA clone of CHV3-GH2 RNA is already available. We will later generate and use infectious cDNA clones of CHV3-GH2 genomic RNA and the accessory RNAs to explore the roles of accessory RNAs in detail.

WVU has completed preliminary testing and analysis of methods of application and the behavior, stability and duration of a hypovirus superdonor strain in the forest (Stauder et al., 2019). While the work was a success and demonstrated the increased penetration of the hypovirus into the local C. parasitica population, there may be efficiency and effectiveness gains to be realized by using a different hypovirus. CHV1-EP713 is regarded as severe, in that the phenotype it causes in the fungal host is significantly debilitating and this may affect the ability of those infected strains to persist and spread. The related hypovirus CHV1-Euro7 has a reduced effect on the fungal host (Chen and Nuss, 1999) and may represent a more effective choice in the compromise between debilitating the host to minimize plant damage yet retaining sufficiently vigorous viability to promote hypoviral spread using the SD328/82 formulation. Additionally, B₃-F₃s being developed by TACF are predicted to have more resistance than C. dentata but less than C. mollissima. WVU hopes to test a superdonor strain with B₃-F₃s planted in the forest.

UMD will explore other CRISPR/Cas-9 editing methods that leave no footprint in the fungal genome except the intended mutation, such as through the use of transiently expressed plasmids containing both the Cas-9 and guide RNA sequences, or through the use of commercially prepared ribonucleoproteins.   Second UMD will work to further characterize the function of the CpSec66 gene by reinserting it with fluorescent tags into one of the CpSec66 knockout strains.  This will demonstrate whether the complementation restores the normal fungal phenotype, as well as to help confirm where the CpSec66 protein accumulates in hyphae. 

MissSU project goals fall into two categories:

Fungal Pathogenicity - Improve the available genome sequence data using long-read technology (MinION) and leverage this advance to identify sequences associated with the trait differences between strains SG2-3 and EP155Vegetative

Incompatibility – pathways and targets. Using the CpVIB-1-target recognition DNA sequences we plan to identify specific targets of the vegetative incompatibility signaling pathway triggered through VIB-1.

For all projects, the basic experimental methods to be employed will be similar and focused on removing target sequences from the genome to test hypotheses about the role of gene products in fungal biology. Gene knockouts will be made in the EP155 strain. The deletion constructs will utilize the more efficient UMD technology where possible but can be prepared using the method of Colot et al (2006) in which the flanking regions were assembled with the Hyg^r marker using a yeast-based recombination system, if necessary. Transformants will then be single-spored for nuclear homogeneity and verified by Southern blot and PCR. Analysis for phenotypic consequences will include chestnut pathogenicity assays and vegetative incompatibility testing.

Objective 3

USFS Delaware and partners will continue to monitor collaborative chestnut plantings to study the long-term success of the chestnuts in forested settings, in particular competitive dynamics among the planted chestnuts and naturally occurring woody vegetation.

Virginia Tech (Hession lab) has collected at TACF Meadowview and begun analyzing Lidar data with the help of J. Resop (University of Maryland). Preliminary results show few coarse scale correlations between genotype and size and growth dimensions phenotype. Further analysis is underway to derive high resolution point cloud-based metrics of branching patterns and percent canopy die-off with the help of additional computational packages. In addition, this group has begun to compare sensing methods which will allow to pinpoint the best timing to use specific sensors. Early results favor leaf-off season for aerial sensing for branching pattern calculation, however ground-based sensing during leaf-on has shown promising results in assessing proportion canopy die-off due its much higher resolution. In upcoming work at VDOF’s Lesesne State Forest, Hession’s group will be joined by Abhilash Chandel for the purpose of coupling Lidar and multi-spectral sensors – the latter of which will be used to search for foliar and stem spectral signatures that may differ among tree ancestries as well as tree stress by disease or abiotic factors.

Following small stem assays in 2023, TACF will be establishing a series of permanent common garden sites to evaluate various breeding and transgenic lines with parentage from across the historic range of C. dentata. By setting up each garden along the latitudinal gradient as a replicate reciprocal transplant, TACF will be able to gauge relative performance (i.e., blight response, growth form, competitive ability, fecundity) of various families, but also assess degrees of local adaptation, plasticity, and combinations of the two based on family origin. At the range boundaries (and potentially beyond, in the North), this reciprocal transplant will shed light on potential fitness trade-offs that could indicate limitations range movement.

With other collaborators, Penn State University (PSU) will with installation of common garden and provenance trial installation and measurement. Affiliates of TACF, PSU, and USFS will continue to update best management practices (BMPs) for private forest landowners, ensuring increased success in outplanting and reintroduction of disease-resistant populations. The results of outplantings established between 10-30 years ago offer ongoing data and results which are regularly incorporated into fact sheets, manuals, and other extension-like documentation made widely available to the public.

Regarding improvement of recommendations for appropriate site selection and habitat suitability of American chestnut, TACF and Villanova University will lead the creation of a publicly accessible on-line tool for forest land-owners. By first combining over one dozen models of habitat suitability for American chestnut, researchers will then contract with web-developers to publish to results in an easy-to-use and interactive format.

To further improve recommendations for reintroduction and reforestation populations which will lead to long-term success and ecosystem integration (Pierson et al 2007), researchers at Penn State and William and Mary combine genomic findings with population spread measured at three naturally regenerating sites of American chestnut.

For over 15 years, naturally regenerating American chestnut stands in Indiana, Vermont, and Maine have been thoroughly studied with over 5,000 individual trees tagged, mapped, and measured semi-annually.

In the summer of 2022, leaf-tissue from a small selection of those trees at all three sites was collected for DNA extraction. Of highest importance for collection were the "founder" trees, the first parents of each of trees that eventually carpeted the sites.

These three sites were specifically chosen because of their small Founder populations: anywhere from 3-5 individuals are thought to have derived these sites. DNA was extracted from those collected samples through the fall of 2022 and, in the winter of 2022-2023, the samples are being sent out for GBS, genotype-by-sequencing.

The data which are then retrieved from the GBS will require extensive reconstruction by a molecular genetics statistician. The result of this research has significant implications for American chestnut restoration. Planting hardwood trees is expensive and can be very difficult; therefore, practitioners will be seeking to plant the fewest trees possible while still ensuring long-term viability of any given population being planted in the forest.

Measurement of Progress and Results

Outputs

• The grand output would be restoration of the American Chestnut Tree to millions of hectares of forest in the Appalachian Mountains and environs. This might be accomplished by traditional breeding, genetic engineering, biocontrol or some mixture of the three. This output will not be achieved for 100 years or more. More immediate outputs during the proposal period are below.
• Public release of genetically diverse populations of blight-resistant chestnuts
• Combining of OxO blight resistance with PRR and blight resistance from Chinese chestnut
• Development of blight resistant chinquapin trees for reforestation
• Release of high-quality, chromosome-scale reference genomes for the Chinese chestnut and American chestnut
• Mapping of QTL for blight and PRR resistance in American chestnut backcross populations
• Development of a genetic marker panel to select for maximal inheritance of C. dentata or C. pumila ancestry, infer locally adapted provenance in C. dentata samples, select against the D58 founder genome, reconstruct pedigrees, and perform genomic selection for blight and PRR resistance.
• Assessment of the relative level of blight resistance in large surviving American chestnuts and elucidation of potential mechanisms of blight resistance in American chestnut
• Identification of biota invading chestnut cankers over time
• Development of individual based models (IBMs) of changes in chestnut canker severity over time
• Testing of the biocontrol potential of biota from chestnut cankers
• Further elucidation of the reasons for survival of LSAs
• A list of fungi growing within healthy tissue of American, Chinese, and hybrid chestnut trees
• Continued evaluation of the superdonor strain of C. parasitica for efficacy in controlling blight in in the forest
• Further characterization of transposons in C. parasitica and their effects
• Characterization of the targets of VIB-1 in triggering apoptosis during anastomosis of vegetatively incompatible colonies of C. parasitica
• Fine map QTL for pathogenicity in C. parasitica.
• Determination of tree establishment and growth at outplantings of blight-resistant chestnut in eastern forests
• Identification of factors impacting establishment of chestnut after planting
• Determination of blight incidence and severity at outplantings of blight-resistant chestnut in managed forests
• Continued assessment of the forest competitiveness of backcross hybrid chestnut as blight progresses at forest planting sites
• Corroboration and/or improvement of recommendations for installation of reintroduction plots of disease-resistant populations based on maximizing within-plot allelic diversity and minimizing founder effect.
• Establishment and ongoing maintenance of on-line landowner site-selection tool
• Assessment of relative performance of Chinese-American backcross, American-x-transgenic, and backcross-x-transgenic genotypes over nearly a decade in randomized controlled trials. This will include inoculation assays in the first year, and well as at 3 and 6 years of age. This will demonstrate whether or not short or longer terms gains are apparent from the combination of Chinese and transgenic source resistance.
• Identify technically, logistically, and economically feasible forms of high throughput remote sensing of tree growth form and/or ancestry and/or abiotic stress and/or disease stress. Allows for future precise and objective tree evaluation across sites, as well as elucidation of any potential growth/competition penalties to Chinese genome introgression in American backcross restoration populations.

Outcomes or Projected Impacts

• Greater and more consistent amounts of mast for animal and human consumption; for wildlife, this results in larger populations (Diamond et al., 2000, Lutts 2004).
• Substantial increase (up to 100%) in rates of timber growth and faster harvest rotations on currently unproductive mountain land (Jaynes and Graves 1963; Kuhlman 1978; Smith 2000).
• Incorporation of PRR resistance will broaden the niche of blight-resistant trees to include areas where PRR eliminated American chestnut prior to blight.
• A broad representation of genetic diversity from the wild population of C. dentata is crucial for adaptation to a large geographic range and a changing climate.
• Planting of millions of American chestnut trees will lead to modest increases in the carbon sequestration potential of hardwood forests in the Eastern U.S. (Gustafson et al., 2017)
• High quality annotated reference genome of C. dentata and C. mollissima will be crucial to better understand the biology of blight and PRR resistance and climate adaptation.
• Prediction of future blight severity in recovering stands of chestnut will identify stands where intervention is indicated.
• Identification and culture of biota in chestnut may provide materials to help control blight.
• Comparison of individual-based models (IBMs) and stands with differing predictions of future recovery may help identify promising biocontrol agents.
• Correlations of biota present in cankers differing in severity may identify promising biocontrol agents.
• Superdonor strains of C. parasitica hold promise for effecting biocontrol of blight on American chestnut similar to that observed on European chestnut because the barrier of multiple vc groups to spread of hypoviruses will have been minimized.
• Superdonors plus blight-resistant stock may yield better blight control than either alone and may help elucidate the relative effect of resistance and vc group on control.
• Transposons in C. parasitica may be important components of their evolution and may be helpful to biocontrol efforts.
• Fine mapping of QTL for pathogenicity in C. parasitica should identify genes for pathogenicity that can be tested by gene knockout. Knowledge of genes for pathogenicity and resistance in the pathogen and host, respectively may help elucidate their roles and lead to better disease control through resistance and pathogenicity modification.
• Characterization of VIB-1 and its gene targets will lead to improved understanding of the vegetative incompatibility process, in particular the molecular signals that lead to apoptosis and interrupt hypovirus transfer.
• Comparison of data from different forest plantings will uncover commonalities and patterns across studies, e.g., linking performance of planted seedlings under similar silvicultural treatments in different regions. Collectively, these plantings should lead to improved guidance for optimal seedling quality and planting conditions, pre- and post-planting management, and performance of hybrid chestnut generations. Tracking regional sources of American parents will help determine possible seed zones and the impacts of adaptation.
• Side-by-side evaluation of Ch-Am backcross, AmxTG, and backcross x TG genotypes will allow researchers and conservationists to make data-driven decisions in implementing single- or multiple-mechanism blight resistance in future work. This will determine the direction of multiple science programs for decades and the accompanying allocation of resources.
• The adoption of remote sensing and other techniques from precision agriculture for chestnut cultivation and evaluation is a matter of time. The studies proposed above are the first steps in calibrating these technologies to chestnut systems for the benefit of both conservation and agriculture alike.
• Federal regulatory approval of transgenic blight-tolerant American chestnuts will facilitate the future use of this and similar technologies to address pressing conservation needs.

Milestones

(2023):Receive federal approval from the USDA, EPA, and FDA for public release of the Darling 58 blight tolerant variety of American chestnut

(2023):Prepare publications on high quality chromosomal American and Chinese chestnut genome sequences. Include results on discovery of candidate genes for resistance to chestnut blight and phytophthora root rot through a combination of QTL mapping, RNA seq, and signatures of natural selection.

(2023):Develop a high throughput and cost-effective genotyping platform to assess species ancestry, perform genomic prediction for resistance, and minimize founder bottlenecks on genetic diversity in outcrosses between transgenic and wild type American chestnuts.

(2023):Directly compare the blight resistance of progeny of D58, best x best backcross trees, and large surviving American chestnuts using small stem assays for blight resistance.

(2023):Complete results on how much climate adaptive diversity from the wild population of C. dentata that TACF has represented in the breeding program.

(2023):Complete assays in order to determine whether endophytes increase resistance to C. parasitica in young American chestnut trees

(2023):Evaluate experiments on biota likely to affect chestnut canker severity, including on LSAs

(2023):Finish evaluating success or failure of superdonor strain first deployed into the forest in 2016

(2023):Complete long-read sequencing of C. parasitica strains EP155 and SG2-3 and begin pathogenicity determinant identification

(2023):Release TACF backcross hybrids to the public and publish

(2023):Analyze Chinese-source vs. TG-source vs. Chinese/TG-source blight response via small stem assays. Analyze Chinese-source vs. TG-source vs. Chinese/TG-source vs. LSA vs. American blight response in larger small stem assays.

(2023):Use machine learning and other advance computation to derive 3-dimensional and multi-spectral reflectance signatures of tree ancestry Am/Ch/hybrid and/or tree abiotic stress and/or tree blight stress.

(2023):Create on-line landowner site selection tool with ongoing improvements from baseline habitat suitability models

(2024):Collect C. parasitica isolates from a diverse selection of chestnut species and hybrids from orchards, wild stands, and restoration sites.

(2024):Analyze study designed to evaluate effect of site quality on chestnut competitive ability est. 2015 and publish

(2024):Analyze inbreeding quotient and founder effect at a minimum of three sites with naturally regenerating American chestnuts. Use those data to create reintroduction population recommendations.

(2025):Assess Chinese-source vs. TG-source vs. Chinese/TG-source blight response via inoculation at 3 years of age.

(2025):Characterize C. parasitica vegetative incompatibility in collected diverse isolates.

(2025):Between 2020-2025, breed D58 progeny with a range wide sample of 200 + wild type and backcrossed American chestnuts to diversify the blight tolerant population. Establish seed orchards composed of 5000+ trees that collectively have the capacity to produce 1 M open pollinated seed by age 10 years.

(2025):Between 2020-2025, breed D58 with PRR resistant backcross trees and establish ‘dual resistance’ orchards at sites where P. cinnamomi is present in the soil.

(2025):Between 2020-2025, conserve 200 + additional American chestnut genotypes through collection of seed and grafting of scion from wild trees

(2025):Between 2020-2025, induce grafted American chestnuts to flower by growing seedlings in long day conditions. Cryostore the pollen for future breeding to diversify blight resistant transgenic varieties of American chestnut.

(2026):Prepare collected diverse C. parasitica isolates for resequencing.

(2027):Assess Chinese-source vs. TG-source vs. Chinese/TG-source blight response via inoculation at 6 years of age.

(2027):Conduct experiments to compare the efficacy of the natural CHV3-GH2 hypovirus with the super hypovirus donor (SD82+SD382/CHV1-EP713) to control the spread of C. parasitica against on a diverse set of C. parasitica isolates.

(2027):Conduct virulence screening of several C. parasitica isolates against highly resistant chestnut germplasm.

(2028):Between 2024-2028 and starting from models trained in 2022-23, apply remote sensing and machine learning techniques to larger scale projects including semi-automated seedling screening, remote ‘uncontacted’ LSA detection, and wholesale orchard assessment.

(2028):Report findings of the mechanisms underlying the high blight resistance of Chinese chestnuts.

(2028):Report findings of the efficacy of hypovirus efficacy against a diverse set of C. parasitica isolates.

(2030):Compare Chinese-source vs. TG-source vs. Chinese/TG-source blight response in orchards and blight + competition response + fecundity in forest setting to help determine direction of future work. In the process, assess longer-term predictive capacity of seedling small stem assays

(2030):Between 2024-2030, generate 3 to 5 additional transgenic founder trees that contain the oxalate oxidase gene. Confirm expression, copy number and insert location. Induce pollen production through high light treatment and breed with wild type trees. Conduct blight resistance screening on the progeny using small stem assays. Apply for federal regulatory approval for founder lines with sufficient resistance.

(2030):Between 2024-2030, refine existing genetic transformation systems for American chestnut other than zygotic embryos, adaptable to diverse genotypes.

Projected Participation

View Appendix E: Participation

Outreach Plan

Since 2014, TACF annually publishes a summary of each NE meeting to showcase high-level advancements from the group into a public forum. In addition, detailed minutes, reports, and recordings from previous meeting are made available via a TACF/Penn State led site (PSU ESM Undated). The easy availability of these proceedings allow students, early-career scientists, as well as citizen-scientists access to a breadth of background information and knowledge otherwise unavailable (Double 2014).

Collaborations between NE-1833 project participants, NGOs, non-profits, and citizen scientists has led not only to research publications and applicable standards for on-the-ground forest tree conservation and restoration, but also strong good-will regarding this project as well a advancement via inclusion of a variety of outside perspectives.

While there are many more to be cited, we cover three major outcomes that have been enjoyed over the course of the NE-1833 project due to the strong collaborations and transparency of scientists participating in this project.

First, because of the major involvement of outside groups and major geographic expansion of American chestnut research ouplantings, the specialist chestnut bee, Andrena rehni, once thought extinct, was found on American chestnuts in Maryland (USGS 2019), Connecticut, and Massachusetts.

Second, the TACF and Virginia Tech-led genomics projects described here were made possible by the efforts of dozens of citizen-scientists participating in finding and documenting wild trees, preserving them ex situ, and then submitting samples for analysis. As described in the previous project, the TreeSnap program developed USFS, University of Tennessee, and University of Kentucky collaborators allowed tremendous expansion of that program. A small snapshot of citizen-scientist involvement was captured in a publication by Fitzsimmons and Alcorn (2022).

Finally, and regarding the process for requesting non-regulated status of the transgenic Darling 58, TACF and ESF embarked on a major campaign to encourage supporters to comment positively during the August 2020 and November 2022 public comment periods (PCP) opened by USDA. The stories, passion, optimism and hope for the future were consistently evident in the positive comments to USDA.  The chestnut community is committed to the mission and demonstrated their support by the sheer number of comments and the quality of many unique responses.

Between the August 2020 Public Comment Period (PCP) and the recently closed PCP regarding the draft EIS and PPRA, over 100 supporting agencies and organizations have submitted supportive comments. Especially noteworthy is the quality of the relationship with Federal and state agencies, particularly with the US Forest Service. In addition, organizations which previously had or still have formalized policies antagonistic toward genetically-modified organisms in general, have since submitted extremely supportive comments toward deregulation of Darling 58.

• The Sierra Club: https://www.regulations.gov/comment/APHIS-2020-0030-4145


• The Nature Conservancy:
  
  
  
  • https://www.regulations.gov/comment/APHIS-2020-0030-3813
  
  
  • https://www.regulations.gov/comment/APHIS-2020-0030-11539
  
  
  
  

A large number of professional scientists, foresters, and naturalists have commented. Of particular note is the outpouring of support and connection to family and place, often incredibly eloquent and heart-warming, that comes from the general public. In the August 2020 Public Comment Period (PCP), the support from unique, individual commenters was strongly supportive (62%), a trend that continued in the most recent period (86% of unique comments are supportive; 56% of comments were supportive when including submitted form letters).

Several common themes have become apparent as ESF and TACF staff and volunteers review comments in the current PCP:

1. Many commenters note a strong connection to both past and future family members, and that they wish to see deregulation of D58 to ensure a legacy or dream can come true for loved ones already lost or yet to come.
   
   
   
   1. In honor of my father who fought in WWII and brought back a love of chestnuts 
   
   
   2. I want my grandchildren to see what my father wanted but didn't live to see 
   
   
   3. I am 88 and remember my father talking about the loss of the American chestnut
   
   
   4. In honor of Sherret Chase
   
   
   
   


2. Many hikers, hunters, and other lovers of the natural world note their connection to the forests in which they regularly recreate, seeing the infected stems of dead and dying American chestnuts, and wanting to see them once again a vibrant part of the forest. (https://www.regulations.gov/comment/APHIS-2020-0030-11928)


3. Because humans spread the disease more rapidly than would have otherwise happened, humans need to solve the problem with all technology available. (https://www.regulations.gov/comment/APHIS-2020-0030-11459)


4. Many embrace the cause as a rare positive environmental story for the future, noting the technology should be used to save other species. (https://www.regulations.gov/comment/APHIS-2020-0030-8623)


5. Dozens of commenters note that, while they are typically skeptical of GMOs, the application of the technology to such an important project as American chestnut restoration, and especially one not directly tied to corporate profiteering, sways them to offer rare support:

(https://www.regulations.gov/document/APHIS-2020-0030-14439

https://www.regulations.gov/document/APHIS-2020-0030-13449

https://www.regulations.gov/document/APHIS-2020-0030-15600

6. Finally, and analogous to the daily calls received by staff persons at The American Chestnut Foundation, several hundred commenters note that they have land on which they want to plant D58 trees

(https://www.regulations.gov/comment/APHIS-2020-0030-8594)

These three anecdotes are only a small snippet if outreach results which have occurred because of the wide-reaching effects made possible by the decades-long scientific collaborative established via the NE chestnut group. Finding once-thought extinct species, engaging the public outdoors, and swaying public sentiment to embrace biotechnological tools for forest health have and will continue to pay dividends for conservation and restoration projects beyond this one focused solely on the American chestnut.

For the next iteration of this program, researchers intend to continue similar outreach and engagement with outside collaborators as well as the general public. The inclusion of stakeholders not within the immediate NE-program researchers has and will continue to be essential not only for research success and progress, but also the ongoing support enjoyed for the related programs.

Outplantings. 

Penn State has several silvicultural field trials, established with wild-type American chestnuts, examining various methods of installation and maintenance. Established in 1997 and 2005, a majority of these trials are now mostly removed by blight, but have provided a wealth of information regarding best establishment methods for seeds, seedlings, site preparation, long-term maintenance, herbivory control, etc. Results from these trials have been collated with findings from USFS partners to create two BMPs, updated in 2022, and made available to the public for review in anticipation of wide-scale American chestnut reforestation efforts.

TACF volunteers, some who are also Master Naturalists or Master Gardeners, are beginning to collect serious data on numerous plantings. With proper guidance, they could be a great resource and a wonderful opportunity to involve citizens in study of numerous aspects our natural world

In addition to Clark, Nelson and Pinchot, USDA Forest Service scientists contributing and being recruited to help install chestnut plantings in the forest include Labonte and Warwell. University personnel installing plantings include Brian McCarthy (Ohio University), Hill Craddock (UTC), Doug Jacobs (Purdue), Harmony Dalgleish (William and Mary), Michael Saunders (Purdue), Marty Cippolini (Berry College), Sunshine Brosi (Frostburg State University), Brian Roth (University of Maine), Tom Klak (University of New England) and Heather Griscom (James Madison University). TACF personnel include Jared Westbrook, Sara Fitzsimmons, Kendra Collins, Jamie Van Clief and Vasiliy Lakoba. Michael French is doing chestnut installations, primarily on minelands and with GFW (Green Forests Work).

Publicity. Members of the project will take advantage of the public outreach specialists at land-grant and non land-grant institutions, especially extension personnel, of whom we have a participant from PSU. Technical publications also are a form of outreach. Interested members of the public, aka stakeholders, were permitted to and have attended group meetings during past iterations of the project and we expect that to continue. A key component of the productivity of the group is the productive working relationship with the American Chestnut Foundation. With several members pf the group directly affiliated with the organization or with projects supported by it, layperson outreach is effectively handled through TACF and their staff in their publications and meetings.

Organization/Governance

The organization of the regional research project was established in accordance with the format suggested in the "Manual for Cooperative Regional Research". One person at each participating agency is designated, with approval of the agency director, as the voting member of the Technical Committee. Other agency individuals and interested parties are encouraged to participate as non-voting members of the committee. Each year, members elect a Chair-elect, whose duties begin the following year as Chair.

Literature Cited

 

Anagnostakis, S. L., and Day, P. R. 1979. Hypovirulence Conversion in Endothia parasitica. Phytopathology. 69: 1226.

Anagnostakis, S.L. and Waggoner, P.E. 1981. Hypovirulence, vegetative incompatibility, and the growth of cankers of chestnut blight. Phytopathology 71: 1198-1202.

Anagnostakis, S.L., Hau, B. and Kranz, J. 1986. Diversity of vegetative compatibility groups of Cryphonectria parasitica in Connecticut and Europe. Plant Disease 70: 536-538.

Aulia A., Hyodo, K., Kondo, H., Hisano, S., Hillman B.I., Suzuki N. 2021. Identification of an RNA silencing suppressor encoded by a symptomless fungal hypovirus, Cryphonectria Hypovirus 4. Biology 10:100.

Aulia, A., Andika, I.B., Kondo, H., Hillman, B.I., and Suzuki N. 2019. A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing. Virology 533: 99-107.

Barakat A., DiLoreto D.S., Zhang Y., Smith C., Baier K., Powell W., Wheeler N., Sederoff R., Carlson. J.E.  2009. Comparison of transcriptome from cankers and healthy stems in American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima). BMC Plant Biol. 9: 51-61.

Biella, S., Smith, M.L., Aist, J.R., Cortesi, P. and Milgroom, M.G. 2002. Programmed cell death correlates with virus transmission in a filamentous fungus.  Proc. R Soc. Lond. B. 269: 2269-2276.

Boland, G.J. 2004. Fungal viruses, hypovirulence and biological control of Sclerotinia species. Can. J. Plant Pathol. 26:6-18.

Brown, S.P., Clark, S.L., Ford, E., Jumpponen, A., Saxton, A.M., Schlarbaum, S. E., Baird, R. 2022. Comparisons of interspecies field performance of Fagaceae (Castanea and Quercus) planted in the southeastern United States with attention to soil fungal impacts on plant performance. Forest Ecology and Management 525: 120569.

Buck, K.W. 1986. Fungal virology-an overview. In: Buck, K.W., editor. Fungal Virology. Boca Raton, FL: CRC Press, p. 2-84.

Burgess T.I., Scott J.K., Mcdougall K.L., Stukely M.J.C., Crane C., Dunstan W.A., Brigg F., Andjic V., White D., Rudman T., Arentz F., Ota N. and Hardy G.E.S.J. 2017. Current and projected global distribution of Phytophthora cinnamomi, one of the world's worst plant pathogens. Glob Change Biol, 23: 1661-1674.

Butler, D. G., Cullis, B. R., Gilmour, A. R., and Gogel, B. J. 2018. ASReml‐R 4 reference manual: Mixed models for S language environments.  Training and development series, No QE02001, QLD Department of Primary Industries and Fisheries, Brisbane, QLD.

Carlson E., Stewart K., Baier K., McGuigan L., Culpepper T. & Powell W. 2022. Pathogen-induced expression of a blight tolerance transgene in American chestnut. Mol Plant Pathology 23: 370– 382.

Case, A. E., Mayfield III, A. E., Clark, S. L., Schlarbaum, S. E. and Reynolds, B. C. 2016. Abundance and Frequency of Asiatic oak weevil (Cyrtepistomus castaneus) on American, Chinese, and hybrid chestnut (Castanea) seedlings. J. Insect Science 16: 29:1–8.

Caswell, H. 2001. Matrix Population Models, 2nd ed. Sinauer Associates, Sunderland, Massachusetts.

Caten, C.E. 1972. Vegetative incompatibility and cytoplasmic infections in fungi.  J. Gen. Microbiol. 72: 221-229. Chen, B. and Nuss, D.L. 1999. Infectious cDNA clone of hypovirus CHV1-Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica. J. Virol. 73: 985-992

Cipollini, M.L., Moss J.P., Walker, W., Bailey, N., Foster, C., Reece, H., Jennings, C. 2021. Evaluation of an Alternative Small Stem Assay for Blight Resistance in American, Chinese, and Hybrid Chestnuts (Castanea spp.). Plant Disease 105: 576-584

Clark S.L., Schlarbaum S.E., Pinchot C.C., Anagnostakis S.L., Saunders M.R., Thomas-Van Gundy M., Schaberg P.G, McKenna, J., Bard, J.F., Berrang, P.C., Casey, D.M, Casey, C.E., Crane, B., Jackson, B.D., Kochenderfer, J.D., Lewis, R.F., MacFarlane, R., Makowski, R., Miller, M.D., Rodrigue, J.A., Stelick, J., Thornton, C.D. and Williamson, T.S. 2014. Reintroduction of American chestnut in the National Forest System. J. Forestry 112: 502–512.

Clark S.L., Schlarbaum, S.E., Pinchot, C.C., Anagnostakis, S.L., Saunders, M.R., Thomas-Van Gundy, M., Schaberg, P.G., Clark, S.L., Schweitzer, C.J., Schlarbaum, S.E., Dimov, L.D. and Hebard, F.V. 2010.  Nursery quality and first-year response of American chestnut (Castanea dentata) seedlings planted in the southeastern United States. Tree Planters’ Notes 53: 13-21.

Clark, S. L., Schlarbaum, S.E., Pinchot, C.C., Anagnostakis, S.L., Saunders, M.R., Thomas-Van Gundy, M., Schaberg, McKenna, J., Bard, J., Berrang, P., Casey, D.M., Casey, C.E., Crane, B., Jackson, B., Kochenderfer, J.,Lewis, R., MacFarlane, R., Makowski, R., Miller, M., Rodrigue, J., Stelick, J., Thornton, C. and Williamson, T. 2014.  American chestnut restoration in the National Forest System. J. Forestry 112: 505-512.

Clark, S.L., Schlarbaum, S.E., and Clark, J.D. 2019a. Restoring a forest icon: could returning the American chestnut remodel our wildlife landscape? The Wildlife Professional 13: 52-56.

Clark, S.L., Schlarbaum, S.E., Crane, B.S., Pinchot, CC., Schaberg, P.G., Thomas-Van Gundy, M. 2020. Restoration of the American chestnut will require more than a blight-resistant tree. In: Pile, L.S., Deal, R.L., Dey, D.C., Gwaze, D., Kabrick, J.M., Palik, B.J., Schuler, T.M., comps. The 2019 National Silviculture Workshop: a focus on forest management-research partnerships. Gen. Tech. Rep. NRS-P-193. Madison, WI: U.S. Department of Agriculture, Forest Service, Northern Research Station: 38-40.

Clark, S.L., Schlarbaum, S.E., Saxton, A.M. and Hebard, F.V. 2016. Establishment of American chestnuts (Castanea dentata) bred for blight (Cryphonectria parasitica) resistance: Influence of breeding and nursery grading. New Forests 47: 243–270. d

Clark, S.L., Schlarbaum, S.E., Saxton, A.M., Baird, R. 2019b. Eight-year blight (Cryphonectria parasitica) resistance of backcross-generation American chestnuts (Castanea dentata) planted in the southeastern U.S. Forest Ecology and Management 433: 153-161.

Clark, S.L., Schlarbaum,S.E., Saxton, A.M. and Hebard, F.V.  2012.  Testing blight resistant American chestnuts [Castanea dentata (Marsh.) Borkh.] in commercial nurseries.  Forestry 85: 589-600.

Colot, H.V., Park, G., Turner, G.E., Ringelberg, C., Crew, C.M., Litvinkova, L., Weiss, R.L., Borkovich, K.A. and Dunlap, J.C. 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA 103: 10352-10357.

Coughlin, E.M., Shefferson, R.P., Clark, S.L., and Wurzburger, N. 2020. Plant-soil feedbacks and the introduction of chestnut hybrids to eastern forests. Restoration Ecology. 29: e13326.

Crouch, J. A., Dawe, A., Aerts, A., Barry, K., Churchill, A. C. L., Grimwood, J., et al. 2020. Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen. Phytopathology 110: 1180–1188.

Davelos Baines, A.L., Eager, E.A. and Jarosz, A.M. 2014. Modeling and analysis of American chestnut populations subject to various stages of infection.  Letters in Biomathematics 1: 235-247.

Davelos, A.L. and Jarosz, A.M. 2004. Demography of American chestnut populations: effects of pathogen and a hyperparasite. J. Ecology 92: 675-685.

Dementhon, K., Iyer, G. and Glass, N.L. 2006. VIB-1 is required for expression of genes necessary for programmed cell death in Neurospora crassa. Eukaryot. Cell 5: 2161-2173.

Diamond, S.J., Giles, R.H., Kirkpatrick, R.L., and Griffin, G.J. 2000. Hard mast production before and after the chestnut blight. Southern Journal of Applied Forestry 24: 196-201.

Double, M. 2014. New Faces at TACF USDA Regional Research Project, NE-1333. Chestnut 28: 4.

Double, M.L., A. M. Jarosz, D. W. Fulbright, A. Davelos Baines, and W. L. MacDonald. 2018. Evaluation of two decades of Cryphonectria parasitica hypovirus introduction in an American chestnut stand in Wisconsin. Phytopathology 108: 702-710.

Dutech, C., Barrès, B., Bridier, J., Robin, C., Milgroom, M. G., and Ravigné, V. 2012. The chestnut blight fungus world tour: successive introduction events from diverse origins in an invasive plant fungal pathogen. Mol. Ecol. 21: 3931–3946.

Elwood, EC, Lichti, NI, Fitzsimmons, SF, Dalgleish, HJ. 2018. Scatterhoarders drive long- and short-term population dynamics of a nut-producing tree, while pre-dispersal seed predators and herbivores have little effect. J Ecology 106: 1191– 1203.

Fitzsimmons, S. and Alcorn, J. Spring 2022. Tracking And Documenting Chestnut Tree Locations. Chestnut. 36: 7-11.

Graziosi, I. and Rieske, L.K. 2013. Response of Torymussinensis, a parasitoid of the gallforming Dryocosmus kuriphilus, to olfactory and visual cues. Biological Control 67:137-142.

Graziosi, I. and Rieske, L.K. 2015. Can plant pathogens advantageously utilize insects as resources? Agricultural and Forest Entomology 17: 366-374.

Griffin, G.J., Elkins, J.R., McCurdy, D., and Griffin, S.L. 2005. Integrated use of resistance, hypovirulence, and forest management to control blight on American chestnut.  Pages 97-107 in: Steiner K.C. and J.E. Carlson, eds.  Restoration of American Chestnut to Forest Lands-Proceedings of a Conference and Workshop. May 4-6, 2004, The North Carolina Arboretum, Natural Resources Report NPS/NCR/CUE/NRR-2006/001, National Park Service, Washington, DC.

Gustafson, E.J., de Bruijn, A., Lichti, N., Jacobs, D.F., Sturtevant, B.R., Foster, J., Miranda, B.R., Dalgleish, H.J. 2017. The implications of American chestnut reintroduction on landscape dynamics and carbon storage. Ecosphere. 8: e01773

Gustafson, E.J., Miranda, B.R., Dreaden, T.J., Pinchot, C.C., and Jacobs, D.F. 2022. Beyond Blight: Phytophthora Root Rot under Climate Change Limits Populations of Reintroduced American Chestnut. Ecosphere 13: e3917.

Hall, C.J., Welch, J., Kowbel, D.J. and Glass, N.L. 2011. Evolution and diversity of a fungal self/non-self recognition locus. PLoS ONE. 5: el4055.

Heiniger, U. and Rigling, D. 1994. Biological control of chestnut blight in Europe. Annu. Rev. Phytopathology 32: 581-599.

Hillman, B. I., Foglia, R., and Yuan, W. 2000. Satellite and defective RNAs of Cryphonectria hypovirus 3, a virus species in the Family Hypoviridae with a single open reading frame. Virology 276: 181-189.

Hillman, B.I. and Suzuki, N.  2004. Viruses of the chestnut blight fungus, Cryphonectria parasitica. Adv. Virus Res. 63: 423-472.

Hillman, B.I., Aulia, A., and Suzuki, N. 2018. Viruses of plant-interacting fungi. Advances in Virus Research 100: 99-116.

Jaynes, R.A., & Graves, A.H. 1963. Connecticut hybrid chestnuts and their culture. Connecticut Agricultural Experiment Station Bulletin 657 New Haven, 29 pp.

Kim, K. S., Min, J.-Y., and Dickman, M. B. 2008. Oxalic Acid Is an Elicitor of Plant Programmed Cell Death during Sclerotinia sclerotiorum Disease Development. Mol. Plant-Microbe Interactions. 21: 605–612.

Knapp, B.O., Wang, G.G., Clark, S.L., Pile, L.S. and Schlarbaum, S.E. 2014. Leaf physiology and morphology of Castanea dentata (Marsh.) Borkh., Castanea mollissima Blume, and three backcross breeding generations planted in the southern Appalachians, USA. New Forests 45:283-293.

Kuhlman, E.G. 1978. The devastation of American chestnut by blight. Pages 1-3 in: W.L. MacDonald, F.C. Cech, J. Luchok, and C. Smith, eds., Proceedings of the American Chestnut Symposium. West Virginia University, Morgantown.

Lefkovitch, L.P. 1965. The study of population growth in organisms grouped by stage. Biometrics 21: 1-18.

Linder-Basso, D., Foglia, R., Zhu, P., and Hillman, B.I. 2001. Crypt1, an active Ac-like transposon from the chestnut blight fungus, Cryphonectria parasitica. Mol Gen Genomics 265: 730-738.

Lutts, R.L. 2004. Like manna from God: The American chestnut trade in southwestern Virginia. Environmental History 9: 497-525.

MacDonald, W.L. and Fulbright, D.W. 1991. Biological control of chestnut blight: Use and limitations of transmissible hypovirulence. Plant Dis.75: 656-661.

Majtnerová, P., and Roušar, T. 2018. An overview of apoptosis assays detecting DNA fragmentation. Mol. Biol. Rep. 45: 1469–1478.

Mittempergher, L. 1978. The present status of chestnut in Italy. Pages 34-37 in: MacDonald, W.L., Cech, F.C., Luchok, J., and Smith, C., eds., Proceedings of the American Chestnut Symposium, West Virginia University Press, Morgantown, WV.

Newhouse A.E., Powell W.A. 2021. Intentional introgression of a blight tolerance transgene to rescue the remnant population of American chestnut. Conservation Science and Practice. 3: e348.

Newhouse, A. E., Coffey, V. C., McGuigan, L. D., Oakes, A. D., Breda, K. M., Matthews, D. F., Drake, J. E., et al. 2020. Petition for determination of nonregulated status for blight-tolerant Darling 58 American chestnut (Castanea dentata). (Petition 19-309-01p_a1). USDA-APHIS-BRS, Washington, DC.

Newhouse, A. E., Polin-McGuigan, L. D., Baier, K. A., Valletta, K. E. R., Rottmann, W. H., Tschaplinski, T. J., et al. 2014. Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny. Plant Sci. 228: 88–97.

Newhouse, A. E., Spitzer, J. E., Maynard, C. A., & Powell, W. A. 2014a. Chestnut leaf inoculation as a rapid predictor of blight susceptibility. Plant Disease. 98: 4–9.

Newhouse, A.E., McGuigan, L.D., Baier, K.A., Valletta, K.E., Rottmann, W.H., Tschaplinski, T.J., Maynard, C.A. and Powell, W.A. 2014b. Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny. Plant Science 228: 88-97

Noah, P.H., Cagle, N.L., Westbrook, J.W., Fitzsimmons, S.F., 2021. Identifying Resilient Restoration Targets: Mapping and Forecasting Habitat Suitability for Castanea dentata in Eastern USA under Different Climate-Change Scenarios. Climate Change Ecology 2:100037.

Penn State University, Ecosystem Science and Management. Undated. USDA CSREES Northeast Regional Projects – Chestnut.

Pierson S.A.M., Keiffer C.H., McCarthy B.C., and Rogstad S.H. 2007. Limited reintroduction does not always lead to rapid loss of genetic diversity: an example from the American Chestnut (Castanea dentata; Fagaceae). Restoration Ecology. 15: 420-429

Pinchot, C., Schlarbaum, S., Tepke, S. 2020b. Using oak silviculture to reintroduce American chestnut. Chestnut: The Journal of The American Chestnut Foundation. 34: 26-28.

Pinchot, C.C., A.A. Royo, Stanovick, J.S., Schlarbaum, S.E., Sharp, A.M. and Anagnostakis, S.L. 2022. Deer browse susceptibility limits chestnut restoration success in northern hardwood forests. Forest Ecology and Management 523: 120481.

Pinchot, C.C., Clark, S.L., Schlarbaum, S.E., Saxton, A.M., Sung, Shi-Jean S., and Hebard, F.V.  2015.  Effects of temporal dynamics and nut weight and size effects on growth of American chestnut, Chinese chestnut, and backcross generations in a commercial nursery.  Forests 6: 1537-1556.

Pinchot, C.C., Royo, A.A., Schlarbaum, S.E., Peters, M.P., Sharp, A.M. and Anagnostakis, S.L., 2020a. The effect of site quality on performance of American Chestnut (Castanea dentata) seedlings bred for blight (Cryphonectria parasitica) resistance. In: Publication: Gen. Tech. Rep. SRS-252. Asheville, NC: US Department of Agriculture Forest Service. Southern Research Station, pp.100-107.

Pinchot, C.C., Schlarbaum, S.E., Clark, S.L., Saxton, A.M., Sharp, A.M., Schweitzer, C.J. and Hebard, F.V. 2017.  Growth, survival, and competitive ability of chestnut (Castanea Mill.) seedlings planted across a gradient of light levels. New Forests 48: 491-512.

Powell W. A., Newhouse A.E., & Coffey V. 2019. Developing blight-tolerant American chestnut trees. Cold Spring Harbor Perspectives in Biology, 1–16: a034587.

Reazin, C., Baird, R. Clark, S.L., and Jumpponen, A. 2019. Chestnuts bred for blight resistance depart nursery with distinct fungal rhizobiomes. Mycorrhiza 29: 313-324.

Rogstad, S. H., and S. Pelikan. 2014.  Restoring the American Chestnut:  optimizing founder spacing to promote population growth and genetic diversity retention. Restoration Ecology. 22: 668-675.

Sandercock A. M., Westbrook J. W., Zhang Q., Johnson H. A., Saielli T. M., Scrivani J. A., Fitzsimmons S. F., Collins K., Perkins M. T., Craddock J. H., Schmutz J., Grimwood J., & Holliday J. A. 2022. Frozen in time: Rangewide genomic diversity, structure, and demographic history of relict American chestnut populations. Molecular Ecology, 31: 4640– 4655.

Saupe, S.J. 2000. Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.  Micobiol. Mol. Biol. Rev. 64: 489-502.

Short, D. P. G., Double, M., Nuss, D. L., Stauder, C. M., MacDonald, W., and Kasson, M. T. 2015. Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States. Appl. Environ. Microbiol. 81: 5736–5742.

Smart, C.D., Yuan, W., Foglia R., Nuss, D.L., Fulbright, D.W. and Hillman, B.I. 1999. Cryphonectria hypovirus 3, a virus species in the family Hypoviridae with a single open reading frame. Virology 265: 66-73.

Smith, D.M. 2000. Ill-fated monarch of the eastern hardwood forest. Journal of Forestry 98:12- 16.

Springer, J. C., Baines, A. L. D., Fulbright, D. W., Chansler, M. T., and Jarosz, A. M. 2013. Hyperparasites Influence Population Structure of the Chestnut Blight Pathogen, Cryphonectria parasitica. Phytopathology. 103: 1280–1286.

Stauber, L., Badet, T., Feurtey, A., Prospero, S., and Croll, D. 2021. Emergence and diversification of a highly invasive chestnut pathogen lineage across southeastern Europe. eLife. 10: e56279. Stauder, C.M., Nuss, D.L., Zhang, D.-X., Double, M.L., MacDonald, W.L., Metheny, A.M. and Kasson, M.T. 2019. Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes. Virology, 528: 1-6.

Steiner, K.C., Westbrook, J.W., Hebard, F.V., Georgi, L., Powell, W.A. and Fitzsimmons, S.  2017. Rescue of American chestnut with extra specific genes following its destruction by a naturalized pathogen. New Forests 48: 317-336.

Suzuki N, Cornejo C, Aulia A, Shahi S, Hillman BI, Rigling D. 2021. In-tree behavior of diverse viruses harbored in the chestnut blight fungus, Cryphonectria parasitica. J Virology 95: e01962-20.

Tabima, J. F., Søndreli, K. L., Keriö, S., Feau, N., Sakalidis, M. L., Hamelin, R. C., et al. 2019. Population Genomic Analyses Reveal Connectivity via Human-Mediated Transport across Populus Plantations in North America and an Undescribed Subpopulation of Sphaerulina musiva. Mol. Plant-Microbe Interactions. 33: 189–199.

VanRaden, P. M. 2008. Efficient Methods to Compute Genomic Predictions. Journal of Dairy Science 91: 4414-4423.

Vijayaraghavareddy, P., Adhinarayanreddy, V., Vemanna, R. S., Sreeman, S., and Makarla, U. 2017. Quantification of Membrane Damage/Cell Death Using Evan’s Blue Staining Technique. Bio-Protocol. 7: e2519.

Westbrook J.W., Holliday J.A., Newhouse A.E., Powell W.A. 2020. A plan to diversify a transgenic blight-tolerant American chestnut population using citizen science. Plants, People, Planet, 2: 84-95.

Westbrook, J. W., Zhang, Q., Mandal, M. K., Jenkins, E. V., Barth, L. E., Jenkins, J. W., et al. 2020. Optimizing genomic selection for blight resistance in American chestnut backcross populations: A trade‐off with American chestnut ancestry implies resistance is polygenic. Evol. Appl. 13: 31–47.

Westbrook, J.W., James, J.B., Sisco, P.H., Frampton, J., Lucas, S., Jeffers S.N. 2019. Resistance to Phytophthora cinnamomi in American Chestnut (Castanea dentata) Backcross Populations that Descended from Two Chinese Chestnut (Castanea mollissima) Sources of Resistance. Plant Disease 103: 1631-1641

Yu, X., Li, B., Fu, Y., Xie, Y., Cheng, J., Ghabrial, S.A., Li, G., Yi, X. and Jiang, D. 2013. Extracellular transmission of a DNA mycovirus and its use as a natural fungicide.  Proc. Natl. Acad. Sci. USA. 110: 1442-1457.

Yuan, W. and Hillman, B. I. 2001. In vitro translational analysis of genomic, defective, and satellite RNAs of Cryphonectria parasitica hypovirus 3-GH2. Virology 281: 117-123.

Zhang, D.X. and Nuss, D.L. 2016. Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes.  Proc. Natl. Acad. Sci. USA. 113: 2062-2067.

 

Attachments

Land Grant Participating States/Institutions

KY, NJ, SC

Non Land Grant Participating States/Institutions

American Chestnut Foundation