Brief Summary of Minutes of Annual Meeting

Summary of Minutes:

The 2022 NE-1833 annual meeting was held live in Charlottesville, Virginia Aug 26-27. A total of 16 presentations spanned topics pertaining to all three of the project’s objectives. Presentations represented research from Georgia, Maine, Maryland, Mississippi, New York, North Carolina, Pennsylvania, South Carolina, Tennessee, West Virginia, and Virginia, as well as from The American Chestnut Foundation, a strong partner in this project.

The project’s Administrative Advisor, Brad Hillman, was unable to participate in the meeting due to illness but communicated with the conference organizers and participants for the NE1833 business meeting.  It was decided that the 2023 annual meeting will be hosted by Hill Craddock at the University of Tennessee, Chattanooga.

The current project expires in 2023. A Request to Write a renewal proposal will be submitted this fall to NERA and a full proposal will be submitted to the USDA through NERA in the proper time frame. The current format for the project has worked well, and the objectives encompass all of the research presented annually. The format/objectives for the renewal will be similar to the current project format/objectives, and three members will take the lead in writing, one member for each objective area.

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

Chestnut research in the Connecticut Agricultural Experiment Station

Dr. Susanna Keriö: Clonal Propagation and Embryogenic Cell Line Updates

Dr. Susanna Keriö currently conducts research that aims to improve clonal propagation techniques for Chinese chestnuts. The project tests the impact of media composition, cold treatments, and silver thiosulfate on somatic embryo conversion rates. The mineral basal media tested include the woody plant medium, Murashige-Skoog medium, and Driver-Kuniyuki walnut medium. Dr. Keriö reported on the preliminary findings from this work during the NE1833 meeting. Preliminary results indicate that media composition may impact cell growth, but it is still too early to say whether media impacts embryo conversion.

Additionally, Dr. Keriö is working to establish new embryogenic cell lines from the trees growing in the CAES farms and chestnut orchards. In collaboration with the Connecticut Chapter volunteers of the American Chestnut Foundation, controlled pollinations were performed to obtain pure Chinese chestnut embryos. Trees from a progeny of Mahogany x Nanking were pollinated in June, and immature burs with the seeds were harvested in July. The tissues are currently maintained on an induction maintenance medium and monitored for the initiation of somatic embryogenesis. These cell lines will be available for collaborators who wish to use them for research.

GEORGIA

University of Georgia

Dr. Scott Merkle: The American chestnut founder line transformation project 

Production of American chestnut trees expressing the wheat oxalate oxidase gene (OxO) to provide resistance to the chestnut blight fungus has been adopted by The American Chestnut Foundation (TACF). The primary path chosen by TACF for spreading the transgene to multiple genetic backgrounds for restoration is via pollinating American chestnut trees with pollen produced by Darling58 transgenic OxO trees. An alternative approach is to directly insert the OxO gene into multiple American chestnut genotypes representing the natural genetic diversity of the tree. The resulting trees would already be adapted for growth in their native regions. We began pursuing this approach by initiating new embryogenic culture lines (“Founder Lines”) from nuts collected by TACF cooperators from large surviving American chestnut trees (LSAs) growing in different parts of the range from Maine to Georgia. In 2020, over 100 new embryogenic cultures representing eight source trees from five regions (New England, Pennsylvania, Maryland, Virginia, and Georgia) were captured. Copies of all the new Founder Lines were placed in cryostorage. Then, the cultures were screened for their abilities to produce abundant somatic embryos and high-quality somatic seedlings, to facilitate choosing those to target for transformation with OxO.  The selected Founder Lines tested so far for sensitivity to the selection agent geneticin showed a range of sensitivities to the antibiotic in a liquid medium, so selection needed to be customized for each line. Transformation experiments with these lines using the pFHI-OXO and pWIN3.12-OXO vectors are underway, and the first putative transgenic events are growing in the selection medium. Once the presence of the OxO transgene in the colonies is confirmed using PCR and an OxO enzyme assay, copies of the transgenic events will be cryostored and multiple events in each background will be grown up for somatic embryo and plantlet production. 

MAINE

University of New England (UNE), Biddeford, Maine

Thomas Klak: Transgenic Chestnut Pollen Production under High-Lights & Field Pollination at the University of New England.

Field Pollinations of July 2022. The major defining feature of this summer was the impact of drought in Maine and throughout New England. Female flowers were significantly less than in 2021. Some saplings that were emerging as producers in 2021 did not flower at all this year. So, the overall yield of nuts from trees could be much lower than in 2021.

UNE Transgenic/Controls Orchard. There is ongoing work to outplant and maintain a permitted orchard comprised of mostly Darling58 (+) seedlings, intermixed with various Chinese and full-sibling controls. The orchard now has about surviving 650 seedlings.

Pollen Production under Hi-Light. UNE is now producing transgenic pollen in unprecedented quantities. The pollen is also coming from a variety of twenty or more seedlings from mother trees located in different parts of Virginia, New York, and Maine. UNE will be prepared to ship out on dry ice as much as 1,000 vials of Darling58 pollen to collaborators across the native range when deregulated.

NEW YORK

SUNY-ESF American Chestnut Research & Restoration Project

Patrícia Fernandes, Andrew Newhouse, Linda McGuigan, Hannah Pilkey, and William Powell

Scientific and Regulatory Updates on Darling 58 Transgenic American Chestnuts

Given the historical, ecological, and economic importance of American chestnuts, there is substantial interest in restoring these trees after their decline due to chestnut blight.  Researchers at SUNY-ESF have developed transgenic trees that tolerate blight infections by degrading oxalic acid.  One line of these trees, known as Darling 58, has been submitted for regulatory review by the USDA, the EPA, and the FDA.  Reviews are in progress by all three agencies, and we expect regulatory approval to allow for initial public distribution to begin in 2023.  Natural cankers have been observed on Darling 58 and related sibling trees, and Darling 58 cankers are consistently small and not lethal (in contrast to lethal cankers on related trees).  Replicated intentional inoculations were initiated in the summer of 2022, which corroborates results observed with natural infections: Darling 58 trees with OxO have consistently smaller and less severe cankers than their non-transgenic relatives.  Discussions are underway to enable the effective distribution of trees (seeds, seedlings, and/or pollen) to various stakeholders next year pending regulatory approval.  ESF researchers are also discussing how the chestnut project will proceed into the future, potentially addressing both other chestnut disease questions and threats to unrelated trees such as the American elm.

Applying American chestnut’s biotechnological efforts to related species: The case of the Ozark chinquapin

With the close deregulation of Darling 58 (D58) by the U.S. regulatory entities, the next logical step is to apply the knowledge from American Chestnut to related species impacted by the chestnut blight, such as the Ozark Chinquapin (OC). Classical breeding and direct transformations are being used in parallel to insert the OxO gene. So far, 55 OxO-positive and 58 OxO-negative OC/D58 hybrids were obtained. These trees will be evaluated for: morphological development, blight tolerance after pathogen inoculation, and OxO expression levels. Also, rapid pollen production methods developed for American chestnut will be used for future backcrosses to restore the OC phenotype. The applicability of the American chestnut’s in vitro protocols was confirmed in OC tissues (somatic embryo multiplication and regeneration; shoot multiplication, regeneration, and rooting). Agrobacterium-mediated transformation protocol was confirmed by obtaining OC somatic embryos expressing a green fluorescent protein (GFP) reporter gene, and transformations with OxO are ongoing. Laboratory and field techniques developed for the AC have shown promising results and are currently being fine-tuned for the future restoration of the OC.

Transgenic Chestnut Breeding and Outcrossing Updates

Continuously outcrossing the transgenic, blight-tolerant, American chestnut (Darling 58), to other surviving wild-type trees has been a large focus of ESF’s field season each year. After six years of pollinating with transgenic pollen in permitted locations, we have outcrossed to over 500 Castanea trees with many genetically unique Darling 58 derived pollen genotypes. Transgenic pollen has been distributed to collaborators in multiple states throughout the chestnut’s range. This effort has been working on diluting the founder genome of Darling 58 and capturing the remaining genetic diversity of the chestnut through the production of blight-tolerant progeny.

This year, ESF reports one of its most productive pollination seasons to date. Over 200 vials of frozen transgenic pollen were sent to our collaborators.  ESF also reports the production of its first tree homozygous for the OxO gene, confirmed by the testing copy number of the OxO gene.

Lastly, as we prepare for the potential distribution of transgenic pollen following the deregulation of Darling 58, ESF has been sending out practice pollination kits. These kits include frozen, wild-type chestnut pollen and pollination materials designed to familiarize citizen scientists with chestnut flower morphology and the controlled pollination process.

NORTH CAROLINA

The American Chestnut Foundation

Dr. Jared Westbrook: Multiple paths to success for American chestnut restoration

The American Chestnut Foundation (TACF) is currently pursuing two strategies in parallel and in combination to improve blight resistance in C. dentata. The approaches are:  A) outcrossing transgenic trees containing the oxalate oxidase (OxO) gene from wheat with wild-type American and backcrossed chestnuts B) and controlled intercrossing between our most blight-resistant backcross selections.

As proof of concept for our multiple approaches to restoration, TACF and collaborators at SUNY-ESF made hundreds of controlled pollinations among trees with enhanced blight and phytophthora resistance in 2022. The cross combinations include best x best blight resistant backcrosses (20 crosses), Darling 58 x wild type American chestnut (>100 crosses), Darling 58 x blight resistant backcross (~60 crosses), Darling 58 x phytophthora resistant backcrosses (15 crosses), Darling 58 x large surviving American chestnuts (3 crosses), and large surviving x large surviving American chestnut (3 crosses). In 2023, we will inoculate a subset of 3000 to 5000 seedling progeny from these crosses with the chestnut blight fungus. We will evaluate their blight resistance in replicated greenhouse trials to be conducted at Meadowview Research Farms, Penn State University, and SUNY-ESF. The results from these trials will be useful for prioritizing our future breeding efforts. If we find that we can achieve much higher levels of blight resistance and American chestnut ancestry with Darling 58 crosses as compared to best x best crosses among our backcross selections, then we will spend more of our efforts on Darling 58 breeding. If we find that best x best and Darling 58 progeny have similar levels of resistance, then we will pursue both D58 and best x best in parallel to be able to offer our members both transgenic and non-transgenic trees. In parallel, we will also conduct an experiment in collaboration with the US Forest Service Resistance Screening Center in Asheville, NC to inoculate 1000+ Darling 58 x phytophthora-resistant backcrosses with Phytophthora cinnamomi (the pathogen that causes phytophthora root rot) and then chestnut blight. This experiment will determine whether the blight resistance conferred by OxO is robust when the trees are challenged by phytophthora root rot.

TENNESSEE

The University of Tennessee at Chattanooga

Alex Perkins and Dr. Hill Craddock

American chestnut (Castanea dentata) and the chinquapins (C. pumila sensu lato) are an evolutionary sister species pair that represents a promising system for studies of admixture and adaptation in wild plants. Since the 1920s, botanists and geneticists have hypothesized that hybridization between American chestnut and the chinquapins is common and that this process has been involved in the origins of some Castanea taxa and populations. Rigorously testing these hypotheses, however, has been difficult until the recent availability of high-throughput DNA sequencing technologies and associated computational tools. Here, we present the preliminary results of our analyses of whole genome sequencing data from 255 plants representing all North American Castanea species and subspecific taxa. Population structure analysis and D statistics tentatively indicate that introgression between C. dentata and the chinquapins has been rare. In contrast, admixture between the different chinquapin taxa—Allegheny chinquapin (C. pumila var. pumila), Ozark chinquapin (C. pumila var. ozarkensis), and Alabama chinquapin (C. alabamensis)—may have been more frequent, but is still limited to only a few sympatric sites. Future work using this data set will include the estimation of local ancestry (i.e., chromosome scale) and tests for evidence of natural selection at introgressed ancestry tracts. Finally, work has continued to be done at the many breeding orchards in TN, making crosses for both blight and PRR resistance.

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: Genetic modification of Cryphonectria parasitica using CRISPR/Cas9

Ph.D. student Bruce Levine reported on his efforts, partially funded through a TACF external grant, to develop a CRISPR/Cas9-mediated system for making genetic modifications to the chestnut blight fungus, Cryphonectria parasitica (Cp).   A well-established method for making genetic modifications to Cp by homologous gene replacement (HGR) already exists and has been used many times to knock out various Cp genes, including by Levine in 2018, when he knocked out the CpSec66 gene and produced a less virulent strain of Cp with an abnormal phenotype.  The potential advantages of applying CRISPR/Cas9 to the process however are: 1) improved transformation efficiency, 2) the ability to knock out multiple genes at once, and 3) the ability to make genetic modifications without leaving an antibiotic marker or other footprints.   Levine attempted two methods, based on work in other ascomycete fungi, to knock out the CpSec66 gene using CRISPR/Cas9.    The first involved transient expression of the Cas9 gene via an introduced plasmid, co-transformed with synthesized guide RNA targeting the first exon of the CpSec66 gene.   This attempt failed for unknown reasons.   The second effort involved using standard HGR to permanently integrate the Cas9 gene into Cp at a harmless locus (Vic4-1).   Levine successfully produced a Cp strain called DC9 in the DK80 Cp background, in which the Cas9 gene and a neomycin resistance marker replaced Vic4-1.  To test its effect, Levine repeated the process of knocking out CpSec66 in fungal spheroplasts derived from DC9 both with and without synthesized guide RNA targeting CpSec66.    The transformation with gRNA appeared to show higher transformation efficiency, not in terms of a greater number of transformations per experiment, but in terms of producing in one "monokaryon" knock-out colonies, in which the wild-type Cp-Sec66 gene was removed from all nuclei in the resulting colonies, in one step.   Transformation by HGR typically produces heterokaryon colonies, in which transformed and non-transformed nuclei co-exist, and they then have to be separated by taking single spore subcultures, which is how the non-gRNA-amended transformation behaved. Levine's next steps will be 1) to attempt to knock out CpSec66 again using DC9 plus gRNA but without an antibiotic resistance marker, 2) to insert the Cas9 gene into the EP155 background to create a CRISPR-ready wild-type Cp strain, and 3) to re-attempt transient expression of the CRISPR/Cas9 system with some modifications compared to the first attempt.

MISSISSIPPI

Mississippi State University

Angus Dawe, Department of Biological Sciences,

Current personnel:

Graduate students –Soum Kundu, Melanie Tran

Current Projects:

1. ARV-1 and its potential role in sterol homeostasis
2. Identifying C. parasitica genes associated with pathogenicity and virulence

Project details

1. Identifying C. parasitica genes associated with pathogenicity and virulence. (Melanie Tran, MS student.)

This project is leveraging a set of progeny from a cross between strains EP155 (considered more virulent) and SG2-3. Virulence phenotyping of the progeny was previously performed by ACF in Meadowview (F. Hebard). Sequencing was completed of all 92 progeny in late 2019 at Mississippi State via the Genomics Core at the University of Mississippi Medical Center in Jackson, MS. Work is ongoing with these data. Melanie is building a pipeline for analysis using the MSU Biological Sciences genomics server in collaboration with Jean-Francois Gout, a computational biologist member of the faculty. Jared Westbrook (ACF) is also contributing to genetic mapping although this has shown that there are deficiencies in some of the data leading to parts of the map that do not resolve properly. There are indications of potential QTL locations on chromosomes 1 and 2 but the size of the regions indicated means that identifying individual genes is not practical. We plan to address this with long-read (Minion) sequencing to improve the genetic map and increase resolution.

1. ARV-1 and its potential role in sterol homeostasis. (Soum Kundu, PhD student).

ARV-1 is a predicted gene in C. parasitica that shares similarities with genes that code for proteins with important roles in sterol homeostasis in other organisms. The knockout of ARV-1, serendipitously made when investigating an unrelated phenomenon, is avirulent and has a heavily impaired vegetative growth phenotype. Soum has confirmed that disrupting the ARV-1-like gene is the cause of the severe phenotype. He has developed and verified an assay for ergosterol production in C. parasitica by modifying published protocols and using a GC/MS system in collaboration with the lab of Todd Mlsna in the Department of Chemistry at Mississippi State. This assay is now quantitative and has been used to analyze the amounts of ergosterol present in different colony extracts. When tested, the hypovirus-infected strain EP713 shows a reduction of ergosterol accumulation similar to that of the mutant, suggesting that a component of the membrane alterations induced by the hypovirus may be due to altered ergosterol presence. Additional work has confirmed prior EM analysis showing increased vesicles in the hypovirus-infected strain.

WEST VIRGINIA

West Virginia University

Amy Metheny, Danielle Mikolajewski, and Matthew Kasson

Optimization of engineered super donor strains of Cryphonectria parasitica to reduce canker expansion in a forest setting

5 years ago, the super donor was released into two forest stands in western Maryland. From these studies, we learned that strain CHV1/Euro7 gave smaller cankers, punch and scratch methods gave smaller cankers, and that one-time application of SD did not give sustained control. During the pandemic, these trees were left to succumb to chestnut blight. After 5 years, the Kasson lab team made our way back out to remove these trees. These trees were removed for several reasons. This is an unregulated organism and according to our APHIS permit, the trees had to be removed and any treated cankers had to be sterilized. 

Felling the study trees allowed researchers to access their canopies. Currently, few published studies have measured cankers above approximately 9 feet in a forest setting where orchard ladders and bucket trucks are impractical. 

 The trees were removed in June 2022. Four bark plugs were taken from each of the three cankers above and below breast height. These samples are currently being processed in the lab. Any wild-type Cryphonectria parasitica or super donor strains will be retained and identified as virulent or hypovirulent. Presumably, super donor strains with their genetic knockouts and arrested growth will not be fit enough to compete in the environment long-term. Measurements of canker width and length were also taken and recorded along with whether the tree was dead or alive, whether there was stroma present, and whether there were stump sprouts present. Overall, 10 trees had survived from the original 75 from both sites over the 5 years. CHV1/Euro7 treated cankers were smaller than CHV1/713 treated cankers (P < 0.05) which still holds after 5 years. Punch and scratch methods were still not significantly different (P > 0.05).

Planting: 

West Virginia University also assisted in the potting and subsequent outplanting of approximately 1200 American chestnuts in collaboration with the WV chapter of TACF. Most of these nuts were installed across the state of West Virginia to establish genetic conservation orchards or GCOs. GCOs serve as an important source of wild-type American chestnut pollen and/or nuts for future breeding and preservation purposes. Notable plantings include an expansion of planting near Sutton, WV which experienced 92% survival from the previous year, another planting expansion in Queens, WV which had 87% survival from the previous year, and a new planting in Preston County, WV in habitat managed for ruffed grouse and American woodcock.

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.

VIRGINIA

Virginia Department of Forestry and Virginia Chapter of The American Chestnut Foundation

Jerre Creighton and John Scrivani:

Lesesne State Forest: 54 Years of Chestnut Breeding, 1968-the present

The 54-year history of chestnut breeding work at Lesesne State Forest in Nelson County, Virginia, was reviewed and the potential of the site for further breeding and research was discussed. The Lesesne backcrossing program primarily used large, surviving Americans (LSAs), in comparison to the TACF backcrossing program which sought no source of resistance from the American parents. The Lesesne program used the 10 surviving grafts in the American orchard and other LSAs from Central and Northern Virginia. The hybrid parents came from the 8 selections, or closely related trees, in the hybrid orchid. Another contrast with TACF backcrossing is that no blight inoculations were used to test the trees at Lesesne, only naturally-occurring blight (endemic and common) was used to challenge backcross trees and evaluate levels of resistance. Potential future work at Lesesne includes “best-by-best” crosses with Lesesne backcrosses and TACF backcrosses, LSA x LSA crosses, crosses with Darling58 pollen from SUNY/ESF, and other studies made possible by the large populations of hybrid, backcross and LSA trees over 24 acres.

Virginia Chapter, The American Chestnut Foundation

Fred V. Hebard,

Dr. Hebard's analysis showed that organic fertilizer is more expensive and bulkier than conventional fertilizer. A summary of the current TACF seed orchards was done and provided support to the argument that seed orchards are a massive resource.  The chapters have captured a tremendous amount of the genetic diversity in American chestnut. Should the B3-F3s have inadequate blight resistance after further testing that is being done, and they do not have as much as Chinese chestnut, we would need more generations of recurrent selection to increase it. In my opinion, that selection could not be accomplished by humans in orchards while maintaining the existing genetic diversity. Natural selection could be accomplished because the B3-F3s now have enough resistance to reproduce for extended periods.  They could resume evolving on their own.  Humans could help by favoring reproduction and by making some selections. This would be in addition to best x best and best x OxO crosses. At its most basic, all it entails is planting chestnuts in the woods.

2022 Publications

Carlson, E., Stewart, K., Baier, K., McGuigan, L., Culpepper, T. & Powell, W. (2022) Pathogen-induced expression of a blight tolerance transgene in American chestnut. Molecular Plant Pathology, 23, 370– 382. https://doi.org/10.1111/mpp.13165

Double, Mark, and Melinda Double. 2022.  Keeping it in the family. Chestnut, The Journal of The American Chestnut Foundation 36 (3):6-7.

Double, Mark. 2022. Second chances. Chestnut, The Journal of The American Chestnut Foundation 36 (3):16-17.

Gustafson, Eric J., Brian R. Miranda, Tyler J. Dreaden, Cornelia C. Pinchot, and Douglass F. Jacobs. "Beyond blight: Phytophthora root rot under climate change limits populations of reintroduced American chestnut." Ecosphere 13, no. 2 (2022): e3917. https://doi.org/10.1002/ecs2.3917

Newhouse, Andrew E., Anastasia E. Allwine, Allison D. Oakes, Dakota F. Matthews, Scott H. McArt, and William A. Powell. 2021. Bumble Bee (Bombus Impatiens) Survival, Pollen Usage, and Reproduction Are Not Affected by Oxalate Oxidase at Realistic Concentrations in American Chestnut (Castanea Dentata) Pollen. https://doi.org/10.1007/s11248-021-00263-w.

Noah PH, Cagle NL, Westbrook JW, Fitzsimmons SF (2021). Identifying resilient restoration targets: Mapping and forecasting habitat suitability for Castanea dentata in Eastern USA under different climate change scenarios. Climate Change Ecology 2, 100037, https://doi.org/10.1016/j.ecochg.2021.100037

Onwumelu, A., Powell, W.A., Newhouse, A.E. et al. Oxalate oxidase transgene expression in American chestnut leaves has little effect on photosynthetic or respiratory physiology. New Forests (2022). https://doi.org/10.1007/s11056-022-09909-x

Pinchot, Cornelia C., Alejandro A. Royo, John S. Stanovick, Scott E. Schlarbaum, Ami M. Sharp, and Sandra L. Anagnostakis. "Deer browse susceptibility limits chestnut restoration success in northern hardwood forests." Forest Ecology and Management 523 (2022): 120481. https://doi.org/10.1016/j.foreco.2022.120481

Schaberg, P. G., Murakami, P. F., Collins, K. M., Hansen, C. F., & Hawley, G. J. (2022). Phenology, cold injury and growth of American chestnut in a Range-Wide provenance test. Forest Ecology and Management, 513, 120178. https://doi.org/10.1016/j.foreco.2022.120178

Sandercock, AM, Westbrook JW, Zhang Q, Johnson HA, Saielli, TM, Scrivani JA, Fitzsimmons SF, Collins K, Perkins MT, Craddock JH, Schmutz J, Grimwood J, Holliday JA (2022). Frozen in time: Rangewide genomic diversity, structure, and demographic history of relict American chestnut populations. Molecular Ecology 31(18) 4640-4655. https://doi.org/10.1111/mec.16629  

Wright, James R., Stephen N. Matthews, Cornelia C. Pinchot, and Christopher M. Tonra. "Preferences of avian seed-hoarders in advance of potential American chestnut reintroduction." Forest Ecology and Management 511 (2022): 120133. https://doi.org/10.1016/j.foreco.2022.12013