NE2333: Biological Improvement of Chestnut through Technologies that Address Management of the Species and its Pathogens and Pests

(Multistate Research Project)

Status: Active

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SAES-422 Reports

Annual/Termination Reports:

[11/20/2024] [12/12/2025]

Date of Annual Report: 11/20/2024

Report Information

Annual Meeting Dates: 09/13/2024 - 09/15/2024
Period the Report Covers: 10/01/2023 - 09/30/2024

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

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

Impact Statements

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.
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.
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.
Transgenic Advancements: Darling 54 transgenic chestnut, under regulatory review and research into additional transgenes (ODC, RNAi) aims to further enhance resistance.
Conservation & Genetic Diversity Preservation: Nut-grafting efforts successfully conserve germplasm, with a 66% success rate on Chinese rootstocks.
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.

Date of Annual Report: 12/12/2025

Report Information

Annual Meeting Dates: 09/15/2025 - 09/18/2025
Period the Report Covers: 10/01/2024 - 09/30/2025

Participants

Susanna Keriö (CAES), Elisabeth Ward (CAES), Jack Swatt (TACF), Angus Dawe (MSU), Mark Double (WVU), Patrícia Fernandes (ESF), Fred Hebard (TACF), Bruce Levine (UMD), Dakota Matthews (ESF), Amy Metheny (WVU), Cassie Stark (TACF), Jared Westbrook (TACF), Nathaniel Westrick (CAES), Tracy Zarillo (CAES), Zachary Placzek (UConn), Isabel Munck (USDA Forest Service), Vinny Varsalona (Reinhardt University, TACF), Deni Ranguelova (TACF)
Online: Chuck Bordaine (USFS), Dana Nelson (USFS), John Hempel, Steven Jeffers (Clemson), Hill Craddock (UTC), John Scrivani

Brief Summary of Minutes

Business meeting:
The 2025 NE-2333 meeting was held in Cromwell, Connecticut on September 16-17th, hosted by CAES, Connecticut Agricultural Experiment Station. Presentations represented research from Connecticut, Georgia, Maryland, New York, New Hampshire, North Carolina, South Carolina, Virginia, and West Virginia.
This is the second meeting under the 2333 project, and an estimated 30 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.
Cassie Stark will host NE2333 in 2026 in Virgina.

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. CONNECTICUT Connecticut Agricultural Experiment Station Dr. Tracy Zarrillo: The chestnut bee, Andrena rehni: a survival story *published checklist of bee species this year for state of CT Solitary bees, including Andrena rehni, nest underground or in tunnels, with single females constructing nests and provisioning food for their offspring. In Connecticut, approximately 24% of bee species are specialists. The historical range of A. rehni, a mining bee, aligns with that of the American chestnut (Castanea dentata). The project originated in 2018 when, Sam Droege observed an unfamiliar bee in Maryland, later identified as A. rehni. Subsequent surveys were conducted across New England from 2019 to 2024, with bees collected during June and July using aerial netting and traps. Results indicate that A. rehni is present at Lockwood Farms and was found at 10 of 53 sites surveyed, including locations in Massachusetts and Connecticut. Research on chestnut pollination shows that 82 bee species visit male flowers, while no species were observed visiting female flowers. At Lockwood Farms, hybrid and non-hybrid chestnuts were compared; in plot #9 (Humphry Hill), non-hybrid trees received more frequent bee visits, including visits by A. rehni. Future work will focus on obtaining A. rehni designation as a “State Assessment Priority Species,” expanding surveys to better understand its distribution, and investigating how the species disperses to new locations. Connecticut Agricultural Experiment Station The American Chestnut Foundation, Connecticut chapter Jack Swatt: A novel approach to pollinating isolated American chestnut trees Five wild-type American chestnut trees were pollinated using an experimental method in which branches bearing catkins were placed in milk jugs and hung within the tree canopy. This approach increased pollination success, as four out of the five trees produced a greater quantity of nuts compared to previous years when this method was not used. The primary advantages of this technique include the use of low-cost materials and minimal time and labor requirements. However, a disadvantage observed was a lower overall seed yield compared to results achieved through manual or traditional pollination methods. MARYLAND University of Maryland Bruce Levine: CRISPR gene editing of Cryphonectria parasitica Genetic modification of Cryphonectria parasitica (CP) has been pursued through gene knockouts and replacement with antibiotic markers to detect and test virulence. While Cas9-mediated transformations can achieve this, some genes are more difficult to target, and repeated transformations can increase antibiotic resistance. Researchers are exploring whether CRISPR-based methods can bypass these limitations. Initial Cas9 integrations caused untargeted cuts, as observed in control experiments. Reisolated strains were inoculated into new stems and exhibited expected behavior, showing no signs of avirulence. In 2024, ribonucleoprotein (RNP)-mediated transformation was employed, resulting in a high transformation rate confirmed via amplicon sequencing; all ten colonies tested carried the intended mutant gene. Subsequent attempts to transform CE1 using the RNP method achieved a 0.25% transformation rate. Future efforts will focus on identifying mutants without requiring antibiotic resistance and optimizing guide RNA sequences to improve efficiency. CE1 knockout testing revealed that rating cankers are often more effective than measuring them, as small cankers can make size differences difficult to detect. Knockouts generally show reduced virulence, although variation exists among isolates. In conclusion, RNP-mediated transformation is effective and can be efficient, with outcomes dependent on the target gene and RNA design guide. Isolation of monokaryon colonies remains essential, and delayed virulence has been observed with Cas9-based approaches. NEW YORK SUNY ESF Patrícia Fernandes: Development of transgenic blight tolerant Ozark chinquapins (OC) Two approaches have been taken in attempt to create a successful transgenic blight tolerant Ozark chinquapin: 1) backcross breeding chinquapins with OxO American chestnuts, and 2) direct transgenic transformation. Currently, the goal of this research is to get to a BC3 generation and evaluate what percent of American chestnut DNA remains. Transgenic American chestnut × chinquapin hybrids have been evaluated for inheritance, nutritional composition, growth, and breeding performance. The OxO transgene is inherited by approximately 31.4-38% of F1 hybrids in the Darling line and 32-46% of F1 hybrids in the DarWin line. Comparative analyses of Ozark seeds from transgenic and non-transgenic trees show no differences in nutritional composition. Stem inoculation trials were performed on 81 two-year old seedlings to assess blight tolerance. Progression of canker length in transgenic hybrids was slower than non-transgenic, however all died after 4 weeks. Survival measurements indicate no significant differences between transgenic (OxO-positive) and non-transgenic (OxO-negative) trees, with survivability near 100% for OxO-positive trees and approximately 20% for OxO-negative trees. Field study data from 2022 to 2025 reveal that transgenic trees exhibit significantly lower height and diameter at breast height (DBH) compared to non-transgenic trees (n = 39 OxO-positive, n = 39 OxO-negative; p = 0.02). Breeding progress has been notable, with first flowers observed in the field after four years. In high-light grow rooms, embryo rescue of hybrids allowed rapid flowering, with the first pollen collected this year. Fifteen flowers were pollinated using BC1 pollen from these high-light grow room hybrids, demonstrating accelerated reproductive development. Pollen production was evaluated to compare OxO-positive and OxO-negative individuals. NORTH CAROLINA The American Chestnut Foundation, National Dr. Jared Westbrook: Looking into the black box of chestnut blight resistance Efforts are currently focused on breeding chestnut trees for resistance to both blight and Phytophthora root rot (PRR) resistance, as PRR has now been detected in New Hampshire. True success in this program depends on achieving a combination of disease resistance, forest competitiveness, and genetic diversity. The hope with this program is to capture enough disease resistance from Chinese chestnut while retaining the forest competitiveness of American chestnut. To date, The American Chestnut Foundation (TACF) has phenotyped and genotyped approximately 3,500 trees, of which around 500 meet the criteria for disease resistance. However, there is an inherent trade-off between maintaining a high proportion of American chestnut ancestry and achieving strong blight resistance. Genetic mapping has identified roughly 17 loci associated with root rot resistance and an estimated 57 loci total that are associated with blight resistance. To fully map these areas, TACF aims to genotype and phenotype approximately 9,000 trees for blight resistance and 2,500 for PRR. A major focus this year is on Large Surviving American (LSA) crosses. The progeny from these crosses will be inoculated and evaluated to assess the heritability of disease resistance. The breeding program uses a Recurrent Genomic Selection approach, selecting parent trees whose crosses are expected to have an average blight resistance score above 70 and an American ancestry greater than 70%. Some crosses may include individuals with slightly lower resistance to maintain overall genetic diversity. As part of the ongoing regionalization of the TACF breeding program, researchers are also determining how many wild-type (WT) trees must be conserved to capture more than 95% of adaptive diversity. Fewer trees are needed in the southern regions, where genetic diversity per tree tends to be higher. Genomic prediction models estimate breeding value based on the weighted average performance of related individuals. Simulation studies have shown that increasing genetic markers beyond 6,000 does not improve prediction accuracy; therefore, TACF currently uses approximately 6,500 DArT markers for genomic selection. TACF’s overarching goal is to double population-level resistance and forest competitiveness within the next decade. While variation in resistance will always exist, maintaining this variation is considered beneficial for restoration trials, as it supports adaptability and resilience in reintroduced populations. SOUTH CAROLINA Clemson University Dr. Steve Jeffers and Haiying Liang: South Carolina State Report 2025 A multi-state collaboration involving The American Chestnut Foundation (TACF) and USDA Forest Service (USFS) aims to understand host resistance in chestnut and to screen backcross trees for susceptibility to Phytophthora root rot (PRR). From 2003 to 2025, samples have been collected from 12 states, totaling 739 samples, of which 293 (40%) tested positive for Phytophthora species. Sample submissions in 2025 were limited, with only eight contributed by Cassie Stark. Studies on host resistance involve collaborations with Clemson University, USFS, the University of Kentucky, and the University of Tennessee. Researchers including Tatyana Zhebentyayeva and Bert Abbott have inoculated seedlings in five-gallon buckets with zoospores and sampled roots at 3, 6, 12, and 24 hours post-inoculation. RNA analyses are used to identify genes involved in resistance or susceptibility. Research began with American and Chinese chestnuts in 2022 and 2023, expanded to F2 seedlings in 2024, and resumed in 2025 at Clemson following delays due to funding and the indefinite closure of the USDA FS Bent Creek Experimental Forest Center. Seedlings are inoculated with PRR and flooded every 48 hours, with mortality subsequently recorded. Fungicide trials for PRR on American chestnut seedlings identified Aliette as one of the most effective products. In March 2024, Steve requested a Section 2(ee) label for Aliette use on chestnuts, which was approved by September 2024. Field trials were conducted at Warren Wilson College testing Ridomil, Reliant, and untreated controls, with three applications at three-month intervals; hurricane Helene caused a nine-month interval between treatments. A second field trial at Clemson faced challenges from deer and polyphemus caterpillars. Research on adventitious rooting in American chestnut cuttings, led by Haiying Laing, demonstrated nearly 90% rooting in two-month-old seedlings with auxin induction. Cuttings from seedlings older than four years rooted at only 50% with auxin, likely due to age-related changes in hormones and metabolites. Maintaining high humidity (near 100%) was critical for rooting, achieved by covering cuttings with moss and enclosing them under a screen. American chestnut cuttings contained higher cytokin levels than poplar. This study shows that including a cytokinin inhibitor (100nm fluridone) enhanced the rooting rate. VIRGNIA The American Chestnut Foundation, Virginia chapter Dr. Fred Hebard: Adequate levels of blight resistance in B3F2s are confined to three or four parents out of 130 Genetic analyses indicate that specific Chinese chestnut (CH) alleles persist in backcrossed populations. In the Clapper line, CH alleles are most prevalent on chromosomes 5 and 12, while in the Graves line, they are concentrated on chromosomes 7, 10, and 12. Evidence suggests that two or three major genes may be responsible for resistance within each line. When Clapper and Graves are crossed, four key alleles consistently stand out. Overall, results suggest that studying individual crosses provides clearer insights into the genetic basis of resistance. Within the B3F2 generation, individuals with Best Linear Unbiased Predictor (BLUP) scores above 40 are rare in the Clapper line. Among these, families SC80, SC169, and SC163 represent the best performers. These findings suggest that maintaining or restoring genetic diversity may require combining seed from multiple chapters, even at the cost of sacrificing local adaptation. Data from the Johns Creek reintroduction trial show that Clapper trees have lower stem index BLUPs compared to the Graves line; however, these differences may not necessarily translate to reduced resistance in their progeny. It may be fruitful to investigate the inheritance of blight resistance in B3F2s and B3F3s derived from SC169 and SC80. 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. CONNECTICUT Connecticut Agricultural Experiment Station Dr. Susanna Keriö: CAES Chestnut Research Update Elodie Eid, an independent researcher based in Massachusetts, is currently phenotyping chestnut trees, evaluating nut morphology and quality, and collecting scion wood for propagation at the Sleeping Giant orchard. Her work is supported by a grant from the Northern Nut Growers Association. A new partnership was established this year between the Connecticut Agricultural Experiment Station (CAES) and The American Chestnut Foundation (TACF) with the relocation of TACF’s New England regional office to the CAES facility in Griswold, Connecticut. Connecticut’s tallest known chestnut trees include a 72-foot hybrid and a 40-foot American chestnut. Results from a 2024 public questionnaire indicated a strong preference for growing American chestnuts, which inspired a new CAES hypovirulence project. This initiative received funding from the Connecticut chapter of TACF ($5,000) and the USDA Forest Service ($120,000). Project objectives include: (1) utilizing existing 50-year-old hypovirulence trial sites to identify current strains, (2) developing new hypovirulent strains for Connecticut, (3) testing these strains in both greenhouse and field settings, (4) monitoring hypovirus transmission, and (5) hosting educational workshops on biocontrol at Lockwood Farm. This project was inspired by discussions and collaborations stemming from the NE2333 meeting last year, which motivated Susanna to pursue this line of research. Connecticut Agricultural Experimental Station Dr. Nathaniel Westrick: Ecological Implications of Historical and Future Approaches to Chestnut Blight Biocontrol In 1969, scientists at the Connecticut Agricultural Experiment Station (CAES) imported French hypovirulent strains of Cryphonectria parasitica. Between 1975 and 1977, CAES researchers demonstrated that hypovirus is transmissible via RNA. Hypoviridae are double-stranded RNA viruses that require a living fungal host and are spread either through hyphal fusion or via spores. Hypovirus biocontrol has been successful in Europe but less effective in the United States. One key factor is strain selection: the U.S. initially used highly virulent strains that were unable to reproduce effectively, whereas European efforts employed mild strains that slowed disease without affecting overall fungal growth. Another factor is vegetative compatibility: the U.S. has a high diversity of vegetative compatibility groups, which limits virus spread, whereas Europe has fewer groups, making hypovirus transmission more generalizable. Ecological differences also play a role; U.S. chestnut populations are largely wild and widely dispersed, while European chestnuts are often contained in managed orchards, meaning that treating as little as 10% of stands can be sufficient for effective spread. A reassessment of biocontrol is timely due to the development of more resistant hybrid chestnut varieties, increasing threats from beech leaf disease in northern forests, and improved knowledge of strain selection and universal donor strains capable of transferring viruses across CHVs. At Lockwood Farm, chestnut trees planted in 1976 were treated with a slurry of eight hypovirulent strains, including some from Europe. Canker samples were collected, plated, and analyzed via PCR. Results revealed substantial diversity in phenotypes, with most positive cankers found on moderately blighted trees. Remarkably, trees continue to test positive for hypovirus more than 50 years after treatment. Some of the large, healthy chestnut trees are consistently testing negative for hypovirus via PCR, and Cryphonectria parasitica cannot be isolated from them. However, another fungal species is repeatedly present in these trees, suggesting it may play a role in the observed phenotypes. Current investigations are focused on comparing growth and sporulation among hypovirus-infected strains to better understand interactions between the virus, C. parasitica, and other fungal species in long-term biocontrol dynamics. GEORGIA Reindhart University, VA TACF Vinny Varsalona: Hypovirulence research at Lesesne State Forest in Virginia At Lesesne State Forest (LSF) in Roseland, Virginia, hybrid and American chestnut trees were planted in 1960 and inoculated with hypovirus in the 1980s by Gary Griffin. A key research question is whether, and to what extent, hypovirus persists in this forest. To investigate, 160 samples were collected from chestnut cankers, phenotyped, and analyzed using the Zymo Quick RNA Extraction Kit. Results indicate that CHV-1 hypovirus continues to persist throughout LSF. WEST VIRGINIA West Virginia University Amy Metheny: WVU Update Hypovirus infection in Cryphonectria parasitica (CP) is influenced by six unlinked diallelic genes, resulting in 64 described vegetative incompatibility (VIC) genotypes, which prevent virus replication via hyphal anastomosis between incompatible strains. Super-donor strains, developed by Zhang and Nuss (2016) using a Cre-loxP recombination system, have been designed to overcome vegetative incompatibility. However, vegetative incompatibility is not the only barrier: the vic2 locus produces mutants, requiring the use of two super-donor strains for effective virus transmission. For example, certain European strains (EU65 and EU66–74) cannot receive hypovirus from the super-donor but can pair with EU41, which likely carries both vic2 alleles. Moving-forward, they hope to perform full genome sequencing of these strains to better understand their interactions. Endothia gyrosa, often misidentified as a superficial blight infection, has been successfully infected with both hypovirus super-donor strains, indicating that host species differences can affect virus transmission. Temperature also influences hypovirus dynamics, hypovirus isolates show rapid growth at 30°C, while virulent fungal strains perform better between 20–25°C. The fast growth of hypovirus at higher temperatures suggests impaired virus persistence, allowing the fungus to dominate. Objective 3: Investigate chestnut re-establishment 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. CONNETICUT University of Connecticut Zachary Placzek: Post-Fire Microbial Inoculation in American Chestnut Regeneration and Soil Nutrient Cycling This burn study consists of two phases: an initial jack-pile burn followed by a prescribed ground cover fire to prepare the site for planting. The jack-pile burns are all that have been done thus far. Low-intensity fires are beneficial for reducing fuel loads, controlling invasive plants, mobilizing nutrients, altering soil pH, and shifting microbial communities. Chestnut regeneration strategies can mimic red oak, with abundant sprouts occurring in areas with high light availability. However, mesophication in New England has shifted forests from fire-adapted species to fire-sensitive species due to changing climate and the historical absence of fire. Methods included inoculation of chestnut-associated soil into reference sites by creating furrows and transplanting unburned soil containing microbes specialized to chestnuts. Key research questions focused on whether burning and soil inoculation could enhance chestnut regeneration, increase soil microbial diversity, and improve soil quality. Results indicate that cation exchange capacity (CEC) and pH were higher in burned sites, although pH itself did not change significantly due to burning. Soil nutrients, particularly calcium, increased following the burn, and remained elevated in inoculated sites. Fungal community composition shifted in response to both burning and inoculation. These findings suggest that fire paired with soil inoculation may enhance soil fertility and microbial support for chestnut regeneration. Future work will include a low-intensity burn this fall, followed by planting chestnuts and assessing site suitability for long-term growth and survival. Connecticut Agricultural Experiment Station Dr. Elizabeth Ward: Forest Health Research in Connecticut and Opportunities for Collaboration Dr. Ward is a forest ecosystem ecologist who manages Connecticut’s Forest Health Program. Her work focuses on addressing major forest health threats such as spongy moth, beech leaf disease (BLD), and emerald ash borer, with an emphasis on strategies to mitigate the negative impacts of tree decline. One key management question she explores is how to diversify forests affected by BLD through targeted forest management practices. Recommended treatments include enrichment plantings, the installation of deer exclusion fencing, and selective removal of diseased or dominant beech trees. Because beech trees are shade-tolerant and often form monodominant stands, enrichment plantings present valuable opportunities to collaborate by underplanting American chestnuts in forested settings. Notably, Dr. Ward also has access to a 100-year forest inventory dataset—the oldest in the country—which provides a unique opportunity to study long-term forest management outcomes. NEW HAMPSHIRE USDA Forest Service Isabel Munck: Effect of silvicultural treatment and forest type on survivability of chestnut seed The objective of this study is to partner with The American Chestnut Foundation (TACF) to develop silvicultural treatments that support chestnut establishment and growth. Experimental treatments included forest type (oak–pine and northern hardwood) and silvicultural approach (patch cuts >2 acres, group selection ~1 acre, shelterwood, and untreated control). Key response variables measured were seedling emergence, survival, growth, susceptibility to blight, nut production, and wildlife use. Seeds were planted in mid-May containing 3 sites per treatment, with 30 seeds per site (15 hybrid chestnuts and 15 American chestnuts). Results indicate that seedling emergence was higher in northern hardwood forests (83%) compared to oak–pine forests (34%). Among silvicultural treatments, control sites had the highest emergence, likely reflecting the influence of drought conditions during the planting season.   Annual Project Impacts Objective 1: Chestnut Bee Ecology Surveys across New England confirmed persistence of the chestnut-associated bee Andrena rehni at 10 sites, improving understanding of pollination ecology and informing conservation actions that support chestnut restoration. Low-Cost Pollination Method A simple, low-cost pollination technique increased nut production in 4 of 5 wild American chestnuts, giving landowners and volunteers an accessible tool for conserving local genetics. CRISPR Editing of Blight Fungus CRISPR RNP editing produced precise mutations in all tested fungal colonies, improving the ability to identify virulence genes and accelerating development of future blight-control strategies. Transgenic Ozark Chinquapin Progress Transgenic hybrids showed slower canker growth and near-complete survival compared to 20% in non-transgenics, demonstrating meaningful progress toward restoring an endangered chinquapin species. Genomic Selection for Blight & PRR Resistance Genotyping of 3,500 trees and mapping of >70 resistance loci strengthened genomic prediction models, improving breeding efficiency and advancing efforts to produce competitive, disease-resistant restoration stock. PRR Resistance & Propagation Advances Aliette received approval for chestnut use following fungicide trials, providing growers a new PRR management option. Vegetative propagation studies achieved up to 90% rooting, improving capacity to clone elite genotypes. Genetic Basis of Blight Resistance Analysis of B3F2 families showed that only 3–4 parents contribute most resistance, enabling more targeted breeding and more efficient preservation of genetic diversity. Objective 2 Hypovirus Biocontrol Program A new $125K hypovirus project will develop Connecticut-specific strains and reactivate long-term trial sites, expanding regional capacity for biological blight control. Long-Term Hypovirus Ecology Fifty-year-old treated trees still carry hypovirus, demonstrating long-term biocontrol persistence and informing modern deployment strategies. Hypovirus Persistence at Lesesne Sampling of 160 cankers confirmed CHV-1 remains active decades after inoculation, validating early biocontrol efforts and guiding future forest-scale applications. Overcoming Fungal Compatibility Barriers Work with super-donor strains clarified genetic and environmental limits to hypovirus transmission, improving strategies for more reliable biological blight suppression. Objective 3 Fire & Soil Microbial Inoculation for Regeneration Burning and soil inoculation increased nutrient availability and shifted microbial communities, suggesting a combined approach may improve chestnut establishment on fire-suppressed sites. Forest Health Management Opportunities Forest diversification strategies for beech leaf disease—such as enrichment plantings and canopy thinning—create new opportunities to integrate American chestnut into recovering

Publications

2025 Publications summary: NE-2333 Carlson, E. (2025). Advancement of Blight Tolerance Biotechnology in Chestnut and a Tissue Culture-Free Method for the Genetic Transformation and Regeneration of Populus (Doctoral dissertation, College of Environmental Science and Forestry). Charles, M. A., Adams, R. H., Anderson, R. S., Bird, K. M., Johnson, L. R., Kelso, N., ... & McKenna, D. D. (2025). Rediscovery of the greater chestnut weevil highlights the power of digital platforms in biodiversity research and conservation. Current Biology. Cleary, M. S., Horton, J. L., & Filgueiras, C. C. (2025). Physiological Comparison of Pure American and American-Chinese Chestnut Hybrids. Castanea, 90(1), 35-47. Does, J., Duarte, B., Dobson, K., & Bretfeld, M. (2025). Are Leaf Decomposition and Morphology of Hybrid Chestnuts Functionally More Similar to American or Chinese Chestnuts?. Ferguson, M. (2025). Management of an Invasive Wasp and a Native Weevil in Commercial Chestnut Orchards in Michigan (Master's thesis, Michigan State University). Klak, Thomas, Hannah Pilkey, Virginia G. May, Dakota Matthews, Allison D. Oakes, Ek Han Tan, and Andrew E. Newhouse. "Speed breeding transgenic American chestnut trees toward restoration." bioRxiv (2025): 2025-05. Klak, T., Travis, S., May, V. G., Tan, E. H., Chatfield, M. W., & Wheeler, M. (2025). Two-Year Field Trial of Genetically Engineered American Chestnut Reveals Greater Fungal Blight Tolerance Compared to Wild-Type Full-Sibling Trees. bioRxiv, 2025-11. May, V. G. (2025). Speed Breeding and Fungal Blight Testing of The Darling 54 American Chestnut (Castanea dentata). Scott, G., Morris, D., Bialonska, D., & Fadroski, K. A. (2025, March). Effect of chestnut cultivation on soil microbial communities at the Hurricane Creek Field Station. In UNG Annual Research Conference (Vol. 30, No. 1). Westbrook, J. W., Malukiewicz, J., Zhang, Q., Sreedasyam, A., Jenkins, J. W., Lakoba, V., ... & Lovell, J. T. (2025). Improving American chestnut resistance to two invasive pathogens through genome-enabled breeding. bioRxiv, 2025-01. Williams, M. E., Wilson, O. A., & Horton, J. L. Investigating the development and mycorrhizal communities of American, Chinese, and hybrid chestnut seedlings. Wegner, T. M., Newhouse, A. E., Satchwell, S., & Drake, J. E. (2026). Evaluating transgenic Darling 54 American chestnuts for reintroduction: Insights from survivorship, growth, and respiration in a common garden. Forest Ecology and Management, 602, 123361. Zarrillo TA, Stoner KA, Ascher JS. Biodiversity of Bees (Hymenoptera: Apoidea: Anthophila) in Connecticut (USA). Zootaxa. 2025 Feb 12;5586(1):1-138. doi: 10.11646/zootaxa.5586.1.1. PMID: 40174048.