Annual/Termination Reports:

[11/20/2024]

Report Information

Participants

Albert Abott (USFS), Andrew Albert, Kaitlin Breda (ESF), Erik Carlson (ESF), Kendra Collins (TACF), Hill Craddock (UTC), Angus Dawe (MSU), Mark Double (WVU), John Dougherty, Robert Eckenrode, Patrícia Fernandes (ESF), Sara Fitsimmons (PSU), Fred Hebard (TACF), Molly Hickey (SYR), Emily Holliman (WVU), Steven Jakobi, Steven Jeffers (Clemson), Susanna Keriö (CT-AG), Thomas Klak (UNE), Bruce Levine (UMD), William L MacDonald (WVU), Nora MacDonald, Dakota Matthews (ESF), Virginia May (UNE), Brian McClain, Linda McGuigan (ESF), Amy Metheny (WVU), Danielle Mikolajewski (WVU), Mohammad Mostofa (ESF), Charles Dana Nelson (USFS_, Andrew Newhouse (ESF), Maya Niesz Kutsch (ESF), Jacob Olichney (ESF), Hannah Pilkey (ESF), Cassie Stark (TACF), Sophia Suriano (ESF), Jamie Van Clief (TACF), Noah Vincent (PSU), Taylor Wegner (SRY), Jared Westbrook (TACF) and Jeff Zarnowski

Brief Summary of Minutes

The 2024 NE-2333 meeting was held in Syracuse New York on September 13th-14th, hosted by SUNY College of Environmental Science and Forestry. Presentations represented research from Connecticut, Kentucky, Maine, Maryland, Mississippi, New York, North Carolina, South Carolina, Tennessee, Virginia, and West Virginia.

This is the first meeting under the 2333 project, and an estimated forty people attended. Members still need to sign up, and an effort should be made to encourage new researchers working on chestnuts to join. Those interested should make sure to apply to Appendix E.

Susanna Keriö at the Connecticut Agricultural Experiment Station is hosting next year in mid-August and is now chair-elect.

Accomplishments:

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

Kentucky

USFS, Forest Health Research and Education Center, Lexington, KY

Dana Nelson: SRS chestnut genetics update: blight resistance mapping, conservation through grafting, and satellite imaging

Genetic mapping blight resistance for marker-assisted selection (MAS): We published one paper on this in 2024 (Fan et al. 2024) and have another one in the later stage of preparation (Thomas et al. in prep). The later analysis includes two advanced GWAS models (FarmCPU and BLINK) that adjust for false positive associations. These models identified 20 QTNs (significant SNPs after multiple comparison adjustment) for stem canker size. We then estimated the QTL size around each QTN by testing adjacent SNPs for linkage disequilibrium (LD). This resulted in 20 QTLs (with left and right borders mapped to the Ellis genome), accounting for ~6% of the genome and 23% of the variation in canker size. With this information in hand, we are developing a model-free MAS method. With this method, for any tree of American (AC) and Chinese chestnut (CC) hybrid ancestry, we genotype its markers within the 20 QTLs and predict its blight resistance based on the allelic content of these markers (i.e., the higher the CC content the more blight resistant). We can also weight the prediction based on the effect size of the QTL. At the same time markers outside of the QTLs can be genotyped to assess their AC vs. CC content, aiming to select those trees with high predicted blight resistance and high AC content outside of the QTLs. We are collaborating with Jared Westbrook (TACF) to test this method in an independent data set.

Nut-grafting for American chestnut germplasm conservation: We completed our third year of nut grafting to conserve surviving American chestnut genotypes of Mississippi and northwest Alabama origin. Results were similar to the first two years (Burdine et al. 2023) with grafts performing significantly better on Chinese chestnut (CC) rootstock than on American (AC). The overall graft take rate was 66% of CC rootstock and 35% on AC rootstock. The CC rootstocks were collected by mother-tree cultivar and we found significant difference among them with ‘Hong Kong’ performing the best (88% take) and ‘Jersey Gem’ the worst (38% take). Over the years the correlation in percent take for cultivars is positive but not significant, so more testing will be needed to confidently evaluate these cultivars for their use as root stocks for American chestnut germplasm conservation.

Cytogenetic comparison of the rDNA regions of American and Chinese chestnut: Dr. Faridi and collaborators published an important paper in plant cytogenetics this year (Islam-Faridi et al. 2024) cytogenetically characterizing the major rDNA locus (nucleolar organizing region, NOR) and its associated satellites in American (AC) and Chinese (CC) chestnuts. This locus encodes the major ribosomal RNA genes whose transcripts are structural and functional elements of ribosomes, the macromolecular machines of protein synthesis. The CC NOR appears to contain more rDNA copies than the AC. Furthermore, the results clearly suggest that the CC satellite contains a substantial amount of euchromatin (i.e., gene-rich DNA) compared to AC that appears to have very limited amounts. In addition, the CC has a large satellite region distal to its NOR, while AC does not. Whether these large structural and compositional differences between the species’ satellites have an impact on backcross or intercross hybrid performance (two copies of AC vs. one or none) remains to be determined. Molecular methods are needed test to a large number of hybrid trees to evaluate the satellite’s effect on tree performance.

USFS, Forest Health Research and Education Center, Lexington, KY

Albert Abbott: An update on Phytophthora cinnamomi and resistance studies in chestnut

To explore the genetic architecture of Phytophthora cinnamomi resistance in Chinese chestnut, researchers conducted QTL mapping on 957 hybrid progenies derived from three Chinese/American hybrid crosses. Using sequence-based markers, they constructed eight parental linkage maps, identifying 17 QTLs on four linkage groups, with the most consistent QTLs located on LG_E and LG_K. Candidate genes within these QTL intervals were identified through RNA sequencing (RNAseq) of root tissues subjected to Pc zoospore inoculation, revealing differential expression patterns associated with resistance.

RNAseq analysis of root samples showed distinct gene expression patterns between Chinese (resistant) and American (susceptible) chestnuts. Chinese chestnut roots exhibited an early, robust defense response, with most genes differentially expressed between 6–12 hours post-inoculation, tapering off by 24 hours. Conversely, American chestnut roots had a delayed response, with increasing gene expression changes continuing through 24 hours. This suggests that Chinese chestnut rapidly recognizes and arrests Pc development, while American chestnut fails to initiate an effective early defense, allowing Pc to progress to a necrotrophic phase.

Based on these findings, researchers identified four candidate resistance genes in Chinese chestnut, including those encoding receptor-like protein kinases, a tetrahydroberberine oxidase, and an ethylene-forming enzyme. In American chestnut, no strong susceptibility genes were identified, though potential interaction factors were noted, such as a JA-signaling repressor and an acyltransferase. These candidates are undergoing validation through RT-PCR and protein-protein interaction studies to better understand host-pathogen interactions, laying the groundwork for advanced Pc resistance strategies in chestnuts.

MAINE

University of New England (UNE)

Virginia May: Comparative blight (Cryphonectria parasitica) resistance of the American chestnut (Castanea dentata) in Maine transgenic orchard

In an effort to determine Cryphonectria parasitica resistance of transgenic Darling 54 American chestnuts, we utilized an established procedure from Cipollini et al.'s 2021 paper. I inoculated 208 saplings, Chinese, with negative, and positive transgenic siblings, with cultured blight and measured their response over 90 days. My results found a significant reduction in the orange zone and canker size of positive transgenic American chestnuts compared to non-transgenic siblings. There was no significant difference between the positive or negative transgenics compared to the Chinese chestnut controls which have natural blight tolerance. However, we did see significant differences in heights, measured in August 2024, between positive transgenic trees and Chinese chestnuts. The Chinese chestnuts were significantly shorter than the blight resistant American chestnuts.

Overall, I will continue my analysis to see if there are any significant differences in blight resistance among American mother groups. I will also measure my 90-day post inoculation measurements of the eight DarWin saplings inoculated to see how they compare to the transgenics and controls.

NEW YORK

The State University of New York, College of Environmental Science and Forestry

SUNY-ESF American Chestnut Research & Restoration Project

Andy Newhouse: ESF Transgenic Chestnut Update

The transgenic American chestnut project at ESF has made significant strides, including updates on the Darling line. The transgenic line previously referred to as ‘Darling 58’ is now corrected to ‘Darling 54,’ reflecting a difference in the transgene insertion site, with regulatory agencies notified accordingly. Regulatory reviews with the USDA, FDA, and EPA continue, with nearly 7,000 pages of documentation submitted. Preliminary assessments from APHIS confirm no risks associated with Darling 54, consistent with earlier findings.

Studies show that Darling 54 trees grow more slowly than their non-transgenic relatives, though the difference varies by family. Research indicates this growth difference is not simply due to the metabolic cost of transgene expression, and ongoing efforts aim to better understand and address this issue. Seasonal and multi-family studies confirm stable transgene expression without silencing over at least five generations. Additionally, homozygous Darling 54 trees have been successfully produced via tissue culture and embryo rescue, and pollinations in 2024 resulted in inheritance rates exceeding 99%.

Environmental and safety research continues, focusing on soil microbial communities, land use changes for restoration, and fire ecology. Controlled inoculation studies demonstrate that Darling 54 trees respond better to chestnut blight, producing blocky cankers with less impact compared to non-transgenic trees. Additional research includes backcrossing Darling 54 with Ozark chinquapin, testing transgenic elm trees for tolerance to Elm Yellows, and collaborating with the NYS Department of Environmental Conservation to address Beech Leaf Disease.

Challenges remain in monitoring canker development due to the decline of non-transgenic trees, though variability in canker appearance suggests less severe impacts on transgenic trees. Restoration efforts prioritize diversifying genetic lines to adapt to site-specific variability. Future goals include refining oxalate oxidase (OxO) expression, selecting and improving families for growth and resistance, and expanding work to other species like beech and elm. These advancements underscore the project’s progress and its contributions to forest restoration and tree health.

Erik Carlson: Investigating new transgenes for chestnut blight tolerance

Research on enhancing resistance to chestnut blight continues to explore various mechanisms, including oxalic acid detoxification and RNA interference (RNAi). Oxalate oxidase (OxO), alone produces hydrogen peroxide, which can trigger a hypersensitive response. Researchers are exploring the effects of stacking OxO with additional enzymes, such as oxalate decarboxylase (ODC), which breaks down oxalic acid without generating hydrogen peroxide. Potential sources for ODC include the fungus Flammulina velutipes, a non-toxic, saprophytic species.

Host-induced gene silencing (HIGS) via RNAi is another promising avenue. This approach uses plant-expressed double-stranded RNA to target and suppress specific pathogen genes, reducing virulence. Studies show that knocking out the oxaloacetate hydrolase (OAH) gene in CP reduces its virulence, but regulatory challenges arise with pathogen gene insertions. Instead, researchers are investigating alternative targets, including fitness-related genes like Arv-1, whose knockout debilitates fungal colonies, and virulence-related genes such as GPI-AP, Cyp1, and CPLC.

At SUNY ESF, new genetic resistance strategies are under development, including additional oxalate-detoxifying enzymes and transgenic chestnut lines expressing dsRNA for RNAi-mediated resistance. These approaches aim to improve tolerance to chestnut blight while overcoming regulatory hurdles. The integration of multiple strategies, including PRR resistance mechanisms, offers the potential for broader applications against other diseases, such as gall wasp infestations, brown rot, and laurel wilt. Current efforts also include testing the uptake of RNAi sprays using fluorescent markers and examining their efficacy in fungal disease management.

Patrícia Fernandes: Ozark chinquapin and Phytophthora cinnamomi research update

Patrícia reported on the advances to develop blight-tolerant Ozark chinquapin. The study of F1 Ozark x Darling hybrids suggests that introgressing the oxo gene from transgenic chestnuts may be a successful approach to increase blight resistance in Ozark chinquapins. Controlled inoculations of 2-year-old seedlings with Cryphonectria parasitica showed that the progression of canker length over time and stem survival in transgenic hybrids was significantly slower and higher, respectively, in transgenic hybrids compared to non-transgenic. Pollen from transgenic hybrids was produced and used to pollinate Ozark chinquapins to obtain the first backcross generation, which will be the next focus of our study. We are also studying 27 transgenic Ozark chinquapin events obtained via Agrobacterium-mediated transformation. Patrícia also reported results from a recent publication focused on understanding susceptible and resistant chestnut responses to Phytophthora cinnamomi.

NORTH CAROLINA

The American Chestnut Foundation, National

Dr. Jared Westbrook: Optimized breeding and genomic selection to improve disease resistance in American chestnut

Significant progress has been made in breeding chestnuts with enhanced resistance to Phytophthora root rot (PRR) and Cryphonectria parasitica while capturing genetic diversity without regulatory constraints. Researchers have identified trees with high levels of American chestnut traits and blight tolerance, aiming for an average blight index of 65. However, challenges remain, as Chinese chestnut, the primary source of resistance genes, lacks the height and competitive traits of American chestnuts. PRR resistance, a growing concern, is more complex, involving both major genes and polygenic factors. Currently, about 30% of the Chinese chestnut genome is linked to resistance, and some trees with limited Chinese ancestry show promising resistance. Testing for PRR remains limited due to its patchy spread.

Advances in genomic tools, such as DArT for cost-effective genotyping across ~12,000 markers, have supported the evaluation of ancestry, resistance traits, and genetic compatibility. Genomic prediction models show 95% accuracy for blight resistance and 75% for PRR, with speed breeding being explored to accelerate progress. Modeling suggests two generations are needed for blight resistance, one for PRR, and three for both traits. Male sterility, caused by cytoplasmic-nuclear gene incompatibility, is a minor complication. Trials will be needed across the chestnut’s range, and local TACF chapters may play a key role in scaling these efforts. The work leverages substantial investments in genotyping to support a long-term strategy for chestnut restoration.

SOUTH CAROLINA

Clemson University

Steve Jeffers: South Carolina Report, Chestnut Research at Clemson University

Clemson University’s has ongoing research into Phytophthora root rot (PRR) in collaboration with The American Chestnut Foundation (TACF), and USDA (though some partnerships are temporarily paused). The work focuses on isolating Phytophthora cinnamomi from soil and dead trees, refining techniques to improve detection. Using less soil in experiments has proven effective, as zoospores struggle to reach the surface. Data collected since 2003 across 12 states revealed a 40% positive detection rate, identifying four Phytophthora species and some unknown isolates. The team is also exploring PRR impacts in a white oak study and has identified three phenotypes of  P. cinnamomi. Fungicide trials on genetically engineered chestnuts (GCOs) have begun, testing products like Reliant, Aliette, Subdue MAXX, and Ridomil in greenhouse and field settings.

Efforts to understand host resistance involve inoculating plants and analyzing responses at specific time points (3, 6, 12, and 24 hours). Trials include soil solution inoculation, studies comparing Chinese (CH) and American (AM) chestnuts, and freezing roots for detailed analysis by Kentucky State University (KYU). F2 populations are being tested in batches to evaluate resistance traits further. These multi-faceted approaches aim to improve detection, develop effective fungicides, and deepen understanding of host resistance to support chestnut restoration.

 

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.

MARYLAND

The University of Maryland

Bruce Levine: High-efficiency marker-free gene editing in Cryphonectria parasitica

Bruce Levine from the University of Maryland reported on efforts to improve the efficiency of gene editing in Cryphonectria parastica using (Cp) CRISPR/Cas9.   Established methods of gene editing in Cp are very inefficient and require the use of antibiotic resistance genes or other selectable markers to to distinguish transformed from colonies.  This makes it very difficult to make multiple edits in one strain, and has generally limited reverse genetics research in Cp to single-gene knockouts.  Transformation of various species of fungi using synthesized CRISPR ribonucleoproteins (RNPs), has been shown to be highly efficient.  Levine described his successful effort to make a knockout strain of Cp using RNPs.  He has not yet been able to estimate the exact percentage of nuclei transformed using this method, but noted that in each of three transformation attempts, he was able to isolate mutants from samples of 10 randomly selected colonies, all without selectable markers.  The transformed colonies contained numerous versions of the targeted genetic sequence, including the wild-type sequence, the exact engineered edit, and variants of the edit.   Single genome isolates must be subcultured from single spores or hyphal tips, and sequencing of the target region is required to confirm the exact edit.  Further refinements to the method may increase transformation efficiency further or decrease the number of sequence variants in resulting colonies.

 

Objective 3: Investigate chestnut reestablishment in orchard and forest settings with special consideration of the current and historical knowledge of the species and its interaction with other pests and pathogens.

CONNECTICUT

Connecticut Agricultural Experiment Station

Dr. Susanna Keriö:  Chestnut Research at CAES

Research into Mahogany and Nanking offspring focuses on blight incidence, tissue culture, and drought tolerance. Among full-sibling Chinese chestnuts, 35% show signs of blight. The presence of mycorrhiza appears to reduce mortality under conditions of drought and blight, though unexpectedly, trees with mycorrhiza also show increased dye uptake. These findings suggest a complex interaction between mycorrhizal fungi, tree health, and stress resistance.

A Connecticut survey on the future of chestnuts gathered 114 responses through 13 questions, with results to be presented at an upcoming meeting. Feedback was overwhelmingly positive, including from respondents outside The American Chestnut Foundation (TACF). Most participants expressed a preference for restoring a native tree, followed by interest in its value for wildlife and animal consumption. Researchers invite others to visit the Connecticut Agricultural Experiment Station (CAES) to view breeding materials, including LSAs, Clapper, Graves, Mahogany, and interact with expert staff.

NEW YORK

The State University of New York, College of Environmental Science and Forestry

SUNY-ESF American Chestnut Research & Restoration Project

Maya Niesz Kutsch: Fire tolerance and pollination distance of the American chestnut

Maya presented on two ongoing research projects involving the American chestnut. The first focuses on the effective pollination distance of American chestnut trees, though progress has been delayed due to the slow establishment of the study trees. The second project examines the fire tolerance of chestnut bark through a bark insulation experiment, which aims to quantify heat resistance and provide insights into fire tolerance levels.

The fire resistance study involves American, Chinese, and hybrid chestnuts in an open field dominated by goldenrod, where the trees are approximately 1.5 years old. Efforts are being made to compare trees of similar size and age, with measurements including bark moisture, density, and weight. A prescribed burn was conducted in the spring, though it was not very intense, and no fall burn has occurred yet. These experiments aim to deepen our understanding of both pollination dynamics and fire resilience in chestnut species.

Jacob Olichney: How does the growth and survival of Darling 54 transgenic American chestnut compare to other varieties in open and forested conditions?

As part of a USDA biotechnology risk assessment grant, common garden plots were established in New York, Pennsylvania, and Virginia to evaluate the performance of hybrid, backcross, irradiated, transgenic, and non-transgenic American chestnut varieties. Annual measurements of height and diameter revealed that transgenic trees (OxO+) exhibited the slowest growth rates, similar to hybrids, while non-transgenic American chestnuts showed the fastest growth. Survival rates were lower for transgenic trees, particularly during the establishment phase, likely due to their smaller size at planting. However, survival varied significantly by site and among the two transgenic families studied.

These findings highlight that the reduced growth and survival of transgenic trees during early establishment are comparable to challenges observed with other chestnut varieties. While these factors impact restoration efforts, their effects diminish over time as trees are established. This research underscores the importance of site-specific approaches and genetic diversity to improve restoration outcomes.

TENNESSEE

The University of Tennessee at Chattanooga

Dr. Hill Craddock: The Tennessee Report

Chestnut restoration faces the challenge of balancing conservation with the reality that forests will never fully replicate their historical state. Efforts hinge on locating and preserving Southeastern Castane populations, though planting of pure Americans in the South is not viable due to vulnerability to Phytophthora root rot (PRR). Grafting techniques, guided by detailed protocols and supported with proper pruning, fertilization, and care, have proven successful. PRR screening is underway in Tennessee using local isolates, while efforts continue to focus on backcross breeding (BxB) for PRR resistance. Conservation sites have now been established with 45,000 trees harvested and replanted.

Significant genetic findings include the identification of Castanea alabamensis as a distinct species and validation of these results by Alexander Sandercock. Research suggests that American chestnut C. dentata and C. pumila rarely hybridize, with principal component analysis showing distinct genetic differentiation between the two.

VIRGINIA

The American Chestnut Foundation, Virginia Chapter

Fred Hebard: Growth and nut production of B3-F3 chestnut in two 12-year-old forest progeny tests

Analysis of Clapper and Graves backcrosses revealed distinctive patterns of large, introgressed genomic blocks that persist through generations due to limited recombination, especially in Clapper progeny. These blocks, identified through genotype scans, highlight the monolithic nature of introgressed regions, though recombination within them does occur. The Clapper line, a B1 backcross, has a more uniform genetic contribution compared to the Graves line, an F1 with multiple parentage sources. Selection pressures during breeding for American type and blight resistance have further shaped allele frequencies, with Clapper progeny averaging 90% American and Graves 88%.

The study also explored deviations from Hardy-Weinberg Equilibrium (HWE) to assess recessive lethal alleles and their interaction with artificial selection for blight resistance. Observations showed homozygote deficiencies in Chinese alleles due to recessive lethals, balanced by selection favoring heterozygotes and some Chinese homozygotes. Resistance loci were identified across several chromosomes, with notable findings on chromosomes 7, 10, and 12 in both lines. Efforts to isolate quantitative trait loci (QTLs) for resistance revealed promising regions, though large linkage disequilibrium from introgressed blocks complicates fine-scale analysis. Further generations may offer more opportunities to refine these regions for targeted breeding. This research underscores the intricate interplay of genetics, selection, and recombination in chestnut restoration efforts.

MAINE

University of New England (UNE)

Tom Klak: Seven ongoing projects at the University of New England toward restoration

The University of New England is currently engaged in seven projects aimed at advancing American chestnut restoration. First, EP-155 branch inoculation and canker tests are being conducted on Darling 54s, wild-type, and Chinese chestnuts in an orchard setting. Second, a speed-breeding initiative is underway to enable year-round pollen and nut production in the lab, with D54 and Darwin lines producing over 100 nuts on the T6 generation under high-light conditions.

A third project seeks to determine whether Northeastern Germplasm Conservation Orchard (GCO) trees are American or hybrids, with results showing that 11 out of 96 tested were some type of hybrid. Fourth, new methods for field pollination are being explored, including July 2024 pollinations with Darwin pollen in GCOs across New England, where trees are maturing rapidly. Fifth, a Maine orchard comparing transgenic and control chestnuts has encountered challenges, including heavy rodent activity under mats and some naturally occurring blight causing bark deterioration.

The sixth project involves three transgenic silviculture trials. While one trial has failed, the remaining two indicate better performance in a white pine-shaded site compared to an open site. TRICO deer repellent is being applied every two years to support these trials. Lastly, LIDAR technology is being used to measure biomass in silviculture trials. While the practical applications of this technique are still being determined, a UNE student is refining its use with promising results. These diverse projects collectively contribute valuable insights toward American chestnut restoration efforts.

WEST VIRGINIA

West Virginia University

Amy Metheny: WVU Update

GCOs: Still able to work with Mark Double to establish and maintain GCOs with the WVTACF chapter. There are currently 28 GCOs. In the local WVU orchard, facing many challenges with terrible soil and invasives. Tracking mortality within the families and planting methods.

Hypovirulence: 4 known families of hypoviruses, CHV1 is best known to reduce growth, and sporulation and is pigmented. Hypovirus is highest in isolates grown between 15-25c while CP grows best at 30C. The test was to grow different CP isolates w/ and w/o hypervirulent strains, some being “super donors” at a range of temperatures. 30 appears to be too hot, and 10 is too cold on traditional isolates, but hypovirulent strains thrive at 30.  The virus may not be able to “hold” back the CP in these strains. Testing hypo strains CHV1 is not as aggressive as EURO 6 is.Moving forward: to test dsRNA concentrations, test EP155 as there have been many issues with the strain and look at storage.

Danielle Mikolajewski: Is Cryphonectria radacalis more aggressive than Cryphonectria parasitica on red and white oak species?

Danielle Mikolajewski’s research investigates the comparative aggressiveness of two fungal pathogens, Cryphonectria radacalis and Cryphonectria parasitica, on red and white oak species. C. parasitica, the causative agent of chestnut blight, has occasionally been found on oaks, but its impact is generally minimal, apart from some localized swelling at the base of the trunk (butt swell). In contrast, C. radacalis was previously considered extinct but has been rediscovered, prompting new studies into its virulence and potential threat to forest ecosystems.

Preliminary findings suggest that C. radacalis poses no significant risk to the health of red or white oak forests. While its aggressiveness has been tested on oaks, its effect on American chestnuts remains unstudied, leaving open the question of its impact on this critical species. Further research could provide insights into whether C. radacalis interacts differently with chestnuts or other hardwood species, but current data suggests it is unlikely to be a major ecological concern. This work is an important step in understanding the broader implications of fungal pathogens within North American forests.

Accomplishments

Publications

<p><strong>2024 Publications summary: NE-2333</strong></p><br /> <p>Burdine, C. S., Parker, C. K., Nance, W. L., Galeano, E., &amp; Nelson, C. D. (2023). Conserving Mississippi-origin American chestnut genotypes: Initial results with a modified nut-grafting method. <em>Proceedings of the 37th Southern Forest Tree Improvement Conference</em>, June 20&ndash;23, 2023, Knoxville, TN, 28&ndash;30. https://sftic.org</p><br /> <p>Double, M. (2024). The chestnut blight fungus. <em>Chestnut: The Journal of The American Chestnut Foundation</em>, 38, 32&ndash;34.</p><br /> <p>Evans, G. R., Burton, J. I., Powell, W. A., &amp; Drake, J. E. (2023). Comparative growth and physiological performance of American chestnuts, oaks, hickories, and sugar maple across a silvicultural gradient in overstory retention. <em>Forest Ecology and Management,</em> 536, 120908. https://doi.org/10.1016/j.foreco.2023.120908</p><br /> <p>Fan, S., Georgi, L. L., Hebard, F. V., Zhebentyayeva, T., Yu, J., Sisco, P. H., Fitzsimmons, S. F., Staton, M. E., Abbott, A. G., &amp; Nelson, C. D. (2024). Mapping QTLs for blight resistance and morpho-phenological traits in inter-species hybrid families of chestnut (<em>Castanea</em> spp.). <em>Frontiers in Plant Science</em>, 15, 1365951. https://doi.org/10.3389/fpls.2024.1365951</p><br /> <p>Fernandes, P., Pimentel, D., Ramiro, R. S., Silva, M. D., Fevereiro, P., &amp; Costa, R. L. (2024). Dual transcriptomic analysis reveals early induced <em>Castanea</em> defense-related genes and *Phytophthora cinnamomi* effectors. <em>Frontiers in Plant Science</em>, 15, 1439380.nhttps://doi.org/10.3389/fpls.2024.1439380</p><br /> <p>Henderson, A. F., Santoro, J. A., &amp; Kremer, P. (2023). Impacts of spatial scale and resolution on species distribution models of American chestnut (<em>Castanea dentata</em>) in Pennsylvania, USA. <em>Forest Ecology and Management</em>, 529, 120741. https://doi.org/10.1016/j.foreco.2023.120741</p><br /> <p>Islam-Faridi, N., Hodnett, G. L., Zhebentyayeva, T., Georgi, L. L., Sisco, P. H., Hebard, F. V., &amp; Nelson, C. D. (2023). Cyto-molecular characterization of rDNA and chromatin composition in the NOR-associated satellite in chestnut (<em>Castanea</em> spp.). <em>Scientific Reports</em>, 14, 980. https://doi.org/10.1038/s41598-023-45879-6</p><br /> <p>Islam-Faridi, N., Zhebentyayeva, T., Hodnett, G. L., Georgi, L. L., Sisco, P. H., Hebard, F. V., &amp; Nelson, C. D. (2023). Cytogenomic characterization of rDNA and the chromatin composition of the NOR-associated satellite in American and Chinese chestnuts. <em>Proceedings of the 37th Southern Forest Tree Improvement Conference</em>, June 20&ndash;23, 2023, Knoxville, TN, 31&ndash;33. https://sftic.org</p><br /> <p>Jacobs, D. F., Dumroese, R. K., Brennan, A. N., Campbell, F. T., Conrad, A. O., Delborne, J. A., ... Nelson, C. D., et al. (2023). Reintroduction of at-risk forest tree species using biotechnology depends on regulatory policy, informed by science, and public support. <em>New Forests</em>, 54, 587&ndash;604. https://doi.org/10.1007/s11056-023-09980-y</p><br /> <p>Sandercock, A. M., Westbrook, J. W., Zhang, Q., &amp; Holliday, J. A. (2023). The road to restoration: Identifying and conserving the adaptive legacy of American chestnut<em>. bioRxiv</em>, 2023&ndash;05. https://doi.org/10.1101/2023.05</p><br /> <p>&nbsp;</p><br /> <p>&nbsp;</p>

Impact Statements

1. Genomic Insights & Marker-Assisted Selection (MAS): Identified 20 quantitative trait loci (QTLs) linked to blight resistance, improving predictive breeding models. Genome-wide association studies (GWAS) refining resistance gene mapping, explaining 23% of variation in canker size.
2. Biological Approaches to Blight Control: High-efficiency, marker-free CRISPR/Cas9 editing in Cryphonectria parasitica enhances genetic studies. Studies on hypovirulence highlight temperature-dependent viral efficacy, informing biocontrol applications.
3. Phytophthora Root Rot Resistance: Mapping of 17 resistance loci and RNA sequencing reveal defense mechanisms in Chinese chestnut roots. Field trials support breeding for improved root rot resistance.
4. Transgenic Advancements: Darling 54 transgenic chestnut, under regulatory review and research into additional transgenes (ODC, RNAi) aims to further enhance resistance.
5. Conservation & Genetic Diversity Preservation: Nut-grafting efforts successfully conserve germplasm, with a 66% success rate on Chinese rootstocks.
6. Optimized Breeding & Restoration Strategies: Genomic selection models achieve 95% accuracy for blight resistance and 75% for Phytophthora resistance. Speed breeding accelerates progress, with two generations needed for stable blight resistance. Site-specific trials validate restoration strategies across diverse landscapes.

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