NE1833: Biological Improvement of Chestnut through Technologies that Address Management of the Species and its Pathogens and Pests

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[08/05/2020] [08/18/2021] [02/17/2022] [11/30/2022]

Date of Annual Report: 08/05/2020

Report Information

Annual Meeting Dates: 09/05/2019 - 09/08/2019
Period the Report Covers: 10/01/2018 - 09/30/2019

Participants

Matthew Kasson ; John E. Carlson ; Laura Barth ; Bradley Hillman ; Kendra Collins ; Angus Dawe ; Kara Dobson ; Jason Payne ; Fred Hebard ; Emily P Dobry ; Bruce Levine ; Michael A Campbell ; Tetyana Zhebentyayeva ; Hannah Carter Pilkey ; Kirsten Hein ; Sandy Anagnostakis ; mkm5562 ; Yurij Bihun ; Trent Deason ; Kim Steiner ; Chuck Ray ; Linda McGuigan ; Don Nuss donaldnuss47@gmail.com ; Mark Double ; Rita.lcosta ; William MacDonald ; Hill Craddock ; Andrew Jarosz ; ben.jarrett@acf.org ; Revord, Ronald ; Steven Jeffers ; Tom Saielli ; Sara Fitzsimmons ; Amy Metheny

Brief Summary of Minutes

The 2019 NE-1833 annual meeting was held at Alpine Lake Resort in Terra Alta, WV. A total of 16 presentations representing nine state experiment stations spanned topics pertaining to all three of the project’s objectives. A field trip was hosted by Matt Kasson, Amy Metheny, and Bill MacDonald, who brought meeting participants to Savage Rivers State Forest in Western Maryland to see the two-year results of the NE1833-supported field study led by Amy Metheny examining biological control of chestnut blight in a natural American chestnut setting, using Super Donor strains of the fungus.

At the NE1833 business meeting, it was decided that the 2020 annual meeting will be held in Charlottesville Virginia with Tom Saielli and Jared Westbrook hosting.

Accomplishments

Objectives of NE1833:   Objective 1: Develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome.   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.   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.   Reports:   Sandra L. Anagnostakis, The Connecticut Agricultural Experiment Station Topic: Breeding Chestnut Trees for Gall Wasp Resistance (all Objective 1) Summary: <ul> <li>Dryocosmus kuriphilus has distribution across range of chestnut with new detection in CT in 2011</li> <li>Breeding efforts are underway using C. sativa, C. ozarkensis, C. pumila, and C. henryi</li> <li>First crosses, commercial trees x C. henryi, 2011; Second crosses, commercial trees x C. ozarkensis, 2013</li> <li>Crosses showing promise (starting to see growth beyond the galls)</li> </ul>   Fred V. Hebard, TACF Topic: The “what’s left from Chinese” question: can enumerating what remains from Chinese chestnut in the genome of selected B3-F2s help characterize genes for blight resistance? (all Objective 1) Summary: <ul> <li>Current Clapper BC3F2 selections range from 0% to 39% Chinese. Median 13% Chinese</li> <li>It is only this year that chromosome-level DNA sequences became available for chestnut. The data were voluminous, millions of data. The markers were distilled to about 5000 loci, depicted here.</li> <li>To explore the usefulness of testing the “What’s left from Chinese” hypothesis on the full dataset <ul> <li>Only seven of 163 B3-F2s had more than one locus homozygous Chinese.</li> <li>Forty-seven of 163 selections were homozygous Chinese on at least one locus among the five gut chromosomes.</li> <li>Ranking by the mean frequency of American alleles across the most Chinese locus on each chromosome spotlighted five putative loci for blight resistance that had shown signal in previous QTL searches.</li> </ul> </li> <li>The comparison of expected to observed genotype frequencies yielded suggestions of dominant and recessive gene action.</li> <li>The great virtue of this approach is that it depends solely on data intrinsic to DNA sequence. It does not depend on measurements of blight resistance</li> <li>Backcrossing was key to removing irrelevant loci.</li> </ul>   Brad Hillman, Rutgers University Topic: Viruses and transposons of Cryphonectria parasitica, the chestnut blight fungus (all Objective 2) Summary: <ul> <li>At least three independent horizontal transmission events were responsible for the four known hypovirus species of C. parasitica</li> <li>Infection of the reovirus MyRV2 from West Virginia C. parasitica strain C-18 is stabilized by coinfection with the hypovirus CHV-4</li> <li>The C. parasitica mitochondrial virus CpMV1 can be introduced into different fungal strains, species, genera and families by protoplast fusion, is stable in many but not all</li> <li>Through RNAseq and PCR, we examined expression of the 9.2 kb C. parasitica helitron transposable element, and found that only the 5’-terminal 0.8 kb of the element appears to be expressed in culture</li> </ul>   Linda McGuigan and Kristen Stewart, SUNY ESF Topic: Transgenic American chestnut Update (all Objective 1) Summary: <ul> <li>Laccase-like gene from Chinese chestnut (cisgene); Laccase-like gene expression much higher in resistant Chinese chestnut vs. the susceptible American chestnut</li> <li>Phytophthora Resistance Genes?</li> <li>Quick Leaf Assay Survey of T2 offspring</li> <li>Initiating Ozark Chinquapin Embryos in Tissue Culture</li> <li>New Ozark chinquapin embryo lines have been established in tissue culture for future transformations</li> <li>First transformation with embryos from OK is complete</li> <li>Optimizing multiplication, rooting, and acclimatization of Ozark chinquapin in anticipation of a transgenic OC</li> <li>Use genetic engineering to develop American elms with resistance/tolerance to: <ul> <li>Dutch elm disease</li> <li>Elm yellows</li> <li>Phytophthora</li> <li>Other vascular wilt diseases</li> </ul> </li> </ul>   Steve Jeffers, Clemson Topic: NE1833 Chestnut Research at Clemson University Summary: <ul> <li>Focus at Clemson: Phytophthora root rot (PRR) <ul> <li>PRR is the other major disease that kills American chestnut (Objective 2)</li> </ul> </li> <li>Collaborating with TACF to screen backcross hybrid chestnut seedlings for resistance to Phytophthora cinnamomi (Objective 1)</li> <li>Evaluating the virulence of different populations of P. cinnamomi to hybrid chestnut seedlings (Objective 1)</li> <li>Excised twig assay to identify resistance in hybrid chestnut trees</li> <li>Detection of Phytophthora spp. in soils where chestnut are growing or might be planted (Objective 2)</li> <li>Efficacy of oomycete-specific fungicides for Phytophthora root rot (PRR) on American chestnut seedlings (Objective 2)</li> </ul>   Angus Dawe, Mississippi State University Topic: Mississippi Report Summary: (all Objective 2) <ul> <li>Description of predicted LysM proteins that play a role in regulation of growth and development in C. parasitica</li> <li>Genome sequencing of 90+ progeny from crosses of two standard C. parasitica strains, SG2-3 x EP155</li> </ul>   Amy Metheny and Matt Kasson, WVU Topic: WVU Report: Super Donor 2.0 (all Objective 2) Summary: <ul> <li>Application method and hypovirus both impact biocontrol efficacy using field deployed super donor strains in a forest setting</li> </ul>   Paul Sisco, Carolinas Chapter - TACF Topic: Results of Using F1’s as controls in the Carolinas Chapter Seed Orchard Summary: <ul> <li>We would like Restoration Chestnut trees to be as least as blight resistant as F1’s (Objective 3)</li> <li>Male-sterile F1’s will not contaminate seed orchard (Objective 3)</li> <li>New sources of resistance can be introduced by collecting seed of F1 x B3F2 trees selected for blight resistance “Better B1 trees” (Objective 2)</li> <li>Phytophthora resistance can be added by collecting seed of F1 x B3F2 selected for blight resistance “Better B1 trees” (Objective 2)</li> </ul>   Tom Saielli, TACF Topic: Engaging Citizen Scientists to help find surviving American chestnut Summary: <ul> <li>The more trees that are found, the more trees that can be used in the (3BUR) breeding program (Objective 1)</li> <li>To preserve native germplasm (in GCO’s) (Objective 1)</li> <li>Harvest open-pollinated American chestnut seeds (Objective 3)</li> <li>Cultural significance, educational opportunities, fun and interesting (Objective 3)</li> </ul>   Jared Westbrook, TACF Topic: Genome-wide ancestry inference in American chestnut backcross hybrids With application for mapping loci associated with resistance to Phytophthora cinnamomic Summary: Discover regions of BC3F2 mother trees’ genomes associated with variation in mortality of their BC3F3 after infection with P. cinnamomic (Objective 1)   Rita Costa, INIAV Topic: Understanding the Interaction of Phytophthora cinnamomi Rands with Castanea spp. Summary: (all Objective 1) <ul> <li>A breeding program for resistance of chestnut to Phytophthora cinnamomi was initiated in 2006, based on controlled crosses, using the Asian resistant species, C. crenata and C. mollissima, as donors of resistance and C. sativa as female parent.</li> <li>The progenies were root phenotyped through inoculation with the pathogen.</li> <li>Under the scope of the research program, candidate genes and QTLs were identified.</li> <li>The comprehension of the host-pathogen system in Castanea spp using histopathology is being performed and the functional validation through genetic transformation is being implemented</li> <li>The ultimate goal is to select molecular markers linked with resistance genes to expedite selection of genotypes with improved resistance to the pathogen</li> </ul>   Tetyana Zhebentyayeva, John Carlson, Penn State Topic: Update on Genetics of Resistance to Phytophthora cinnamomi in Chestnut: an Integrated Genetic and Genomic Approach for Candidate Gene Discovery Within QTL Interval Summary: <ul> <li>Examination of quantitative trait loci for resistance to Phytophthora cinnamomi (Objective 1)</li> <li>Update on genome sequencing of Castanea mollissima and Castanea dentata (Objective 1)</li> </ul>   Dana Nelson, University of Kentucky Topic: QTL Mapping Resistance to Cryphonectria parasitica in Chinese × American Chestnut Hybrid Families Summary: <ul> <li>Description of recent progress with mapping of quantitative trait loci for resistance against Cryphonectria parasitica in Castanea mollissima X Castanea dentata progeny (Objective 2)</li> </ul>   Andrew Jarosz, Michigan State University Topic: Michigan report Summary: <ul> <li>Perspective from the Midwest Chestnut Producers Council (Objective 3)</li> <li>Broad/Preliminary look at hypovirulence treatments from 2013 to 2019 at two large commercial orchards in Michigan.  (Objective 2)</li> <li>Utilizing tree ring analyses to determine the age of chestnut blight cankers. (Objective 2)</li> </ul>

Publications

Westbrook, J.W., James, J.B., Sisco, P.H., Frampton, J., Lucas, S. and Jeffers, S.N., 2019. Resistance to Phytophthora cinnamomi in American chestnut (Castanea dentata) backcross populations that descended from two Chinese chestnut (Castanea mollissima) sources of resistance. Plant disease, 103(7), pp.1631-1641.   Zhebentyayeva, T.N., Sisco, P.H., Georgi, L.L., Jeffers, S.N., Perkins, M.T., James, J.B., Hebard, F.V., Saski, C., Nelson, C.D. and Abbott, A.G., 2019. Dissecting resistance to Phytophthora cinnamomi in interspecific hybrid chestnut crosses using sequence-based genotyping and QTL mapping. Phytopathology, 109(9), pp.1594-1604.   Perkins, M.T., Robinson, A.C., Cipollini, M.L. and Craddock, J.H., 2019. Identifying host resistance to Phytophthora cinnamomi in hybrid progeny of Castanea dentata and Castanea mollissima. HortScience, 54(2), pp.221-225.   Stauder, C.M., Nuss, D.L., Zhang, D.X., Double, M.L., MacDonald, W.L., Metheny, A.M. and Kasson, M.T., 2019. Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes. Virology, 528, pp.1-6.   Staton, M., Addo-Quaye, C., Cannon, N., Sun, Y., Zhebentyayeva, T., Huff, M., Fan, S., Bellis, E., Islam-Faridi, N., Yu, J. and Henry, N., 2019. The Chinese chestnut genome: a reference for species restoration. bioRxiv, p.615047.   Perkins, M.T., Zhebentyayeva, T., Sisco, P.H. and Craddock, J.H., 2019. Genome-wide sequence-based genotyping supports a nonhybrid origin of Castanea alabamensis. BioRxiv, p.680371.   Sato, Y., Miyazaki, N., Kanematsu, S., Xie, J., Ghabrial, S.A., Hillman, B.I. and Suzuki, N., 2019. ICTV Virus Taxonomy profile: megabirnaviridae. Journal of General Virology, 100(9), pp.1269-1270.   Aulia, A., Andika, I.B., Kondo, H., Hillman, B.I. and Suzuki, N., 2019. A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing. Virology, 533, pp.99-107.   Shahi, S., Eusebio-Cope, A., Kondo, H., Hillman, B.I. and Suzuki, N., 2019. Investigation of host range of and host defense against a mitochondrially replicating mitovirus. Journal of Virology, 93(6).   Crouch, J.A., Dawe, A., Aerts, A., Barry, K., Churchill, A.C., Grimwood, J., Hillman, B.I., Milgroom, M.G., Pangilinan, J., Smith, M. and Salamov, A., 2020. Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen. Phytopathology, pp.PHYTO-12.   Mcguigan, L., Chartrand, A., Northern, L., Russell, K., Powell, W. and Maynard, C., 2019, August. Use of a Temporary Immersion Bioreactor System to Transform American Chestnut Somatic Embryos. In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT (Vol. 55, No. 4, pp. 483-484). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.   Pilkey, H.C., McGuigan, L.D. and Powell, W.A., 2019, August. Genetic Transformation of the Ozark Chinquapin (Castanea ozarkensis). In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT (Vol. 55, No. 4, pp. 487-488). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.   Brown, A.J., Newhouse, A.E., Powell, W.A. and Parry, D., 2019. Comparative efficacy of gypsy moth (Lepidoptera: Erebidae) entomopathogens on transgenic blight‐tolerant and wild‐type American, Chinese, and hybrid chestnuts (Fagales: Fagaceae). Insect Science.   Powell, W.A., Newhouse, A.E. and Coffey, V., 2019. Developing blight-tolerant American chestnut trees. Cold Spring Harbor Perspectives in Biology, 11(7), p.a034587.   Oakes, A.D., Pilkey, H.C. and Powell, W.A., 2019, June. Improving Ex Vitro Rooting and Acclimatization Techniques for Micropropagated American Chestnut. In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL (Vol. 55, pp. S71-S71). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.   Goldspiel, H.B., Newhouse, A.E., Powell, W.A. and Gibbs, J.P., 2019. Effects of transgenic American chestnut leaf litter on growth and survival of wood frog larvae. Restoration Ecology, 27(2), pp.371-378.   Cipollini, M., Wessel, N., Moss, J.P. and Bailey, N., 2019. Seed and seedling characteristics of hybrid chestnuts (Castanea spp.) derived from a backcross blight-resistance breeding program. New Forests, pp.1-19.

Impact Statements

Tens of thousands of backcross hybrid chestnut trees have been planted throughout the range of the native American chestnut, and the effort has led to massive public engagement

Date of Annual Report: 08/18/2021

Report Information

Annual Meeting Dates: 09/17/2020 - 09/17/2020
Period the Report Covers: 10/01/2019 - 09/30/2020

Participants

Bradley Hillman
Tom Saielli
Sara Fitzsimmons
Amy Methany
Matt Kasson
Andrew Newhouse
Thomas C. Klak
Dana Nelson
Fred Hebard
Jared Westbrook
Alex Sandercock
Hill Craddock
Steven Jeffers
Ellen Crocker
Monique Sakalidis
Stacy Clark
Leila Pinchot
Angus Dawe
Sandra Anagnastokis

Brief Summary of Minutes

Minutes Attachment:

NE1833 Annual Meeting Report 2020-Summary.docx

Accomplishments

NE1833 Annual Meeting Report 2020   Station Reports:   CONNECTICUT Sandra L. Anagnostakis, Emeritus, The Connecticut Agricultural Experiment Station, New Haven, CT, web page  <a href="https://www.ct.gov/caes/sla">https://www.ct.gov/caes/sla</a> Projects fall under Objective 1, 2 and 3:   During the past year I sent all of the important chestnut documents and materials housed at the Connecticut Agricultural Experiment Station to the National Agricultural Library in Maryland for preservation.  This included all of the USDA Plant Importation and Distribution cards, with records of where imported trees and seeds were sent within the US and to other countries. Planting records from Bell, MD included information on the germination of seeds received, and their designations.  I have recorded some of this on my web page as “Chestnut Importations into the U.S.”  There were boxes of photographs and negatives from USDA work and many from R. K. Beattie who traveled in the early 1900’s in Japan.  We had several extensive reports from the plant explorer Peter Liu, which were illustrated with photographs that he took in his searches for chestnuts in China. I also sent all of the USDA breeding records, the CAES breeding records, all of the laboratory books (fungal stock records) and all theses in my collection, including those of Nienstaedt, Jaynes, Grente, Anagnostakis, and Hebard. I now have grant funds to scan all of the distribution cards and make them available to anyone who wants the information.  However, the Library is closed, and no work can be done until it reopens.  The person in charge of the Special Collection (“The Chestnut Collection”) is: Amy Morgan Special Collections Librarian National Agricultural Library 10301 Baltimore Avenue, Room 304 Beltsville, MD  20705 Phone: 301-504-5876 Fax: 301-504-7593  Email: <a href="mailto:NALSpecialCollections@ars.usda.gov">NALSpecialCollections@ars.usda.gov</a> If others have historical documents and items relating to chestnuts, I recommend sending them to the Library’s Special Collection for keeping.   MAINE Thomas Klak, University of New England Project falls under Objective 3:   Transgenic Chestnut Pollen Production under High-Lights & Field Pollination  The Chestnut restoration team at the University of New England has had good success producing transgenic (blight-tolerant) pollen from seeds and seedlings obtained from SUNY-ESF. We collected and froze T2 pollen from eight lines and distributed that to six collaborating institutions for pollination of wild type chestnuts in July 2020. The UNE team is expanding this pollen-production project in the coming year. We are attempting to produce T3 transgenic pollen during the same year of harvest, with hopes that it could be available for July 2021 pollination season. We also took our transgenic pollen to the field and pollinated about 1500 flowers on wild type trees, and this yielded approximately 600 fertile transgenic nuts. The other collaborating sites also had success numbering in the hundreds of transgenic nuts each. So in total we have increased our transgenic nut production and diversity considerably, as we continue to work under federal permits in hopes of eventual deregulation.  Quantifying and prioritizing the determinants of wood quality in chestnut variants.    MICHIGAN Monique Sakalidis Michigan State University Research Update   Projects fall under Objective 2:   Brown rot is a disease of chestnuts that has been reported globally in nut production areas particularly in Europe and Australia and can result in up to 91% of chestnuts infected. It has been detected annually since 2017 in Michigan. The fungus Gnomoniopsis smithogilvyi (G. smithogilvyi) causes brown rot on chestnuts pre- and post-harvest. The disease is characterized by soft, brown lesions on the kernel and is only detected when nuts are cut open. The presence of brown rot in nuts leads to unsalable nuts. Additionally, as the disease develops overtime after harvest, this can lead to the degradation of nuts that have passed quality check at harvest. Using nuts sampled in fall 2019 and 2020 we have evaluated the diversity of nut rotting organisms, particularly Gnomoniopsis species, and evaluated the effect of cold storage and host cultivar on disease incidence and severity. We have found that cold storage results in decreased disease severity and incidence and that the cultivar Colossal is most susceptible to natural and artificial infection. Future research will focus on the timing of spore production in the field and the infection pathway into the host.   MISSISSIPPI Angus Dawe, Department of Biological Sciences, Mississippi State University   Current personnel: Graduate students –Soum Kundu, Melanie Tran Research Associate – Gisele Andrade (part time) Visiting Scientist – Kum-Kang So   Projects fall under Objective 2: <ol> <li>Identifying parasitica genes associated with pathogenicity and virulence</li> <li>ARV-1 and its potential role in sterol homeostasis</li> <li>Impact of MAPK signaling pathways on fungal phenotype, virulence, and hypovirulence</li> </ol> Project details <ol> <li>Identifying parasitica genes associated with pathogenicity and virulence. (Melanie Tran, MS student.)</li> </ol> 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. In total, the run generated >800 million reads (PE 150), with QC30>81.7%. Coverage is estimated at >15-30X for almost all the samples with only minor quality issues with source DNA for a small number. 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 involved and will be assisting in relating the sequence data to the phenotype data. <ol start="2"> <li>ARV-1 and its potential role in sterol homeostasis. (Soum Kundu, PhD student).</li> </ol> ARV-1 is a predicted gene in C. parasitica that shares similarity with genes that code for proteins with important roles in sterol hemeostasis 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 been working to develop an assay for sterol production in C. parasitica by modifying published protocols and using a GC/MS system in conjunction with Todd Mlsna in the Department of Chemistry at Mississippi State. Recent success using derivatization techniques to tag the appropriate class of compounds are indicating that sterol production is affected by hypovirus infection, although these results require further study and confirmation. <ol start="3"> <li>Impact of MAPK signaling pathways on fungal phenotype, virulence, and hypovirulence. (Kum-Kang So, visiting scientist)</li> </ol> Dr. So came to us from South Korea, arriving in early 2020 and unfortunately was unable to accomplish much before the COVID-related disruptions began. Her time in the US is limited to a year so she is now working hard to make up for lost time. She has a number of mutants in various components of the MAPK signaling pathway of C. parasitica from her PhD work with Dr. Dae-Hyuk Kim and she is further investigating these and transcription factors that mediate their expression. She is also looking into any correlations between the impact of these mutations and the hypovirulent phenotype caused by infection with CHV1 hypovirus. NEW JERSEY Bradley Hillman, Rutgers University Projects fall under Objective 2: We continued to examine effects of related and unrelated viruses on each other during mixed infections in C. parasitica. Coinfection with different viruses may have varying effects on the fungal host, and understanding those effects helps evaluate the potential effectiveness of viruses as biocontrol agents. Infection of the mycoreovirus MyRV2 from West Virginia C. parasitica strain C-18 was reported last year to be potentiated by coinfection with the unrelated hypovirus CHV4; MyRV2 required coinfection to be stable through repeated serial passage in culture. The N-terminal protein encoded by the CHV4 has now been confirmed to be a 24 kDa protease (p24) and has been identified as the protein responsible for potentiating co-infection of the fungus with MyRV2 by acting as a suppressor of RNA silencing. p24 is therefore similar to the well-studied p29 protein of the prototypical hypovirus CHV1 in serving as a self-cleaving protease and as a suppressor of RNA silencing. Unlike CHV1 p29, CHV4 p24 does not serve as a symptom determinant. It is not yet known whether p24 is part of an internal ribosome entry site (IRES) element, as CHV1 p29 is. This project is a collaboration with Dr. Nobuhiro Suzuki, Okayama University. <ol> <li>We proceeded with a project mapping of trees and environmental sampling of chestnut blight cankers in New Jersey, and discovery of new viruses associated with hypovirulence and biological control. We have begun to collect C. parasitica isolates from northern New Jersey, near the New York border, to look for hypovirulent isolates and compare them to isolates from the Atlantic Highlands/Middletown NJ region that we’ve collected over many years. Due to COVID-19, only three isolates were collected in 2020 from a single infected tree before collected stopped. </li> <li>To examine factors promoting virus invasion and adaptation to C. parasitica, we expanded a project comparing codon use among different viruses to codon use in the common host organism. The degeneracy of the genetic code dictates that the 20 amino acids are encoded by 61 codons, and many factors contribute to which codons are preferred in a given organism. Codon bias can be high (at extreme, only 20 codons used to encode the 20 amino acids) or low (at extreme, all 61 codons used equally. The expectation is that the host organism has evolved to optimize its codon use, and that viruses that are well adapted to that host organism evolve toward a similar codon use profile. Especially with RNA viruses, codon preference for translation in a given host is only one driver of nucleotide preference at a given position – RNA structure/function relationships are critical. Our hypothesis is that viruses that have been resident in C. parasitica for a longer time will display greater codon adaptation and thus grater similarity to the fungal host. Through use of a variety of metrics, our analysis of codon usage of six viruses of C. parasitica whose genomes have been completely sequenced supports the hypothesis of a long-term association with host C. parasitica of three members of the family Hypoviridae: CHV1, CHV2, and CHV4; and sorter-term association of one Hypoviridae member, CHV3, and of the two members of the Reoviridae family, MyRV1 and MyRV2. These results are generally consistent with current knowledge of the biogeography of these viruses: CHV1 and CHV2 are closely related to one another and both have been found in Asia, presumably having been there before the fungus invaded North America. In contrast, MyRV1, MyRV2, and CHV3 all have been found only in North America and presumably infected the fungus only after its arrival in North America in the late 19th Century. The unexpected result was that CHV4, which is well established in North America, has never been found in Asia. Our results suggest that CHV4 may have a longer association with the fungus than previously thought, and Asian isolates of C. parasitica should be examined specifically for its presence.</li> </ol> NEW YORK Andy Newhouse - SUNY-ESF American Chestnut Research & Restoration Project   Projects fall under Objective 1 and 3:   Summary: Transgenic American chestnuts have been created to express oxalate oxidase, an enzyme found naturally in a wide variety of plants and other organisms.  Preliminary tests have shown it is effective at reducing damage (canker size) on stems compared to wild-type American chestnuts.  Blight damage on young Darling 58 transgenic trees is approximately similar to that seen on Chinese chestnuts, which can get blight but typically tolerate it well enough to keep growing.  The lack of a direct pesticidal mechanism suggests it should be relatively evolutionarily stable.  ESF has completed a variety of environmental & nutritional tests that collectively show a lack of enhanced risks compared to traditional breeding.  Larger-scale, longer-term ecological tests are in progress.  Regulatory review in the US consists of three agencies: USDA, EPA, & FDA.  The USDA review has begun, and the public comment period for their Plant Pest Risk Assessment is currently open.  TACF has instructions and details on their website for how to make comments on the Federal Register.  So far, most of the comments are positive, in contrast to more typical GE agricultural crops that often draw large numbers of negative comments.  Next research steps are focusing on enhancing genetic diversity through outcrossing with wild-type American chestnuts, and incorporating Phytophthora resistance (possibly through backcrossing).     PENNSYLVANIA Charles D. Ray, Penn State Gary Carver, TACF Sara Fitzsimmons, Penn State/TACF Michael Wiemann, USDA Forest Products Lab Projects fall under Objective 3: The objective of this project is to determine which wood properties in chestnut variants result in statistically significant differences between variants. Based on our findings, these properties and the statistical parameters of investigation will be prioritized for future, long-term chestnut wood studies.  The criteria for prioritization will include importance to wood quality for commercial applications and relationship to desirable growth characteristics. Findings to date include: 1) chestnut color is distinctly differentiable from other common wood species, 2) juvenile and mature wood in chestnut are distinctly differentiable, allowing determination of chestnut specimens to be categorized as being either from a mature tree or from an immature stem, and 3) within juvenile/mature wood classifications, chestnut color is not differentiable between Castanea species. This hints that at least one major reason that chestnut specimens culled from restoration plantations look different than traditional chestnut lumber is that they are from juvenile wood, and that mature wood from these same plantations may grow to closely resemble and assume other properties of traditional C. dentata.   SOUTH CAROLINA Steven N. Jeffers – Dept. of Plant & Environmental Sciences, Clemson University Projects fall under Objective 1 and 2: Screening Hybrid Chestnut Seedlings for Resistance to P. cinnamomi <ul> <li>Previously, screening was conducted in SC for 14 years (2004-2017) in collaboration with TACF <ul> <li>All trials were conducted at Chestnut Return Farms in Seneca, SC using local isolates</li> </ul> </li> <li>Operation turned over to TACF in 2018 <ul> <li>Moved to USDA Forest Service Resistance Screening Center at the Bent Creek Experimental Forest in Asheville, NC</li> <li>Katie McKeever (USDA FS) and Jared Westbrook (TACF) are now supervising this project</li> <li>Our lab at Clemson provides isolates used as inoculum and assists with inoculation and final evaluation of seedlings</li> </ul> </li> <li>2019: New TACF strategy for screening – Use different isolates of cinnamomi each year <ul> <li>Each year, survivors from the screening trials are planted outside in the field in predetermined locations were cinnamomi is already present to determine if seedlings will survive under natural environmental conditions</li> <li>Use isolates of cinnamomi from the out-planting field location to ensure that we do not move unique genotypes of pathogen to new locations</li> <li>Field location identified during fall/winter prior to annual screening, and isolates from soil are recovered, identified, and stored at Clemson</li> </ul> </li> <li>Sources of isolates used as inoculum for screening and field location for out-planting <ul> <li>2004-2018: Two SC isolates from Chestnut Return Farms in Seneca, SC</li> <li>2019: Two NC isolates from Mountain Island Educational State Forest; Stanley, NC</li> <li>2020: One GA isolate from the Austin Flint North Ridge site in GA</li> </ul> </li> <li>Advantage of new strategy: <ul> <li>Each year, hybrid seedlings are screened with different isolates, so seedlings are exposed to diverse genotypes of cinnamomi</li> </ul> </li> <li>Potential disadvantage of new strategy: <ul> <li>Virulence of isolates may vary from year to year; therefore, screening rigor may vary from year to year – so this needs to be evaluated</li> </ul> </li> </ul>   Evaluating Virulence of P. cinnamomi Isolates <ul> <li>Previous experiment in the Jeffers lab on American chestnut showed similar virulence among five groups of isolates of cinnamomi from chestnut trees collected at different geographic locations in the Southeast</li> <li>Experiment conducted in 2019 & 2020 in collaboration with TACF & USDA-FS and conducted at the Bent Creek RSC</li> <li>Experimental design <ul> <li>Eight cinnamomi inoculum treatments—including the five treatments used previously on American chestnut plus two additional treatments composed of isolates from ornamental plants and a non-inoculated control treatment</li> <li>2-3 isolates in each treatment with each treatment in a separate, isolated tub</li> <li>Number of hybrid chestnut families inoculated: 8 in 2019 and 33 in 2020</li> <li>All plants scored weekly by recording days to mortality = survival time</li> </ul> </li> <li>Results from 2019: Data have not been analyzed statistically there may be significant differences among inoculum treatments <ul> <li>Hybrid chestnut families have different levels of resistance, which may help identify differences in virulence among isolates</li> </ul> </li> <li>Experiment is being repeated in 2020 using different hybrid chestnut families</li> </ul>   Detection of Phytophthora spp. in Chestnut Samples <ul> <li>Conducted In collaboration with TACF: We continue to assay soils and symptomatic chestnut seedlings for Phytophthora – including soils where chestnuts are growing or might be planted</li> <li>Protocol is a simple baiting bioassay or direct isolation</li> <li>In the past year: Sep 2019 – Aug 2020 <ul> <li>Samples received from 6 states: AL, GA, NC, PA, SC, TN – including nine submissions that contained 28 soil samples; this number of samples received is down compared to previous years</li> <li>Phytophthora detected in 4/28 samples = 14%; all isolates recovered were P. cinnamomi</li> </ul> </li> </ul>   Fungicides for Phytophthora Root Rot (PRR) on American Chestnut Seedlings <ul> <li>Rational: TACF is encouraging the planting of Germplasm Conservation Orchards (GCO) to collect and preserve existing American chestnut genotypes <ul> <li>Trees planted in GCOs in states where cinnamomi is present are at risk of infection and mortality, so fungicides could be used to protect susceptible seedlings and trees</li> </ul> </li> <li>Objective: Evaluate the efficacy of registered oomycete-specific fungicides to manage PRR on American chestnut seedlings</li> <li>Trial conducted in a greenhouse at Clemson University in 2019-2020 <ul> <li>Experimental Design: 10 treatments: 2 controls + 8 fungicides with 8 replicate seedlings/treatment; Fungicides were applied following label rates & application intervals</li> </ul> </li> <li>Results were published in Plant Disease Management Reports, an online publication</li> <li>Overall conclusions: Fungicides vary in efficacy at protecting American chestnut seedlings <ul> <li>Promising active ingredients: K salts of phosphorous acid (phosphonates), fosetyl-AL (similar to phosphonates), mefenoxam</li> <li>Experiment is being repeated in 2020 but results do not appear to be consistent, which may be due to “modified operations” at Clemson during the pandemic</li> </ul> </li> <li>Therefore, this experiment will be conducted again in 2021</li> </ul>   Understanding Host Resistance in the Chinese Chestnut–P. cinnamomi Pathosystem <ul> <li>This is a multi-state/university collaboration involving Clemson, Penn State, USDA-FS, Univ. of Kentucky, and Univ. of Tennessee</li> <li>A grant proposal was submitted to NSF/NIFA Plant Biotic Interactions Program in 2019 <ul> <li>Project was led by Tatyana Zhebentyayeva at Penn State</li> <li>Our proposal was not funded – in part because we needed more preliminary data</li> </ul> </li> <li>Therefore, an experiment was conducted in Aug 2020 at Clemson University to examine the early steps of the infection process and determine when plants become infected and what genes are being regulated <ul> <li>Roots of American and Chinese chestnut seedlings in an aqueous environment were inoculated with zoospores of cinnamomi</li> </ul> </li> <li>Current status: Frozen root samples are still waiting to be analyzed</li> <li>We have plans to repeat this experiment with minor adjustments to the protocol</li> </ul>   TENNESSEE Hill Craddock, The University of Tennessee at Chattanooga Department of Biology Geology and Environmental Science Projects fall under Objective 1: Breeding for Disease Resistance <ul> <li>Finished selections at Ruth Cochran Orchard and Dave Cantrell Orchard</li> </ul> <ol> <li> dentata collected from underrepresented areas in Alabama and Tennessee</li> </ol> <ul> <li>Clonal collections maintained in field plots in Indiana and in container nursery in Tennessee</li> <li>Germplasm Conservation ex situ</li> </ul> Phylogeography of Castanea in the southern US <ul> <li>Collection trip (with Sisco and Paillet) to S. Missouri and NW Arkansas</li> <li>Annotations of 900 herbarium sheets for Perkins et al</li> </ul> UTC graduates <ul> <li>Masters Theses: Meg Miller & Trent Deason</li> <li>Undergraduate Honors Theses: Hannah Crawford, Colton Jones (Peyden Valentine)</li> </ul> Works in Progress <ul> <li>Herbarium vouchers prepared for SERNEC imaging and digital data capture</li> <li>Nursery production of BnF2s for TN seed orchards</li> <li>Nursery production of C. dentata germplasm for GCOs</li> </ul>   Stacy L. Clark (USDA Forest Service, Southern Research Station, Knoxville, TN), Leila Pinchot (USDA Forest Service, Northern Research Station, Delaware, OH), and Scott E. Schlarbaum (The University of Tennessee, Department of Forestry, Wildlife, and Fisheries, Knoxville, TN) Projects fall under Objective 1 and 3: The University of Tennessee’s Tree Improvement Program (UT-TIP) chestnut activities include evaluations of historic chestnut plantings at the Norris Reservation (Tennessee Valley Authority) in TN and collaborating with the USDA Forest Service Southern and Northern Research Stations. The collaborative work includes implementation and long-term comprehensive field evaluations of chestnut research test plantings (ca. 2009-2017) in NC, PA, TN, and VA. Experimental material represents 7500 trees from various breeding generations (BC1F3, BC2F3, BC3F3, BC3F2) and parental species (American and Chinese chestnut) from The American Chestnut Foundation’s and the Connecticut Agricultural Experiment Station’s breeding programs. Evaluations of survival, growth, blight resistance, deer herbivory, and competitive ability within different silvicultural prescriptions have been conducted. Results indicate chestnuts bred for blight resistance exhibit superior competitive ability and intermediate blight resistance, but performance varies depending on seedling quality, vegetation competition, site quality, and deer browse pressure at the time of planting.   THE AMERICAN CHESTNUT FOUNDATION Tom Saielli and1 Sara Fitzsimmons2 1The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902 2The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802 Ecological Studies on American chestnut hybrids   Projects fall under Objective 1 and 3:   Summary: The most up to date research on traditionally bred hybrid chestnuts indicates that there is a tradeoff between blight resistance and percent American chestnut germplasm, how do we balance “resistant enough” with “American enough”? We propose ecological studies to assess ecological fitness among various hybrid genotypes with a range of American vs. Chinese germplasm. Studies could be performed in labs and greenhouses and at least one or more field studies. Variables of interest may include, but will not be limited to: leaf and seed herbivory, wood and leaf litter decomposition, associations with shoot and root microbes, etc. TACF seeks to fully understand how ecologically fit various hybrids may or may not be and use that information to inform future breeding strategies. TACF seeks partners to assist with these studies. Fred Hebard, The American Chestnut Foundation   Projects fall under Objective 1 and 3:   The American Chestnut Foundation has been conducting a vigorous program of backcrossing the blight resistance of Chinese chestnut into American chestnut.  At the main breeding facility in Meadowview, VA, around 60,000 nuts of B3-F2 progeny in about 30 American backgrounds from two sources of blight resistance were planted in two seed orchards.  Blight resistance of B3-F2s and their B3-F3 progeny was intermediate between that of Chinese and American chestnut, as opposed to equaling the resistance of Chinese chestnut (Steiner et al 2017).   The following study sought to find reasons resistance was not higher. The lower-than-expected level of blight resistance found in B3-F2s might be explained, at least in part, by two complementary hypotheses.  The first is that there are numerous genes for blight resistance and many were lost during backcrossing, so that B3 parents were not heterozygous at all loci conferring resistance.  This would make it impossible to obtain B3-F2s homozygous resistant at those loci.  The second hypothesis is that chestnut has a preference to be heterozygous, and that homozygotes are disfavored by recessive lethal genes and other mechanisms.  Again, this would result in fewer loci homozygous resistant.  Such homozygous deficiency might be exacerbated because the B3-F2s were identical by descent at all Chinese loci, being bred within two sources of blight resistance both derived from a single B1. To test these hypotheses, and others, it would be very helpful to infer the male parent of the open-pollinated B3-F2 progeny.  The 60,000 B3-F2 seed had been winnowed down by mortality and by selection using phenotyping and progeny testing. Two-thousand, six-hundred, seventy-one were genotyped by sequencing (GBS).  An additional 189 potential parents were genotyped.  For this study, 938 SNP markers (single nucleotide polymorphisms) were used for Ellis’ method of hierarchal clustering. The objective was to infer the most likely male parent of chestnut B3-F2 progeny resulting from open pollination of B3s.  About 80% of likely male parents were from the same source of blight resistance as the female parent.  These were the subjects of the study.  They included 826 B3-F2s from the Clapper source of blight resistance, 699 from the Graves source, and 139 B3 parents. The GBS data had been further processed to declare whether or not 100-kb bins along the chromosomes were derived from Chinese or American chestnut.  Under the assumption that blight resistance was conferred by Chinese alleles, this transformation made the data easier to process and interpret.  I owe profound thanks to Jared Westbrook for assembling and leading the team of researchers who prepared these datasets.  Jared furthermore made further selections in B3-F2 progeny based on tests of their B3-F3 progeny, field traits of the B3-F2s themselves, and genomic selection based on SNPs identified by GBS.  Two-hundred, fifty were selected and 1275 rejected. RESULTS The largest Clapper family had 31 progeny, with the number of members of the second largest family declining to 20, the third to 14, with the tenth largest family having only 5 progeny.  Those numbers of family members were inadequate to test directly for homozygote deficiency, even though there were 825 Clapper progeny.  Thus, tests for heterozygous excess had to be conducted with aggregates of families The location on a chromosome containing the largest number of Chinese chestnut alleles would be most associated with a resistance gene, assuming these all came from Chinese chestnut, other things being equal.  Taking the median position of the locus with the largest number of Chinese alleles was considered a good first approximation of loci associated with blight resistance.   The mean number of Chinese alleles varied considerably within and between chromosomes.  There were three chromosomes in Clapper progeny that had frequencies of Chinese alleles greater than 0.2, chromosomes 5, 10 & 12, and five chromosomes in Graves progeny, 1, 7, 9, 10 & 12.  These can be considered to be relatively major _chromosomes_ associated with resistance.  Interestingly, two chromosomes, 5 in Clapper and 10 in Graves, with high frequencies of Chinese alleles did not have a significant incease after the latest round of selection.  Although two chromosomes with high frequencies of Chinese alleles were shared between Clapper and Graves, the closest were 8 Megabases apart, so most likely did not contain homologous genes If there had been no loss of resistance alleles during backcrossing, the B3 parents would have been exclusively heterozygous for resistance and it would have segregated 1:2:1 in B3F2s.  The expected segregation in B3-F2s was calculated from the allele frequencies in their B3 parents. The counting occurred at one locus per chromosome for the two sources of blight resistance.  There were massively fewer Chinese alleles, 67% less, than would have occurred under 1:2:1 segregation.  Thus, loss of alleles during backcrossing had a major influence on the decrease in blight resistance of B3-F2s compared to expectation. Comparing the observed segregation of Chinese alleles in B3-F2s to that expected from allele frequencies in B3 parents would reveal the effect of homozygous deficiency on allele frequency.  It revealed only slight further erosion of the number of Chinese alleles.  But the frequency of loci homozygous for resistance declined 46% further.  It had already declined by 87% comparing expected to 1:2:1 segregation.  Recovery of progeny homozygous for resistance alleles would double their number compared to heterozygotes, and would be the means for increasing their number once most progeny were heterozygous.  Thus homozygous deficiency is likely an important factor limiting high levels of blight resistance. It is not an insurmountable problem however, since homozygotes were recovered.   VIRGINIA Alex Sandercock, Virginia Tech Landscape Genomics of American chestnut   Project falls under Objective 1 and 3:   Blight-resistant American chestnut trees have been developed from gene insertion methods and backcross breeding programs. These trees can reduce the impacts of C. parasitica, but they are not in great enough numbers to capture the genetic diversity present in the native population. Additionally, it is unknown which genes in the American chestnut genome are related to local adaptation. Introducing blight-resistant American chestnut trees that are locally adapted to their planting site will give them the best chance of survival. So, the development of a conservation and breeding strategy is necessary to instill blight-resistance into the American chestnut population while maintaining genomic diversity. This study seeks to re-sequence the genomes of ~500 American chestnut trees, describe the population structure and demographic history, and identify the genes related to local adaption. Thus far, 96 samples have been sequenced and used to develop a dataset consisting of ~47 million SNPs and INDELs, which shows evidence of a genetically diverse population. A DAPC and an ADMIXTURE analysis were performed to estimate population structure, and preliminary results show a two population and three population structure respectively. Though, both analyses are in agreement with an independent northeastern population beginning in New York and extending through Maine. Finally, an SMC++ analysis was used to estimate the demographic history of C. dentata. This showed a steep decline in the effective population size beginning ~2.7 million years ago followed by subsequent bottleneck events. The next steps will be to sequence the remaining American chestnut samples and complete a genotype-environment association analysis.

Publications

Impact Statements

• Widespread public discussion of the value of transgenic, disease-resistant pure American chestnut, Castanea dentata, as a component of forest restoration is now underway. (Objective 3)

Date of Annual Report: 02/17/2022

Report Information

Annual Meeting Dates: 09/10/2021 - 09/10/2021
Period the Report Covers: 10/01/2020 - 09/30/2021

Participants

Sandra L. Anagnostakis, Emerita, The Connecticut Agricultural Experiment Station, New Haven, CT

Stacy L. Clark, USDA Forest Service, Southern Research Station, Knoxville, TN

Hill Craddock, Dept. of Biology Geology and Environmental Science, UT Chattanooga

Angus Dawe, Department of Biological Sciences, Mississippi State University

Sara Fitzsimmons, Forest Resources Lab, Pennsylvania State University, and TACF

Jill Hamilton, Department of Ecosystem Science and Management, Pennsylvania State University

Fred V. Hebard, Virginia Chapter, The American Chestnut Foundation, Charlottesville, VA

Bradley Hillman, Rutgers University, New Brunswick, NJ

Steven N. Jeffers, Dept. of Plant & Environmental Sciences, Clemson University, South Carolina

Thomas Klak, University of New England, Portland, Maine

Andy Newhouse, SUNY-ESF, New York

Taylor Perkins Dept. of Biology Geology and Environmental Science, UT Chattanooga

Leila Pinchot, USDA Forest Service, Northern Research Station, Delaware, OH

Bill Powell, SUNY-ESF, New York

Charles Ray Department of Ecosystem Science and Management, Pennsylvania State University

Laurel Rodgers, Shenandoah University, Virginia

Tom Saielli, The American Chestnut Foundation, Charlottesville, VA

Monique Sakalidis, Michigan State University, East Lansing, MI

Scott E. Schlarbaum, Dept. of Forestry, Wildlife, and Fisheries, UT Knoxville, TN

Kim Steiner, Department of Ecosystem Science and Management, Pennsylvania State University

Tetyana Zhebentyayeva, Department of Ecosystem Science and Management, Pennsylvania State University

Brief Summary of Minutes

Summary of Minutes:

Because of the COVID-19 pandemic, the 2021 NE-1833 annual meeting was held virtually via Zoom. A total of 13 presentations spanned topics pertaining to all three of the project’s objectives. Presentations represented research from Maine, Michigan, Mississippi, New Jersey, New York, Pennsylvania, South Carolina, Tennessee, and Virginia, as well as from The American Chestnut Foundation, a strong partner in this project.

At the NE1833 business meeting, it was decided that the 2022 annual meeting will again be hosted by Tom Saielli and Jared Westbrook and will be held in person if possible in Charlottesville Virginia.

The current project expires in 2023. It was decided that a Request to Write a renewal proposal would be submitted to the USDA through NERA in 2022. The current format for the project has worked well, and the objectives encompass all of the research presented annually. It was decided that the format/objectives for the renewal would be similar to the current project format/objectives, and that three members would take the lead in writing, one member for each objective area.

Accomplishments

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.   NEW YORK Andy Newhouse, Bill Powell - SUNY-ESF, Syracuse, NY American Chestnut Research & Restoration Project Work continues toward potential future release of transgenic American chestnuts created to express oxalate oxidase (OxO), an enzyme found naturally in a wide variety of plants and other organisms. Blight damage on young Darling 58 transgenic trees is approximately similar to that seen on Chinese chestnuts, which can get blight but typically tolerate it well enough to keep growing.  The lack of a direct pesticidal mechanism suggests it should be relatively evolutionarily stable.  ESF has completed a variety of environmental & nutritional tests that collectively show a lack of enhanced risks compared to traditional breeding.  Larger-scale, longer-term ecological tests have continued.  Regulatory review in the US consists of three agencies: USDA-APHIS (submitted Jan. 2020, currently in review), EPA (submitted Sept. 2021), & FDA (to be submitted fall 2021). Regulatory review in Canada consists of two agencies, the Environmental & Livestock Feed Assessments (CFIA) and Novel Food assessments (Health Canada), both submissions in preparation for 2022.  The USDA-APHIS review proceeded in 2020-21, including a public comment period for their Plant Pest Risk Assessment.  Overall, comments were strongly positive, including qualified support from major environment groups including The Nature Conservancy, Sierra Club, and Environmental Defense Fund, in contrast to more typical GE agricultural crops that often draw large numbers of negative comments. The USDA-APHIS recently posted a notice of intent to write an environmental impact statement, with a proposed due date of Aug. 2023. This statement will include reference both to the environmental impact of releasing and also of not releasing transgenic, blight-resistant trees.  Next research steps are focusing on combining OxO with other breeding and biocontrol strategies, enhancing genetic diversity through outcrossing with wild-type American chestnuts and also by introducing genes from related chestnut species, and incorporating Phytophthora resistance (possibly through backcrossing).   PENNSYLVANIA Jill Hamilton, Department of Ecosystem Science and Management, PSU Charles Ray, Department of Ecosystem Science and Management, PSU Tetyana Zhebentyayeva,  Department of Ecosystem Science and Management, PSU Chinese Chestnut Resistance to Phytophthora cinnamomi (Tetyana Zhebentyayeva and chestnut root rot research group). This year research was focused on understanding structural organization genomic regions underlying QTL intervals for resistance to Phytophthora cinnamomi (Pc) in susceptible American and resistant Chinese chestnut, and developing procedures and protocols for gene expression analysis.  Taking advantage of a high-quality genome of Castanea dentata assembled and annotated by the Hudson Alpha Institute, the QTL intervals were delineated with flanking markers from the previous study and mined for gene composition. Comparative analysis between American and Chinese (Vanuxem) chestnut genomes, revealed a multiple-gene family of cysteine-rich receptor-like kinases (CRKs, DUF26/PF001657) associates with a membrane immune complex and apoplastic reactive oxygen species (ROS) signaling in plants. Based on previous and current results, this gene family and associated molecular networks have been prioritized for an in-depth transcriptome profiling in chestnut roots in response to Pc invasion. Treatment of roots with pathogen conducted this growing season in a test mode was used to finalize design of the RNA sequencing experiment (by establishing time intervals after inoculation, number of biological replicates and non-inoculated controls). Results will be used to enhance preliminary data in collaborative proposal “Understanding host resistance in the Chinese chestnut-Phytophthora cinnamomi pathosystem” at re-submission to the NSF Plant-Biotic interaction program. Wood phenotyping and extension (Ray):  Work on identifying, labeling, and classifying the Penn State Xylarium continues. Specimens of the Xylarium were utilized to compare wood quality in early 20th century American chestnut wood to specimen samples produced by hybrids in modern plantation trials around the region. A research project entitled “Wood Quality in Hybrid Chestnuts” supported by the American Chestnut Foundation was completed and reported on in a TACF “Chestnut Chat” webinar. Results will be reported in the Winter 2022 issue of the TACF Chestnut Journal. Implied in the conclusions of the work, which will be published shortly, is that mature wood quality of the second-generation hybrids should be similar to original American chestnut, especially when grown in natural forest conditions.   SOUTH CAROLINA Steven N. Jeffers – Dept. of Plant & Environmental Sciences, Clemson University Screening Hybrid Chestnut Seedlings for Resistance to P. cinnamomi <ul> <li>In Fall 2021, we recovered two isolate of cinnamomi from sites in Maryland: Maryland sites: BARC and WSSC 1</li> <li>These isolates were put into permanent storage in our lab and then used in the 2021 TACF hybrid chestnut seedling screening effort at the USDA Forest Service Resistance Screening Center at the Bent Creek Experimental Forest in Asheville, NC</li> </ul> Understanding Host Resistance in the Chinese Chestnut–P. cinnamomi Pathosystem <ul> <li>We continue to collaborate with colleagues at Penn State, USDA-FS, Univ. of Kentucky, and Univ. of Tennessee on this project, but, so far, we have not been able to secure grant funds</li> <li>Therefore, a preliminary experiment was conducted in Aug 2020 to examine the early steps of the infection process by cinnamomi on chestnut roots using RNA sequencing <ul> <li>results from this first experiment were inconclusive due to experimental inconsistencies</li> </ul> </li> <li>An experiment to more accurately identify when zoospores infect American chestnut roots is being planned—before conducting the RNA-seq experiment again</li> </ul>   THE AMERICAN CHESTNUT FOUNDATION Tom Saielli and1 Sara Fitzsimmons2 1The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902 2The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802 Improved blight resistance among regional hybrid chestnuts Advanced generation hybrid chestnut trees throughout TACF orchards in Virginia and Maryland, and Virginia Department of Forestry hybrid trees at the Lesesne State Forest, have been assessed for long-term blight tolerance and American-type traits for at least four years post inoculation, and sometimes ten years or more. Long-term measurement of the chestnuts’ physiological response to blight infection is required to truly assess the level of resistance for each tree and among various families. This is a uniquely different strategy than the former strategy established by TACF in 1983, in which selections were made at one year post inoculation and open-pollinated seeds were planted directly in seed orchards. We now realize that blight resistance is more complicated and may take years to evaluate. Over time, the trees that appear to be the “best of the best” are crossed via controlled pollinations and the seedlings pre-screened for resistance with a greenhouse assay. The best seedlings are planted in an orchard at Fortunes Cove in Central Virginia. Evaluate the correlation between canopy dominance and blight resistance among backcross chestnuts at Lesesne State Forest Mature hybrid chestnut stands at the Lesesne State Forest (VDOF) in Central Virginia were established in the late 1980’s and early 1990’s, are now canopy trees averaging 70 feet tall. Within the first few decades after the orchard was established, the most susceptible trees succumbed to blight infections and died, leaving only the more resistant trees to form a forest stand. However, after four decades, there is an additional level of variation among the survivors: trees with high levels of blight resistance dominate the canopy; whereas, trees with moderate blight resistance have survived, but are now suppressed in the understory of the more resistant trees. Many of the suppressed trees are showing signs of stress and some dieback, while the dominant trees are healthy and produce large annual masts of seeds. We hope to quantify the correlations between blight resistance and canopy dominance, and the projected implications for growth and survival, recruitment of progeny, and carbon sequestration among reintroduced hybrid chestnuts.   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. MICHIGAN Monique Sakalidis, Michigan State University, East Lansing, MI Brown rot is a disease of chestnuts that has been reported globally in nut production areas particularly in Europe and Australia and can result in up to 91% of chestnuts infected. It has been detected annually since 2017 in Michigan, at somewhat lower levels than in Europe and Australia. In high disease incidence years, cold storage decreases disease incidence and severity; in low disease incidence years, cold storage may decrease disease incidence, but not in all cases. In 2020, brown rot was detected in up to 9% of nuts, caused by the fungus Gnomoniopsis smithogilvyi (G. smithogilvyi) among other species. The most susceptible chestnut cultivar was European X Japanese (Colossal). In addition to its role as a postharvest nut pathogen, G. smithogilvyi  was also found to be associated with canker disease in chestnut populations in Michigan and Wisconsin.   MISSISSIPPI Angus Dawe, Department of Biological Sciences, Mississippi State University Current personnel: Graduate students –Soum Kundu, Melanie Tran Research Associate – Gisele Andrade Current Projects: Note, due to COVID-19 limitations, progress has been negatively impacted on all projects. <ol> <li>ARV-1 and its potential role in sterol homeostasis</li> <li>Identifying C. parasitica genes associated with pathogenicity and virulence</li> </ol> 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. In total, the run generated >800 million reads (PE 150), with QC30>81.7%. Coverage is estimated at >15-30X for almost all the samples with only minor quality issues with source DNA for a small number. 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 involved and will be assisting in relating the sequence data to the phenotype data. Limited progress was made on this project this year due to student challenges. ARV-1 and its potential role in sterol homeostasis. (Soum Kundu, PhD student). ARV-1 is a predicted gene in C. parasitica that shares similarity with genes that code for proteins with important roles in sterol homeostasis in other organisms. The knockout of ARV-1, serendipitously made when investigating an unrelated phenomenon, is avirulent and has a heavily impaired vegetative growth phenotype. Soum has been working to verify which of two possible genes originally knocked out is the source of the debilitated phenotype (the updated genomic arrangement was apparent with a new genome annotation). He has also 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. Recent success using derivatization techniques to tag the appropriate class of compounds show that ergosterol accumulation is much reduced in the ARV-1 mutant. When tested, the hypovirus infected strain EP713 shows a reduction of ergosterol accumulation very similar to that of the mutant, suggestion that a component of the membrane alterations induced by the hypovirus may be due to altered ergosterol presence.   NEW JERSEY Bradley Hillman, Rutgers University, New Brunswick, NJ <ol> <li>Discovery of new viruses associated with hypovirulence and biological control in New Jersey forests proceeded with laboratory characterization of isolates that were collected from a large recovering American chestnut tree in northern New Jersey, near the New York border in 2020. An undergraduate student, Rebecca Bright, began the characterization of the isolates, and the work has continued under another student, Zarja Miovic. The following experiments were performed: 1) Isolation of the chestnut blight fungus, C. parasitica, from the recovering tree. Three new fungal isolates were collected. 2) Examination of fungal colony morphology/phenotype in culture. Two of the three new isolates were fast growing, similar to virulent, virus-free cultures, but one of the cultures grew more slowly and had less aerial mycelium in culture, similar to some hypovirulent, virus-infected cultures in our collection. 3) Isolation of isogenic cultures from single conidia (asexual spores) of the slow-growing culture and determination of colony morphology/phenotype of the single conidial isolates (SCIs). Of the total of 40 SCIs that were isolated and examined for colony morphology, 31 colonies showed rapid growth, orange coloration, and abundant aerial mycelium typical of virulent, virus-free colonies and 9 colonies showed slow growth, brown coloration, and suppressed aerial mycelium typical of hypovirulent, virus-containing colonies. 4) Initial assessment of virulence was performed on one of the rapidly-growing colonies (designated strain R1V2) and one of the slow-growing colonies (designated strain R1V1) by inoculation of apple fruits, including known virus-free virulent (strain EP155) and virus-containing hypovirulent (strain EP713) cultures. In two replicate trials, lesions resulting from slow-growing strain R1V1 were similar in size to control hypovirulent strain EP713, whereas lesions resulting from fast-growing strain R1V2 were similar in size to control virulent strain EP155. 5) Pairings of R1V1 and R1V2 allowing the two colonies to grow together in contact resulted in conversion of the R1V2 phenotype to the R1V1 phenotype, consistent with transmission of a virus. This step completes a modified Koch’s postulates, demonstrating that the infectious agent associated with hypovirulence in strain R1V1 is transmissible and is curable, although the specific virus in R1V1 has not yet been characterized. This represents the first hypovirulence-associated virus from northern NJ forests and could lay the groundwork for investigating its prevalence and impact on natural chestnut populations in the region.</li> </ol>   <ol start="2"> <li>We worked to finish the project comparing codon usage among different viruses in C. parasitica to codon use in the host fungus, as summarized in detail last year. We are continuing to explore the hypothesis that the hypovirus CHV4, which is the most prevalent virus of the chestnut blight fungus in North America, may have a longer association with the fungus than previously thought, and may have entered C. parasitica in Asia, before its invasion to North America in the late 19th century. The virus has never been found in Asia. An undergraduate student in the lab, Abhishek Kashalikar, has expanded the comparisons among the viruses examined last year in the study, and has overlaid details about the geographic origins of those viruses and their different fungal hosts. In other words, he addressed the question of whether there is any notable geographic grouping associated with related viruses in the virus family Hypoviridae, which contains most of the viruses used for biocontrol of chestnut blight disease. Aside from known worldwide movement of CHV1 in C. parasitica, no pattern of phylogeographic association was identified among 51 other members of the family Hypoviridae in this study.</li> </ol>   SOUTH CAROLINA Steven N. Jeffers, Dept. of Plant & Environmental Sciences, Clemson University Evaluating Virulence of P. cinnamomi Isolates <ul> <li>We worked with Dr. Jared Westbrook at TACF to review and evaluate the data collected in 2019 and 2020 variation in virulence among 7 subsets of isolates of cinnamomi</li> <li>Data analysis is in progress, but preliminary results suggest: <ul> <li>there may be significant differences among inoculum treatments</li> <li>differences appear to be related to age of cultures or time in storage</li> <li>the current strategy of screening hybrid chestnut genotypes using fresh isolates from out-planting sites seems reasonable and sound</li> </ul> </li> </ul> Detection of Phytophthora spp. in Chestnut Samples <ul> <li>In the past year, Sep 2020-Aug 2021, samples were received from 6 states: GA, MD, MO, TN, VA, WV</li> <li>In all, there were 9 submissions and 39 soil samples received</li> <li>Phytophthora detected in 13 samples = 33% <ul> <li> cinnamomi was detected in 8 samples from 5 states: GA, MD, MO, TN, VA</li> <li>Phytophthora in 5 samples from WV</li> </ul> </li> </ul>   THE AMERICAN CHESTNUT FOUNDATION Tom Saielli and1 Sara Fitzsimmons2 1The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902 2The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802 Fungicide research Currently, controlled pollinations take place both in situ, on wild chestnut trees and, on wild-type American chestnut established in germplasm conservation orchards (GCO). Unlike the advanced hybrid chestnuts, the GCO trees are highly susceptible to chestnut blight, often leading to significant dieback and mortality. Therefore, we are experimenting with various fungicides and biofungicides to determine if chemical treatments can control blight infections and enable GCO chestnut trees to survive and grow. Experiments are being conducted in collaboration with the Jeffers Lab at Clemson University, greenhouse experiments in Central Virginia, and field trials in GCO’s in Northern Virginia and other locations.   VIRGINIA Laurel Rodgers, Shenandoah University Comparison of the fungal microbiome in chestnut trees resistant to C parasitica to those that are not resistant to C parasitica We sampled a total of 45 trees from each orchard, pulling two plugs from each for a total of 180 bark plugs. A combination of American, Chinese, and hybrid trees were collected from each orchard, with the addition of F1 trees from The Ranch (Table 1). Table 1. Number of plugs collected from sample sites <table> <tbody> <tr> <td width="89"> Location </td> <td width="89"> American </td> <td width="89"> Chinese </td> <td width="89"> Hybrid </td> <td width="89"> F1 </td> <td width="89"> # Trees Sampled </td> <td width="89"> #  Bark Plugs </td> </tr> <tr> <td width="89"> The Ranch </td> <td width="89"> 10 </td> <td width="89"> 10 </td> <td width="89"> 15 </td> <td width="89"> 10 </td> <td width="89"> 45 </td> <td width="89"> 90 </td> </tr> <tr> <td width="89"> Mt. Zion </td> <td width="89"> 4 </td> <td width="89"> 8 </td> <td width="89"> 33 </td> <td width="89"> n/a </td> <td width="89"> 45 </td> <td width="89"> 90 </td> </tr> <tr> <td width="89">   </td> <td width="89">   </td> <td width="89">   </td> <td width="89">   </td> <td width="89">   </td> <td width="89"> 90 </td> <td width="89"> 180 </td> </tr> </tbody> </table>   We isolated a total of 361 fungi samples from all plugs collected. From the Ranch, we isolated 39 fungi samples from Chinese, 34 from American, 46 from hybrids, and 39 from F1 chestnut trees. At Mount Zion, we isolated 39 fungi from Chinese, 20 from American, and 144 from hybrid chestnut trees (Table 2). So far, we have sequenced and identified about twelve samples from the Ranch and thirty-seven from Mount Zion, but we still need to sequence approximately 100 from the Ranch and 150 from Mount Zion. Table 2. Number of fungi isolated <table> <tbody> <tr> <td width="104"> Location </td> <td width="104"> Chinese </td> <td width="104"> American </td> <td width="104"> Hybrid </td> <td width="104"> F1 </td> <td width="104"> Total </td> </tr> <tr> <td width="104"> The Ranch </td> <td width="104"> 39 </td> <td width="104"> 34 </td> <td width="104"> 46 </td> <td width="104"> 39 </td> <td width="104"> 158 </td> </tr> <tr> <td width="104"> Mt. Zion </td> <td width="104"> 39 </td> <td width="104"> 20 </td> <td width="104"> 144 </td> <td width="104"> n/a </td> <td width="104"> 203 </td> </tr> <tr> <td width="104">   </td> <td width="104">   </td> <td width="104">   </td> <td width="104">   </td> <td width="104">   </td> <td width="104"> 361 </td> </tr> </tbody> </table>   Species identification was interrupted by the issues with our protocols in 2019 as we experienced turn over in our undergraduate researchers and again in 2020 due to the Sars-Cov2 pandemic. Identification of samples is once again in progress, but some samples have been lost due to time and contamination. Of the species we have identified thus far at Mount Zion, a majority of the fungi are parasitic fungi. However, based on brief literature searches, three species of fungi we isolated, Fimetariella rabenhorstii, Hypoxlon submonticulosum, and Albifimbria verrucaria, have been documented as being endophytes in other plant species (Table 3). However, the hybrid trees in which these samples were collected proved to have very little resistance to C. parasitica. Though the sample size is low, it seems unlikely that these three species of fungi serve as endophytes in the chestnut tree. We were surprised to find the fungus Gnomoniopsis smithogilvyi growing within one of our hybrid trees. This is a fungus known to cause cankers on chestnuts in Europe and has begun to be documented within the US. A more extensive study will need to be completed in order to determine how wide spread G. smithogilvyi is within the United States and whether it will act as a pathogen in the American chestnut tree. Table 3. Fungi species identified thus far at Mt. Zion (potential endophytes highlighted in blue) <table> <tbody> <tr> <td width="208"> American Chestnut </td> <td width="208"> Chinese Chestnut </td> <td width="208"> Hybrid trees </td> </tr> <tr> <td width="208"> Biscogniauxia mediterranea </td> <td width="208"> Biscogniauxia mediterranea </td> <td width="208"> Biscogniauxia mediterranea </td> </tr> <tr> <td width="208"> Fusarium proliferatum </td> <td width="208"> Biscogniauxia atropunctata </td> <td width="208"> Biscogniauxia atropunctata </td> </tr> <tr> <td width="208"> Coniochaeta </td> <td width="208"> Coniochaeta </td> <td width="208"> Gnomoniopsis smithogilvyi </td> </tr> <tr> <td width="208">   </td> <td width="208"> Diplodia seriata </td> <td width="208"> Diplodia corticola </td> </tr> <tr> <td width="208">   </td> <td width="208"> Aspergillus niger </td> <td width="208"> Botryosphaeria dothidea </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Fimetariella rabenhorstii </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Hypoxylon submonticulosum </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Xylariales </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Albifimbria verrucaria </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Aspergillus niger </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Botryosphaeria dothidea </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Curvularia </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Penicilium </td> </tr> <tr> <td width="208">   </td> <td width="208">   </td> <td width="208"> Sordariomycetes </td> </tr> </tbody> </table>   Table 4: Fungi species identified thus far at The Ranch. <table> <tbody> <tr> <td width="156"> American Chestnut </td> <td width="156"> Chinese Chestnut </td> <td width="156"> F1 trees </td> <td width="156"> Hybrid Trees </td> </tr> <tr> <td width="156"> Aspergillus brasiliensis </td> <td width="156"> Biscogniauxia mediterranea </td> <td width="156"> Fusarium </td> <td width="156"> Aspergillus brasiliensis </td> </tr> <tr> <td width="156">   </td> <td width="156"> Coniochaeta </td> <td width="156"> Fusarium </td> <td width="156"> Biscogniauxia mediterranea </td> </tr> <tr> <td width="156">   </td> <td width="156"> Coniochaetales </td> <td width="156">   </td> <td width="156"> Fusarium solani </td> </tr> <tr> <td width="156">   </td> <td width="156"> Fusarium solani </td> <td width="156">   </td> <td width="156">   </td> </tr> </tbody> </table>     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.   MAINE Thomas Klak, University of New England, Portland, ME Transgenic Chestnut Pollen Production under High-Lights & Field Pollination The Chestnut restoration team at the University of New England continues to have success producing transgenic (blight-tolerant) pollen from seeds and seedlings obtained from SUNY-ESF. Pollen production in chestnut has been inconsistent year-to-year, but the UNE team expanded the pollen-production project in 2021. Green and white aphids, spider mites, mealy bugs, fungus gnats, and powdery mildew fungus are major barriers to pollen production. The total number of fertile transgenic chestnuts produced from UNE-produced pollen in 2021 exceeded the 5500+ total of 2020.   PENNSYLVANIA Kim C. Steiner, Department of Ecosystem Science and Management, PSU Sara F. Fitzsimmons, TACF and PSU     Breeding and field trials (Steiner): Dr. Steiner has a long-time partnership with The American Chestnut Foundation (TACF) on breeding for blight-resistance in American chestnut and on research towards restoring the species to Appalachian forests. TACF's Northcentral Regional Breeding Coordinator is based in the Steiner lab, and the Pennsylvania TACF Chapter’s statewide and regional breeding programs have been coordinated by Steiner’s PhD student S.F. Fitzsimmons. Dr. Steiner has provided oversight to the national TACF plan for breeding and restoration, as Chair of the TACF Science Cabinet from 2007 to 2012, and currently as Chair of the TACF Board of Directors. Dr. Steiner and Sara Fitzsimmons have established a large field trial, on the Penn State campus, of many families of TACF 3rd back-cross generation progeny that are being evaluated for blight resistance and form.   TENNESSEE Hill Craddock, Taylor Perkins, The University of Tennessee at Chattanooga Evolutionary genetics of chinquapin and American chestnut <ul> <li>Morphological diversity of American chestnut and chinquapin</li> <li>Evidence of hybridization between the species</li> <li>Adaptive significance of gene introgression between the species is under investicgation</li> </ul> Breeding for Disease Resistance <ul> <li>Finished selections at Ruth Cochran Orchard and Dave Cantrell Orchard</li> </ul> <ol> <li> dentata collected from underrepresented areas in Alabama and Tennessee</li> </ol> <ul> <li>Clonal collections maintained in field plots in Indiana and in container nursery in Tennessee</li> <li>Germplasm Conservation ex situ</li> </ul> Phylogeography of Castanea in the southern US <ul> <li>Collection trip (with Sisco and Paillet) to S. Missouri and NW Arkansas</li> <li>Annotations of 900 herbarium sheets for Perkins et al</li> </ul> Works in Progress <ul> <li>Herbarium vouchers prepared for SERNEC imaging and digital data capture</li> <li>Nursery production of BnF2s for TN seed orchards</li> <li>Nursery production of C. dentata germplasm for GCOs</li> </ul>   Stacy L. Clark (USDA Forest Service, Southern Research Station, Knoxville, TN), Leila Pinchot (USDA Forest Service, Northern Research Station, Delaware, OH), and Scott E. Schlarbaum (The University of Tennessee, Department of Forestry, Wildlife, and Fisheries, Knoxville, TN): The University of Tennessee’s Tree Improvement Program (UT-TIP) chestnut activities include evaluations of historic chestnut plantings at the Norris Reservation (Tennessee Valley Authority) in TN and collaborating with the USDA Forest Service Southern and Northern Research Stations. The collaborative work includes implementation and long-term comprehensive field evaluations of chestnut research test plantings (ca. 2009-2017) in NC, PA, TN, and VA. Experimental material represents 7500 trees from various breeding generations (BC1F3, BC2F3, BC3F3, BC3F2) and parental species (American and Chinese chestnut) from The American Chestnut Foundation’s and the Connecticut Agricultural Experiment Station’s breeding programs. Evaluations of survival, growth, blight resistance, deer herbivory, and competitive ability within different silvicultural prescriptions have been conducted. Results indicate chestnuts bred for blight resistance exhibit superior competitive ability and intermediate blight resistance, but performance varies depending on seedling quality, vegetation competition, site quality, and deer browse pressure at the time of planting.   THE AMERICAN CHESTNUT FOUNDATION Tom Saielli and1 Sara Fitzsimmons2 1The American Chestnut Foundation, 900 Natural Resources Drive, Charlottesville, VA 22902 2The American Chestnut Foundation, 206 Forest Resources Lab, University Park, PA 16802 Ecological Studies on American chestnut hybrids   Understory trials at Lesesne State forest hybrid orchard The original hybrid orchard at Lesesne State Forest was established in 1960 with American x Chinese x Japanese hybrids from the Connecticut Agriculture Research Station and now consists of a closed canopy stand of chestnut trees that produce tens of thousands of viable seeds every year. To date, no recruitment has ever been observed under the canopy in this orchard. We hypothesize that significant understory vegetation suppresses the chestnut seeds, preventing seedling establishment. In 2022 we will conduct experimental vegetation management to see if recruitment can be promoted. Treatments include cut and burn understory, cut and herbicide understory, no removal of understory.   Forest silviculture studies: mixed hybrid chestnuts and oaks under three canopy treatments We are evaluating the influence of overstory treatments on three open-source American chestnut genotypes include BC1F2, BC2F2 and BC3F3seedlings, LSA x LSA seedlings (large surviving American chestnuts) and two oak species (white oak and southern red oak). Study locations will include a low elevation site at W. Kerr Scott Dam and Reservoir, in Wilkesboro, NC and a high elevation site at Boone NC (Gilley or Blackburn Vannoy). Silviculture treatments include: old field (open site), pine forest (with release planned in 2-3 years), and shelterwood (60% leaf basal area +/- 10%, with release after 2-3 years). Percent canopy removed for “release will be TBD but will be adequate to provide suitable light availability for established seedlings but may not involve a full cutover. The following measurements will be made: height, diameter and mortality and assessment of blight resistance once natural blight infections become visible. The expected results of this research will be to identify silviculture strategies that lead to successful re-establishment of chestnut populations while minimizing management efforts (i.e. aggressive vegetation management). We also hope to quantify how genotype and levels of resistance influence species establishment and long-term competition, as well as recruitment in the understory. The goal of this research is to contribute towards the development of better silviculture and breeding strategies, with a focus on competitive, timber-type, American chestnut trees that are blight resistant (enough) and significantly ‘American’ in all other traits.   Fred V. Hebard, Virginia Chapter, The American Chestnut Foundation The method of Ellis et al (2018, doi: 10.1111/1755-0998.12782) for identifying parents of open-pollinated progeny was applied to B3-F2s bred at Meadowview.  Optimizing input parameters led to identification of male parents in 45% of progeny.  When the most likely choice of male parent was unidentified, but the second most likely choice, identified, was only slightly less probable, the male parent was identified in 81% of progeny.  It is thought that most of the potential male parents had been genotyped, so that the 81% identification rate was more accurate.  This is based on the relative geographic separation of parents; 95% of genotyped male parents were within 200 meters of the female but ungenotyped parents were 1000 meters away.   The failure of the program to identify more than 45% of parents is thought to be due to the severe linkage disequilibrium in backcross chestnut trees. Preliminary  GWAS were run using 13,750 SNPs against 755 Clapper and 918 Graves B3-F2 trees.  Both B3-F2 groups were segregating for blight resistance but only Graves for PRR resistance.    The populations had been selected for blight resistance and PRR resistance by Jared Westbrook and team at TACF.  The SNP alleles had called as being descended from Chinese or American chestnut.  In these GWAS, the binary trait of selected or not was tested against the binary state of SNP markers.  There were significant associations for blight resistance on five chromosomes in both Clapper and Graves families, but only two chromosomes were in common.  Furthermore, the sites of maxima on one common chromosome were 31 Mbp apart.  So blight resistance for Clapper and Graves was associated with eight of the 12 chromosomes in chestnut.  In contrast, PRR resistance was found on only one chromosome in Graves, the same result as obtained by Zhebentyayeva with QTLs. The GWAS were preliminary in that a separate association of resistance and marker state was run for each marker-disease-Chinese ancestor combination rather than a more inclusive model.  GWAS analysis with more inclusive models and further data cleansing is indicated There were no tall, narrow peaks in Manhatten plots of the results.  This is attributed to the strong linkage disequilibrium in many of the B3 parents.  This should be dissected further.  There is scant evidence for or against multiple loci for resistance within a chromosome.

Publications

2021 Publications Aulia, A., Hyodo, K., Hisano, S., Kondo, H., Hillman, B.I., Suzuki, N. 2021. Identification of an RNA silencing suppressor encoded by a symptomless fungal hypovirus, Cryphonectria hypovirus 4. Biology 10, 100. <a href="https://doi.org/10.3390/biology10020100">https://doi.org/10.3390/biology10020100</a> Callahan AM., Zhebentyayeva T.N., Humann JL, Saski CA, Galimba KD, Georgi L.L, Scorza R., Main D, Dardick CD (2021) Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica). Horticulture Research 8:8. <a href="https://doi.org/10.1038/s41438-020-00438-2">https://doi.org/10.1038/s41438-020-00438-2</a>. Groppi A, Liu S, Cornille A, Decroocq S, Bui QT, Tricon D, Cruaud C, Arribat S, Belser S, Marande W, Salse J, Huneau C, Rodde N, Rhalloussi W, Cauet S, Istace B, Deni E,, Carrère S, Audergon J-M, Roch G, Lambert P, Zhebentyayeva T., Liu W-S, Bouchez O, Lopez-Roques C, Serre R-F, Debuchy R, Tran J, Wincker P, Chen X, Pétriacq P, Barre A, Nikolski M, Aury J-M, Abbott AGA, Giraud G, Decroocq V (2021) Population genomics of apricots unravels domestication history and adaptive events. Nature Communication 12, 3956. <a href="https://doi.org/10.1038/s41467-021-24283-6">https://doi.org/10.1038/s41467-021-24283-6</a>. Hillman, B.I., and Milgroom, M.G. 2021. The ecology and evolution of fungal viruses. pp. 139-182 in: Studies in Viral Ecology, 2nd Ed. C.J. Hurst, editor. John Wiley & Sons, NY.  <a href="https://doi.org/10.1002/9781119608370.ch5">https://doi.org/10.1002/9781119608370.ch5</a> (this book chapter was cited last year as in press) Perkins M.T., Zhebentyayeva T.N., Sisco P., Craddock H (2021) Genome-wide sequence-based genotyping supports a nonhybrid origin of Castanea alabamensis.  Systematic Botany 46(3): (in press); preprint BioRxiv.org <a href="https://www.biorxiv.org/content/10.1101/680371v1">https://www.biorxiv.org/content/10.1101/680371v1</a>. Perkins M.T., Zhebentyayeva T.N., Sisco P., Craddock H.(2021) Castanea alabamensis: rediscovery of a lost American chestnut relative. Chestnut (The Journal of the American Chestnut Foundation) 35(3):30-33. Pina A, Irisarri P., Errea P., Zhebentyayeva T. (2021) Mapping quantitative trait loci (QTLs) associated with graft (in)- compatibility in apricot (Prunus armeniaca L.). Front. Plant Sci. 12: 622906. doi: 10.3389/fpls.2021.622906. Suzuki, N., Cornejo, C., Aulia, A., Shahi, S., Hillman, B.I., Rigling, D. 2021. In-tree behavior of diverse viruses harbored in the chestnut blight fungus, Cryphonectria parasitica. Journal of Virology 95 (6), e01962-20. <a href="https://doi.org/10.1128/JVI.01962-20">https://doi.org/10.1128/JVI.01962-20</a> Yu J., Bennett D., Dardick C., Zhebentyayeva T., Abbott A., Liu Z., Staton M. (2021) Genome-Wide Changes of Regulatory Non-coding RNAs Reveal Pollen Development Initiated at Ecodormancy in Peach. Frontiers in Molecular Biosciences, 8:612881. doi: 10.3389/fmolb.2021.612881.

Impact Statements

• Widespread public discussion of the value of transgenic, disease-resistant pure American chestnut, Castanea dentata, as a component of forest restoration is now underway. (Objective 3)

Date of Annual Report: 11/30/2022

Report Information

Annual Meeting Dates: 08/26/2022 - 08/27/2022
Period the Report Covers: 10/01/2021 - 09/30/2022

Participants

Hill Craddock, Dept. of Biology Geology and Environmental Science, UT Chattanooga

Angus Dawe, Department of Biological Sciences, Mississippi State University

Sara Fitzsimmons, Forest Resources Lab, Pennsylvania State University, and TACF

Fred V. Hebard, Virginia Chapter, The American Chestnut Foundation, Charlottesville, VA

Steven N. Jeffers, Dept. of Plant & Environmental Sciences, Clemson University, South Carolina

Matt Kasson, West Virginia University

Susanna Kerio, Connecticut Agricultural Experiment Station, New Haven CT

Thomas Klak, University of New England, Portland, Maine

Bruce Levine, University of Maryland

Amy Metheny, West Virginia University

Danielle Mikolajewski, West Virginia University

Patricia Morales, SUNY-ESF, New York

Dana Nelson, Southern Research Station, Lexington, KY

Andy Newhouse, SUNY-ESF, New York

Taylor Perkins Dept. of Biology Geology and Environmental Science, UT Chattanooga

Hannah Pilkey, SUNY-ESF, New York

Linda Polin, SUNY-ESF, New York

Charles Ray Department of Ecosystem Science and Management, Pennsylvania State University

Laurel Rodgers, Shenandoah University, Virginia

Tom Saielli, The American Chestnut Foundation, Asheville, NC

John Scivani, The American Chestnut Foundation, Asheville, NC

Jared Westbrook, The American Chestnut Foundation, Asheville, NC

Brief Summary of Minutes

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.

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 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: <ol> <li>ARV-1 and its potential role in sterol homeostasis</li> <li>Identifying C. parasitica genes associated with pathogenicity and virulence</li> </ol> Project details <ol> <li>Identifying C. parasitica genes associated with pathogenicity and virulence. (Melanie Tran, MS student.)</li> </ol> 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. <ol> <li>ARV-1 and its potential role in sterol homeostasis. (Soum Kundu, PhD student).</li> </ol> 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.

Publications

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. <a href="https://doi.org/10.1111/mpp.13165">https://doi.org/10.1111/mpp.13165</a>   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. <a href="https://doi.org/10.1002/ecs2.3917">https://doi.org/10.1002/ecs2.3917</a>   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.<a href="https://doi.org/10.1007/s11248-021-00263-w"> https://doi.org/10.1007/s11248-021-00263-w</a>.   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, <a href="https://doi.org/10.1016/j.ecochg.2021.100037">https://doi.org/10.1016/j.ecochg.2021.100037</a>   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).<a href="https://doi.org/10.1007/s11056-022-09909-x"> https://doi.org/10.1007/s11056-022-09909-x</a>   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. <a href="https://doi.org/10.1016/j.foreco.2022.120481">https://doi.org/10.1016/j.foreco.2022.120481</a>   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. <a href="https://doi.org/10.1016/j.foreco.2022.120178">https://doi.org/10.1016/j.foreco.2022.120178</a>   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. <a href="https://doi.org/10.1111/mec.16629">https://doi.org/10.1111/mec.16629</a>     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.<a href="https:/doi.org/10.1016/j.foreco.2022.12013"> https://doi.org/10.1016/j.foreco.2022.12013</a>

Impact Statements

Widespread public discussion of the value of transgenic, disease-resistant pure American chestnut, Castanea dentata, as a component of forest restoration is now underway. (Objective 3)