Brief Summary of Minutes of Annual Meeting

Report of NE-1333 Technical Committee Meeting, Biological Improvement of Chestnut through Technologies that Address Management of the Species, its Pathogens and Pests, at Marion, VA, Sept 10-12, 2015, Fred Hebard, Chair OBJECTIVE 1. Develop and evaluate blight-resistant chestnut trees for food and fiber through traditional and molecular techniques that incorporate knowledge of the chestnut genome SUNY-ESF is moving forward to get permission from the U.S. Government to release transgenic American chestnut into the wild. The subject trees contain a gene from wheat encoding oxalate oxidase (OxO). Testing of additional OxO transformants with different promotors continues. Thirty other candidate genes are being examined; 27 are from Chinese chestnut. Seven exhibited blight resistance in the detached-leaf assay; the most notable was a gene encoding acid phosphatase. More than 21,000 chestnut shoots have been produced by micropropagation; the intent is to improve regeneration success. With the current technique, only 1.5% of micropropagated shoots are outplanted successfully. Controlled crosses are being made to confirm inheritance of transgenes conforms to expectations. West Virginia University, in cooperation with The American Chestnut Foundation (TACF), installed a test of whether hypoviruses effect better disease remission in chestnut with intermediate than with low levels of blight resistance. Main effects for resistance and hypovirus treatment were apparent, but too few trees have been tested thus far to detect substantial interaction between genotype and hypovirus treatment. TACF reported on genotyping by sequencing (GBS) of two bulks of chestnut progeny resistant and susceptible to blight that was performed by Clemson University. dCAPS markers were developed for one SNP in each of the three QTL's for blight resistance in 'Mahogany' F2 progeny. One dCAPS from the QTL on linkage group G (Cbr3) mapped to within 12 cM of the QTL peak. Pollen cryostored for 20 years in liquid nitrogen was still viable and produced viable offspring. It was observed that some B3F2 trees are resistant to blight. This suggests that major genes for blight resistance have been retained in these trees, which are expected to be 94% American chestnut. During the field tour, it was pointed out that the populations of B3F3 trees have an estimated effective population size of about 70. Currently, B3F3 trees are intermediate in blight resistance between Chinese and American chestnut, significantly more resistant than American chestnut. Average blight resistance at B3F3 is expected to increase after selections at B3F2 are complete. There is residual heritability for blight resistance in selected B3s (5-10%), indicating that resistance alleles may have been lost in some backcross lines. Penn State (PSU) reported that the assembly of the Chinese chestnut cultivar ‘Vanuxem’ genome has been significantly improved over the past year. The current status of the genome sequence is a set of 14,358 scaffolds representing 784Mb of genome sequence, or app. 98% of the estimated genome size (improved from 724.4 Mp in 41,270 scaffolds in our last report). The size of the largest individual scaffold is now 3.17Mb, with average scaffold size of app. 55Kb (improved from N50 of 39.6Kb and largest scaffold of 429 Kb in 2014). Another major step forward has been the anchoring of almost 90% of the genome scaffolds to the genetic map, using the chestnut integrated genetic-and-physical map and the BAC-end sequences covering the physical map of cv. Vanuxem. This allowed assembly of the scaffolds into a set of 12 pseudo-chromosome sequences, covering the majority of positions along the 12 linkage groups. The University of Tennessee at Knoxville (UTK) upgraded the hardwoodgenomics.org website for the genome and QTL assemblies to JBrowse format. PSU resequenced additional trees and UTK identified numerous SNPs between the various trees, which are available to others on request. The Southern Institute of Forest Genetics (SIFG) reported that 1000 new SNPs have been added to the Chinese x Chinese chestnut genetic map. The higher map density should assist further refinement of the genome assembly. The Chinese x American F2 mapping populations have been augmented with new progeny and are being mapped with new SNP and SSR markers. Thus far, the three QTL regions on linkage groups B, F and G remain most prominently associated with blight resistance. Examination of other mapping populations for blight resistance QTLs is in progress. 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 New Mexico State University reported a promotor governing gene expression in Cryphonectria parasitica could be controlled by copper in growth media. This allowed gene expression in a hypovirus to be turned on and off; the system is limited because host phenotype changed slowly, over multiple transfers to fresh media. Various tests indicated that virus gene expression was being turned on, but that virion replication was not occurring. The University of Maryland has developed strains of C. parasitica that can transmit hypoviruses to recipient strains with any combination of six vegetative incompatibility or vic genes. This was accomplished by knocking out four vic genes. Two were not removed, Vic4 and Vic2. Vic4 only slowly blocks virus transmission, so is not critical to a superdonor. Vic2 knockouts were unviable. The knockouts had to be done sequentially to eliminate the neomycin selectable marker, which was recycled between knockouts. The neomycin marker was eliminated using the Cre-LoxP recombination systems. The quadruple knockout was crossed into a strain with the complementary allele at Vic2. After selection, quadruple knockouts with both Vic2 alleles were obtained. The next step is to test these in the field. West Virginia University reported that 35% of 517 American chestnut in plots treated with hypovirus remain alive at West Salem, WI. The percentage of living stems in untreated plots is 20%. Vegetative compatibility type WS-1 continues to be the dominant vc type in the stand although its frequency has decreased from 100% in 1995 to 74% in 2014. WS-2 and WS-3 were found at rates of 4% and 7%, respectively. Molecular tools were developed for rapid and precise vic genotyping of numerous isolates. They should significantly improve the ability to predict and evaluate the efficacy of hypovirulence and related management strategies. Michigan State (MSU) reported on an effort to use hypoviruses to control blight in nut-producing chestnut orchards. All non-treated cankers were given the maximum rating of 4, indicating little or no callus, and all treated cankers were rated 1 or 2, indicating the treated cankers had wound tissue and callus present. MSU is trying to get hypovirus GH2 registered as a biopesticide through IR-4; otherwise, nuts cannot be sold from orchards treated the same year. Preliminary results from a controlled experiment testing GH2's efficacy indicated it was efficacious when applied in the five most common v/c types in Michigan orchards. 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 Clemson University and TACF have been testing chestnut backcross hybrids for resistance to Phytophthora root rot (PRR) incited by P. cinnamomi for more than 10 years, primarily by evaluating disease severity in seedling test crosses. Resistance to P. cinnamomi occurs in TACF's trees derived from the 'Graves' source of blight resistance, which is a first backcross to American chestnut (B1). Inheritance of the resistance is compatible with it being controlled by a single gene. It is estimated P. cinnamomi resistance could be fixed in 10 years in B3-F4s while maintaining genetic diversity in the breeding population. Other sources of P. cinnamomi resistance have been examined; it is not present 'Clapper' derivatives but does occur in progeny from 'Nanking.' Resistance is not very prevalent in B2s and B3s from 'Nanking,' presumably because it is controlled by a single gene that is not under selection. Preliminary results from GBS indicate three QTLs for P. cinnamomi resistance are on three linkage groups of B1s from 'Mahogany,' including linkage group E, which previously was associated with resistance. A visiting scientist from Portugal described their investigation of the resistance to P. cinnamomi. The Connecticut Agricultural Experiment Station reported that backcross F2s, either BC2 Chinese x BC3 Japanese or B3-F2 Japanese had good survival, 176/420 and 83/360, respectively. The Chinese x Japanese survival was better than that of the Japanese x Japanese. Foliar mineral analysis revealed deficiencies of boron and manganese. Hybrids were planted in small numbers (50 or 100) at three locations. The 'Lockwood' cultivar yielded progeny with fewer galls than 'Colossal' in crosses with C. henryi. Nutrient content of progeny also varied by pollinator. Shippensburg University reported that, of compounds tested, Naproxen was the most inhibitory to growth and development of C. parasitica in culture. Ethidium bromide, asprin, methylene blue, furosemide, glipzide, oxycodone, ventolin and enoxaparin also were tested, all applied at 3 mg in one location in 20 ml of medium in 90-mm-diameter Petri plates. Substituting multiwell plates with 4 ml of agar in each of 6 wells for the 90-mm plates reduced the space and time needed for the tests. The University of Tennessee at Chattanooga reported that they are planting B3-F2 and B4-F2 seed orchards as part of the breeding program of the Tennessee Chapter of TACF. New sources of blight resistance also are being advanced, but including selection for PRR resistance. A modification of the Clemson-TACF method is being used for P. cinnamomi screening. A Korean C. mollissima x C. crenata cultivar, 'Daebo,' is being evaluated for fruit quality. A large sample of North American Castanea spp is being assembled to study the systematics of the genus using chloroplast and nuclear DNA markers and morphological traits. MSU reported that gall wasp was found at 10 sites in southwestern Michigan in 2015, despite a quarantine that had been in place. The presence of old galls indicated the infestation had been present for more than 2 years. The large numbers of cultivars and genotypes in Michigan orchards may reveal interesting differences in the severity of gall wasp infestation. The European X Japanese hybrid cultivars ‘Marigoule’, ‘Marsol’ and ‘Maraval’ have high levels of cold tolerance and are clearly Michigan’s most cold-tolerant cultivars. The three cultivars had been selected for blight tolerance, Phytophthora root rot resistance, and ability to root, but not for cold tolerance. Michigan has the most chestnut growers, trees and production of the 50 United States. Two recovering American chestnut populations, County Line and Roscommon, continue to exhibit strong tree survivorship and growth. American chestnut at the Frankfort, Leelanau, Missaukee sites all continue to decline due to blight. The University of Kentucky is involved in a fire-disturbance study at Mammoth Cave that is getting underway. Exotic species (Xylosandrus crassiusculus, X. saxesenii, and Euwallaceae validus) comprised 90% of ambrosia beetles recovered in ethanol/canoptheran traps. Those attractants were combined with the repellant, vebanone, in a push-pull control strategy, but it did not affect insect infestation. Colletotricum acutatum infection of gall wasp pupae may help control the wasp. The U.S. Forest Service, Delaware, OH, and the University of Tennessee reported that TACF backcross seedlings were, on average, shorter than American but taller than Chinese chestnut at most sites. Defoliation by Cyrtepistomus casteneus was similar between Chinese chestnut and backcross hybrids. Imidacloprid is recommended for control of defoliating insects. A test of seedlings sorted into four form classes revealed that shorter chestnut seedlings tended to grow better than other forms at mid-story removal sites. The utility of sorting seedlings by form class is being explored further. Comparing 25 sites classified as mesic, intermediate or xeric, chestnut grew best on intermediate sites. Penn State Cooperative Extension reported on 12 outreach efforts in PA. One demonstration orchard was planted and 19 test plantings. Three volunteers were trained in pollination, and 57 volunteers trained in other activities. Several sites were inspected and evaluated. There is a state-wide program on "American Chestnut Restoration," a chestnut mailing list and 2 articles were published in the Extension newsletter, "The Woodlander." At the business meeting, it was decided that Bill Powell, SUNY-ESF, will assume duties as chair in 2016 and host the meeting in New York. Hill Craddock, UT-Chattanooga, agreed to be chair-elect and host the meeting in 2017. ? Attendance: Connecticut: Sandra Anagnostakis (Connecticut Agricultural Experiment Station) Kentucky: Lynne Rieske-Kinney, Anna Conrad (University of Kentucky), Tyler Dreaden (USFS-Lexington) Maryland: Donald Nuss, Dongxiu Zhang (University of Maryland Institute of Bioscience and Biotechnology Research, Shady Grove) Michigan: Andrew Jarosz, Josh Spring, Matt Kolp (Michigan State University) Mississippi: Dana Nelson (Southern Institute of Forest Genetics, Saucier) New Mexico: Angus Dawe (New Mexico State University) New York: Kristen Stewart-Russell, Linda McGuigan (SUNY-ESF); Steve Jakobi (Alfred Station) Ohio: Leila Pinchot (USFS, Delaware) North Carolina: Jared Westbrook, Paul Sisco, Lisa Thomson, Tom Saielli (TACF®, Asheville) Pennsylvania: Gary Micsky (Penn State Extension, Mercer), Mike Marshall (Shippensburg University) Portugal: Rita Costa (Instituto Nacional de Investigação Agrária e Veterinária) South Carolina: Steve Jeffers, Tatyana Zhebentyayeva (Clemson University) Tennessee: Hill Craddock, chair-elect, Taylor Perkins, Conrad Blunck (UT Chattanooga) Virginia: Fred Hebard, chair, Laura Georgi (TACF®, Meadowview), Matt Brinckman (TACF®-Charlottesville), Laurel Rodgers (Shenandoah University) West Virginia: William MacDonald, Mark Double, Cameron Stauder (West Virginia University)

A study involving CHV-1/EP713, CHV-1/Euro7 and newly characterized CHV-1/EP721 reported in J. Virol. 86:12933-12939 revealed unexpected variations in the transcriptional activation of the RNA silencing pathway and in virus-mediated symptom expression in the absence of the RNA silencing pathway. A robust level of antiviral RNA silencing of CHV-1/Euro7 and CHV-1/EP721 was inferred in wild-type C. parasitica, as evidenced by the increase in viral RNA accumulation in the deltadcl2 strain, in the apparent absence of significant induction of dcl2 strain was not accompanied by the debilitating growth phenotype observed for CHV-1/EP713 and CHV-1/Euro7 infections. Moreover, the difference in the virus-mediated delta dcl2-debilitating phenotype could be mapped to a viral coding domain. These results challenge the previous view that the delta dcl2-debilitating phenotype is due simply to highly elevated levels of viral gene expression in the absence of the RN silencing pathway. While providing new insights into the interactions between mycoviruses and host RNA silencing antiviral defense, the combined results also suggest a higher degree of complexity than previously anticipated. A simple and efficient system was developed by adapting the Cre-LoxP recombination system for unlimited recycling of the limited number of available selectable marker genes (SMGs). The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi. Mutational analyses of the infectious CHB-1/EP713 infectious cDNA clone defined the requirements for autocatalytic cleavage of papain-like leader proteases p29 and p48 and the functional importance of autoproteolysis in the context of hypovirus replication. The studies also exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, PCR primer sets were designed that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isoaltes colelcted from five locations in the eastern U.S. revealed 39 unique vic genotypes and generally good argreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies. SUNY-ESF is moving forward to get permission from the US government to release transgenic American chestnut into the wild. More than 21,000 chestnut shoots have been produced by micropropagation shoots; the intent is to improve regeneration success. The assembly of the Chinese chestnut cultivar 'Vanuxem' genome has been significantly improved over the past year. The current status of the genome sequence is a set of 14,358 scaffolds representing 784MB of genome sequence. The University of TN upgraded the hardwoodgenomcis.org website fo the genome and QTL assemblies to JBrowse format. One thousand new SNPs were added to the Chinese X Chinese chestnut genetic map by The Southern Institute of Forest Genetics. A promotor governing gene expression in C. parasitica was found to be controlled by copper in growth media. This allows gene expression in a hypovirus to be turned off and on. Hypovirus strain GH2 was found to efficacious when applied to cankers containing the five most common vc types in Michigan orchards. The OTC drug, naproxen, was found to inhibitory to the growth and development of C. parasitica in culture.

NE-1333 PUBLICATIONS 2014-2015 Carlson, J.E., Staton, M., Islam-Faridi, N., Addo-Quaye, C., Cannon, N., Tomsho, L.P., Ficklin, S.P., Saski, C.A., Burhans, R., Drautz, D., Kane Wagner, T., Zembower, N., Schuster, S., Abbott, A.G., Nelson, C.D., and Hebard, F. 2014. The Chinese chestnut (Castanea mollissima) genome. The American Chestnut Foundation's 31st Annual Meeting. 17-19 Oct. 2014. Northern Virginia 4-H Educational Conference Center, Front Royal, Virginia. Carlson, J.E., Staton, M., Orendovici-Best, T., Zembower, N., Lane, T., Davitt, J., Zhebentyayeva, T., Wu, D., Moses, D., Coggeshall, M., Gailing, O., Liang, H., Romero-Severson, J., Saski, C.A., Schlarbaum, S., Shumaker, K., Schuster, S. and Wheeler, N. 2015. Genomic Resources for North American Hardwoods. Plant and Animal Genome XXIII Conference, San Diego, CA, Jan. 11, 2015, poster #15556. Clark, S.L., Schlarbaum, S.E, Saxton, A.M. and Hebard, F.V. 2015. Establishment of American chestnuts (Castanea dentata) bred for blight (Cryphonectria parasitica) resistance: influence of breeding and nursery grading. New Forests. DOI 10.1007/s11056-015-9512-6. ?urkovi?-Perica, M., Ježi?, M., Karin, V., Idžojti?, M., Sotirovski, K., Risteski, M., Rigling, D., Prospero, S., Kolp, M. and Double, M. 2015. Biological control of chestnut blight: persistence of biocontrol agent Cryphonectria parasitica hypovirus 1 in healed chestnut cankers. International Plant Protection Congress, Berlin, Germany 24-27 Aug. 2015, p. 611. D’Amico, K.M., Horton, T., Maynard, C., Stehman, S., Oakes, A. and Powell, W. 2015. Comparisons of ectomycorrhizal colonization of transgenic American chestnut with those of the wild type, a conventionally bred hybrid, and related Fagaceae species. Appl. Env. Microbiol. 81:100-108. Dane, F. and Sisco, P.H. 2014. Genetic diversity of American chestnut is highest in the southern US: Evidence from nuclear and chloroplast DNA studies. J. Amer. Chestnut Found. 28:9-13. Davelos Baines A.L., Eager, E.A. and Jarosz, A.M. 2014. Modeling and analysis of American chestnut populations subject to various stages of infection. Letters in Biomathematics 1: 235-247. Fulbright, D.W. and Serdar, Ü. 2015. Turkey: Chestnut blight cankers. J. Amer. Chestnut Found. 29:28-33. Georgi, L.L., Zhebentyayeva, T., Islam-Faridi, N., Vining, E., Abbott, A.G., Nelson, C.D., and Hebard, F.V. 2015. The search for genes for resistance to chestnut blight. J. Amer. Chestnut Found., 29:16-22. Gold, M.A. Agroforestry. 2014. Encyclopædia Brittanica Online. Encyclopædia Brittanica Inc. Jakobi, S.R., Werner, A. and Double, M.L. 2015. Nutrient media for the determination of sexual reproduction of Cryphonectria parasitica. J. Applied Sci. Technol. 5:1-7. Jensen, K.S. and Nuss, D.L. 2014. Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation. J. Virology 88:11946-11954. Jose, S. and Gold, M.A. 2014. Agroforestry. In Rowe, D. (Ed.) Achieving Sustainability: Visions, Principles, and Practices. 1st Edition. Macmillan Reference, MI, USA. Medina-Mora, C. 2015. Pollination biology and simple sequence repeat (SSR) genetic identification of chestnut cultivars and their progeny. Ph.D. dissertation, Michigan State University, East Lansing, MI. Newhouse, A.E., McGuigan, L.D., Baier, K.A., Valletta, K.E., Rottmann, W.H., Tschaplinski, T.J., Maynard, C.A. and Powell, W.A. 2014. Transgenic American chestnuts show enhanced blight resistance and transmit the trait to T1 progeny. Plant Science 228:89-97. Newhouse, A.N. Transgenic American chestnut, a new paradigm for restoration. St. Lawrence/Southeast Lake Ontario Regional Invasive Species Symposium, 10 Jun 2015, Pulaski, NY. Newhouse, A.N. Where there be mountains, there be chestnuts. Ozark Chinquapin Foundation, 14 Mar 2015, Rogers, AR. Powell, W.A. Additional tools for solving an old problem: The return of the American chestnut. National Research Council Webinar on GE Trees, 27 Mar 2015, Syracuse, NY. Powell, W.A. Return of the King: The development of a blight-resistant American chestnut tree. Department of Natural Resources Research Symposium. Cornell University, 16 Jan 2015, Ithaca, NY. Powell, W.A. Transgenic American chestnut, a new paradigm for restoration. New Genomic Solutions for Conservation Problems Workshop, 6-9 Apr 2015, San Francisco, CA. Powell, W.A. Where there be mountains, there be chestnuts. Biotechnology Literacy Project, 31 May-4 Jun 2015, Davis, CA. Short, D.P.G., Double, M., Nuss, D.L., Stauder, C.M., MacDonald, W.L. and Kasson, M.T. 2015. Multiplex PCR assays elucidate vegetative incompatibility gene profiles of Cryphonectria parasitica in the United States. Appl. Env. Microbiol. 81:5736-5742. Sisco, P.H., Neel, T.C., Hebard, F.V., Craddock, J.H. and Shaw, J. 2014. Cytoplasmic male sterility in interspecific hybrids between American and Asian Castanea species is correlated with the American D chloroplast haplotype. Acta Hort. 109:215-222. Warmund, M. 2014. Nitrogen fertilization of young Chinese chestnut trees. American Society for Horticultural Science. 28-31 July 2014, Orlando, FL. Zhang, D-X., Spiering, M.J. and Nuss, D.L. 2014. Characterizing the roles of Cryphonectria parasitica RNA-dependent RNA polymerase-like genes in antiviral defense, viral recombination and transposon transcript accumularion. PLoS One 9(9):e108653.