Brief Summary of Minutes of Annual Meeting

OBJECTIVE 1. To 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 The report focused on two genes: Oxalate Oxidase (OxO) and a Laccase-like protein (Lac). Reduced oxalic acid correlates to reduced virulence. On transgenic chestnut, OxO can change the fungal lifestyle from a pathogen to a saprophyte. One of their transgenic trees, ‘Darling 4”, has a lesion similar to Chinese chestnut (< American chestnut). Three-year-old American chestnut trees still maintain OxO in their genome. Laccase is differentially expressed in American and Chinese chestnut trees with Chinese producing 100X more laccase. Laccase-like genes enhance blight resistance, as least partly. Pyramiding the OxO with the Laccase-like gene should provide even more sustainable blight resistance. When comparing metabolites of American, TACF backcross, and transgenic trees, the backcross trees differed by thirteen metabolites while in the transgenic trees, only one metabolite differed. West Virginia University The interaction of host resistance and virulent/hypovirulent strains of Cryphonectria parasitica was assessed using American, BF2, BF3, Chinese, and European chestnuts inoculated with Weekly-2, a moderately virulent strain. Growth, sporulation and canker morphology will be assessed annually. Canker size was assessed in Aug 2014. Canker size was smallest on Chinese and largest on BF3 while death was largest for American and smallest for Chinese. Hypoviruses (Euro 7, COLI, GH2 and Weekly/Ep155/pXHE7) were introduced to all naturally-occurring cankers in hypovirus-treated plots. In Aug 2014, 23 naturally-occurring treated cankers were sampled (4 plugs/canker). Sixty-five percent (15/23) of the cankers yielded at least one hypovirulent isolate. The treated cankers will be sampled and subjectively rated annually. The American Chestnut Foundation, Meadowview The American Chestnut Foundation (TACF) is using a conventional breeding program (backcross method) to transfer the blight resistance of the Chinese into the American chestnut tree. Intercrossing third backcrosses will produce trees (B3-F2) that have a chance of inheriting the genes for blight resistance from both parents. B3-F3 progeny are being set out in forest progeny tests to determine the performance of B3-F3 trees with natural blight and competition. B3-F3 seedlings were transplanted into forest progeny tests. Early results indicate most B3-F3 families resemble American chestnut in metrics of growth. Progeny tests in orchard settings with artificial inoculation with the blight fungus revealed average canker size on B3-F3 trees was similar to F1 Chinese and American hybrids. 'Graves' B3-F3 trees have resistance to both blight and Phytophthora root rot (PRR) (controlled by a single major gene). We plan to fix PRR resistance with two more generations of crossing. Because direct inoculation of B3-F2s cannot be used to distinguish trees with one or two alleles for susceptibility from those with none, some B3-F2s must be progeny tested. Marker assisted selection for disease resistance offers the hope of helping select trees homozygous for blight-resistance factors. Genotyping by sequencing (GBS) using restriction site associated DNA sequencing (RAD-Seq) in a fairly small population of 'Graves' B3-F2s is being initiated. The intent is to see whether a useful whole genome selection model (WGS) can be developed. TACF’s current breeding populations are based on three sources of blight resistance: ‘Graves’ and ‘Clapper,’ are currently producing B3-F3 nuts while the first nuts for a ‘Nanking’ seed orchard were only harvested in 2013. To have a sufficient inbreeding effective size (Nei) to avoid erosion of diversity through genetic drift, additional sources of blight resistance from state chapter and ‘Nanking’ lines will be incorporated into the breeding program. It is still unclear if we will have captured enough resistance from Chinese chestnut for B3-F3 trees to thrive in the forest, or even whether trees with blight resistance equal to that of the best Chinese chestnut trees would do so. Nor have we yet recovered enough of the American genome for our B3-F3 trees to constitute a viable species, but enough trees to do so are in our pipeline. However, evidence to date suggests that many of the trees currently being produced grow as well as American chestnut and have enough blight resistance to be able to survive and reproduce. Pennsylvania State University The sequencing of the Chinese chestnut genome was initiated in 2009 to produce a reference genome sequence for Castanea mollissima cv Vanuxem as a platform for the identification of blight resistance genes and to conduct marker-assisted selection at the whole genome level. By 2011, the first draft of the Chinese chestnut genome was produced. After a second round of sequencing, a new draft genome was completed in early 2013. This represents 90% of the estimated size of the chestnut genome, in which we identified 38,268 genes supported by gene expression data. To ensure that all blight resistance genes could be identified, we also produced deep sequence of pools of BAC clones from the Chinese chestnut physical map contigs covering the three blight resistance QTL. In total we identified 1952 genes in the 3 QTL, from which 15 genes were selected as high priority candidate genes for blight. The scaffolds, gene models, predicted transcripts and proteomes are all available to the community to download at the hardwood genomics website. OBJECTIVE 2. To 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 Institute for Bioscience and Biotechnology Research, University of Maryland, Shady Grove Campus The C. parasitica genome assembly release V 2.0 contains 26 main genome scaffolds totaling 43.9 Mb. Vegetative incompatibility loci, vic1 and vic3, were identified molecularly. Restriction of mycovirus transmission by five C. parasitica vic loci was confirmed. Mutagenesis of the CHV-1/EP713 infectious cDNA clone revealed the autocatalytic processing of the leader proteases p29 (suppressor of RNA silencing) and p48 (viral RNA replication) contributes to optimal virus RNA accumulation. Four RNA-dependent RNA polymerase genes (rdr1-4) were identified and characterized. The possibility of knocking out individual vic genes to create a universal donor was discussed. Rutgers University There are eight different types of transposons in the Cryphonectria genome (four Class I—reverse transcribing and four Class II). One putative helitron, a new class of transposon, has been identified. Flat mutants are deeply pigmented with very little aerial mycelium with spores produced directly on vegetative hyphae and are produced only in virus-infected cultures. Mutant colonies initially contain virus, but is not required to maintain the phenotype. It is a non-reverting nuclear gene mutation. The Castanea microbiome is a teaching tool using chestnut blight to introduce first-year students to research at the NJ AES of Rutgers University. New Mexico State University When Cpvib-1 (a putative transcriptional activator) was deleted from the wild-type strain EP155, enhanced pigmentation, conidiation, and pathogenicity resulted. A role for Cpvib-1 in mediating the incompatibility response and cell death in C. parasitica has been identified. C. parasitica has been found to contain five putative proteins containing LysM motifs which may act as an effector protein, playing a role in virulence. Knockouts of all five of genes have been attempted: two appear to be lethal knock-outs, one led to no discernible changes in phenotype, another led to significant increase in canker size and only one showed significant reduction of virulence and vegetative growth. Carleton University, Ottawa Hyphae in incompatible reactions undergo granualization, vacuolization, and plasmolysis. When two virulent isolates are paired that differ at vic3, 65-80% of the contacts have cell death. When p29 is present, cell death drops to 16-25%. If RNAi is knocked out, there is a decrease in cell death. There are a larger number of differentially expressed genes when an RNAi-deficient strain interacts with a WT strain compared to two WT strains. With just vic3 incompatibility 100-200 genes are transcribed differently. An enrichment analysis was done on only 2/3 of the significantly differentially regulated gene set as 1/3 of those genes are of unknown function. Most are unique to Cryphonectria. It is this gene set that is most interesting. West Virginia University Hypovirus introduction was conducted from 1992-1997 and 2003-2013 in West Salem, WI. Hypovirulent isolates are most readily recovered from treated trees but have spread less effectively to untreated trees. Chestnut sprout populations have increased significantly. The ability of virulent and hypovirulent strains of C. parasitica to grow and sporulate on dead American chestnut and scarlet oak (Quercus coccinea) stems cut from healthy trees was studied. Both species supported significant growth and sporulation on stems inoculated soon after cutting. With later inoculations, colonization and sporulation were greater on scarlet oak. As stems aged, other organisms became significant colonizers. Michigan State University Over 2,000 isolates have been obtained from MI, WI, and MD. More secondary fungi are isolated from non-girdling cankers. In competitive interactions, C. parasitica was grown on Petri plates with other fungi with significant effects of C. parasitica isolate, media, and isolates of non-C. parasitica found. Field tests with Trichoderma (which was best at preventing C. parasitica growth) were conducted. A European X Japanese hybrid, ‘Bouche de Betizac’ is immune to gall wasp. Michigan is still number one in total number of chestnut farms and acreage with nearly 700 acres of chestnuts planted. ‘Colossal’ is resistant/sensitive to winter temperatures because it is grafted onto cold sensitive rootstock. Planting E X J hybrids will give a much larger yield than Chinese chestnuts. University of Wisconsin, La Crosse A model presented by a mathematician revealed the lifetime average number of successful seedlings per individual, r, differed among healthy, diseased and hypovirus-infected American chestnut populations, with healthy populations having 30-times larger r than recovered populations. Hypovirus infection, while being potentially insufficient to allow chestnut populations to persist long term according to these models, is able to provide the population with a boost in the short run. This has given time to pursue alternative conservation measures, which mathematical analyses can guide.? OBJECTIVE 3. To 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 OBJECTIVE 3. To 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 In southeastern states, Phytophthora root rot, PRR (=ink disease) caused by P. cinnamomi, killed a high percentage of seedlings in the first few years after out-planting. The goal was to develop a system to evaluate hybrid chestnut seedlings from families bred for blight resistance for resistance to P. cinnamomi. Screening has revealed that American chestnut seedlings have died while Chinese chestnut seedlings consistently are healthy. Over 200 backcross families have been evaluated; percent mortality ranged from 68% to 99%. Hybrid families selected for resistance to C. parasitica also carry resistance to P. cinnamomi. Genes for resistance to P. cinnamomi and C. parasitica do not appear to be linked. Trees genetically engineered for resistance to chestnut blight do not survive the P. cinnamomi screening. University of Kentucky ACGW (Dryocosmus kuriphilus) is a global pest of chestnut. Given that infestation widely fluctuates, evaluation of ACGW reproductive potential is needed. Understanding invasiveness requires evaluation of fecundity and its correlation to insect size and age. To assess the fecundity of ACGW, the size and age of insects using 4 age-cohorts (0, 1, 2 and 3-days old) was evaluated. Invasiveness is influenced by potential fecundity. General insect size is not a good predictor of egg load. Newly emerged (0 d) wasps carry large egg loads.Egg load decreases with age. Egg volume is greatest the second day after emergence, then decreases. Insect weight and metosomal with can predict egg load. The Connecticut Agricultural Experiment Station Gall wasp has been present in CAES planting since 2011. One tree of C. henryi is the only mature survivor of the species and has no galls. Cultivars ‘Colossal’ and ‘Easton’ were crossed with C. henryi and the resulting seedlings were planted under a heavily galled tree. Within 2 years, all the seedlings had galls. ‘Eaton’ X C. henryi trees had fewer galls than ‘Colossal’ X C. henryi. C. pumila shrubs have occasional galls and C. ozarkensis trees from both Arkansas and Oklahoma have no galls. Crosses using both AR and OK C. ozarkensis with trees of cultivars, ‘Colossal’, ‘Eaton’, and ‘Lockwood’ have been made. The resulting seedlings will be evaluated for resistance to gall wasp invasion. Open pollinated nuts of ‘Colossal’, ‘Lockwood’ and the two C. ozarkensis types vary in protein and fatty acid contents with the C. ozarkensis types having higher protein and fatty acid content. University of Tennessee, Chattanooga There is a good deal of cytoplasmic diversity among chestnut. If an American chestnut as female is crossed by an Asian chestnut as male, the resulting F1 tree is almost always pollen sterile. If the cross is made in the other direction, the offspring are male fertile. This is probably caused by an incompatibility between American chestnut mitochondria and Chinese chestnut nuclear genes, as is found in many other plant species. However, exceptional male-fertile F1 progeny of American (female) x Asian chestnut (male) have been found in the southern U.S. (northern Alabama, northwest GA, northeast MS, and central TN). One way they try to control Phytophthora cinnamomi is with phosphite. The effect of phosphite fungicides on beneficial ectomycorrhizal fungus (ECM) which are necessary to successful chestnut seedling establishment was investigated. Results suggest phosphite-based fungicides may impede root colonization by ECM. Alfred State College The traditional method for producing perithecia in vitro takes time and scratched, autoclaved chestnut stems. An artificial nutrient medium to facilitate sexual production between mating type compatible strains (Mat-1 and Mat-2) of C. parasitica was investigated. Of the 18 nutrient preparations tested, only 4 induced the formation of perithecia: PDA x chestnut bark extract, Leonian agar, Leonian agar amended with 0.01% thiamin and biotin and Leonian agar amended with 1% “light” coconut milk. Twenty-five of twenty-nine crosses produced perithecia. To ensure that the perithecia produced viable ascospores, perithecia from several of the above crosses were examined. Leonian agar with coconut milk produces perithecia with viable ascospores. Penn State Cooperative Extension (submitted report) NE-1333 and TACF personnel and resources have been critical to success in expanding outreach to new audiences and have enhanced the quality of existing extension programming. Programs include: training workshops and field experience, extension newsletters and press releases, grower/site evaluations and pest surveys. Thirty-two on-site test plantings were established and seventy-nine volunteers were trained for roles including host research/demonstration plots and assisting in TACF and other research activities. Outreach efforts include an extension mailing list, chestnut gall wasp fact sheet, and a newsletter.

A Genetics paper describing the identification and characterization of vic1 and vic3 candidate genes was published in 2014, thus completing the goal of identifying and disrupting all 67 genetically defined C. parasitica vic loci (Genetics 197:701-704). A study involving CHV-1/EP713, CHV-1/Euro7 and newly characterized CHV-1/EP721, reported to J. Virol., revealed unexpected variations in the transcriptional activation of the RNA silencing pathways and in virus-mediated symptom expression in the absence of RNA silencing pathway. A robust level of antiviral RNA silencing of CHV-1/Euro7 and CHV-1/EP721 was inferred in a wild-type C. parasitica, as evidenced by the increase in viral RNA accumulation in the delta dcl2 strain, in the apparent absence of significant induction of dcl2 transcript accumulation. The increase in CHV-1/EP721 RNA accumulation in the delta 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-deblitating 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 RNA 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. (J. Virol. 86:12933-12939.) SUNY-ESF reported found that reduced oxalic acid correlates to reduced virulence. Using the oxalate oxidase gene (OXO), genetically inserted into the genome of the American chestnut, OXO has a high level of enhancement (? Chinese chestnut). Intermediate levels of enhancement (Chinese > I > American) can be found in cisgenes, of which Laccase is one. Field assays in New York state demonstrated enhanced blight resistance and validity of the leaf assay prediction, developed at SUNY-ESF. American chestnut has a large lesion in the leaf assay and Chinese chestnut has a small lesion. One of their transgenic trees, ‘Darling 4”, has a lesion similar to Chinese chestnut. It was found that Laccase is differentially expressed in American and Chinese chestnut trees. Chinese chestnut produces more laccase (100X more), whether it is inoculated or non-inoculated trees compared to American chestnut. In forest progeny tests, set out by combined efforts of The American Chestnut Foundation and the US-Forest Service, to determine whether B3-F3 trees can be canopy dominants and co-dominants, it was found that early results indicate most B3-F3 families resemble American chestnut in metrics of growth. Results from 8 years of testing breeding stock indicate that resistance to Phytophthora root rot (PRR) in the ‘Graves’ source of blight resistance is controlled by a single major gene. Browsers for the genome and QTL assemblies were released at the hardwood genomics website (http://www.hardwoodgenomics.org/content/tools). The browsers are fully annotated to model plant genomes. The scaffolds, gene models, predicted transcripts and proteomes are all available to the community to download at the hardwood genomics website. The current draft of the Chinese chestnut genome, and genome browser, were presented to the broader research community through talks and posters at the Plant & Animal Genome Conference XXII, San Diego, CA, on January 12, 2014, and at the PAG-Asia Conference in Singapore, May 19-21, 2014. By examining genome sequence data, C. parasitica has been found to contain five putative proteins containing LysM motifs. These motifs have been recognized using information from the organism’s genome portal. The LysM motif, found in eukaryotes and bacteria but not archaea, has been found to be implicated in many different biological processes. In an agar test to determine vegetative incompatibility, when one of the strains in a pairing contains CHV1, the amount of cell death frequency decreases significantly. This happens with either the virus or simply constructs. Cultivars ‘Nevada ‘and ‘Okei', planted as pollinizers for ‘Colossal’ in Michigan are no longer recommended, as they are too sensitive to cold temperatures. Using a mathematical model to discern the role of hypoviruses in reforestation, it was found that hypovirus infection, while being potentially insufficient in helping chestnut populations persist in the long-run, is able to provide the population with a boost in the short run. For the chestnut pest, Asian Chestnut Gall Wasp, it was found that insect weight and metosomal width can predict egg load. It was found that phosphite-based fungicides may impede root colonization by ectomycorrhizal fungi.

NE-1333 PUBLICATIONS 2013-2014 Anagnostakis, S.L. 2014. A preliminary report on Asian chestnut gall wasp on species and hybrids of chestnut in Connecticut. Acta Hort. 1019:21-22. Anagnostakis, S.L. and Pinchot, C.C. 2014. Restoration of chestnuts as a timber crop in Connecticut. Acta Hort. 1019:17-19. Carlson, J.E., Staton, M.E., Addo-Quaye, C., Cannon, N., Tomsho, L.P., Ficklin, S., Saski, C., Burhans, R., Drautz, D., Wagner, T.K., Zembower, N., Schuster, S.C., Abbott, A.G., Nelson, C.D. and Hebard, F.V. 2014. The Chinese chestnut (Castanea mollissima) genome. PAG-Asia Conference II, Singapore, 19-21 May 2014. Carlson, J.E., Staton, M.E., Addo-Quaye, C., Cannon, N., Tomsho, L.P., Ficklin, S., Saski, C., Burhans, R., Drautz, D., Wagner, T.K., Zembower, N., Schuster, S.C., Abbott, A.G., Nelson, C.D. and Hebard, F.V. 2014. The chestnut genome project. Plant and Animal Genome Conference XXII, San Diego, CA. 21 Jan 2014, Abstract W307. Clark, S.L., Schlarbaum, S.E. and Hebard, F.V. 2014. The first research plantings of third-generation, third backcross American chestnut (Castanea dentata) in the southern United States. Acta Hort. 1019:39-44. Craddock, J.H. 2014. A tree crop archetype. Acta Hort. 1019:45-47 Dale, A. and Galic, D. 2014. Breeding blight resistant American chestnut for Canada. Acta Hort. 1019:49-53. Dane, F., Xiaowei, L, Kocot, K. and Goertzen, L. 2014. Comparative analysis of the transcriptome of Castanea pumila var. pumila. Acta Hort. 1019:55-60. Davelos Baines, A., Fulbright, D.W. and Jarosz, A.M. 2014. Effects of branch size and pathogen virulence on canker development and branch mortality. Acta Hort. 1019:23-29. Dawe, A.L. 2014. Leveraging the genome sequence to identify genes with potential involvement in the virulence of Cryphonectria parasitica. Acta Hort. 1019:61-64. Dawe, A.L. and Nuss, D.L. 2014. Hypovirus Molecular Biology: from Koch’s postulates to host self-recognition genes that restrict virus transmission. Adv. Virus Res. 86: 109-147. Donis-González, I.R. Guyer, D.E., Pease, A., D.W. Fulbright and Barthel, F. 2014. Visualizing internal characteristics of fresh chestnuts (Castanea spp.) using traditional and ultrafast limited-angle-type x-ray computed tomography (CT) imaging. Acta Hort. 1019:65-71. Donis-González, I.R., Guyer, D.E., Burns, J. and Leive-Valenzuela, G.A. 2014. Quality assessment of sliced chestnut (Castanea spp.) using color images. Acta Hort. 1019:73-80. Double, M.L, MacDonald, W.L. and Taylor, G.M. 2014. Evaluation of Cryphonectria parasitica isolates collected from the Great Smoky Mountains National Park. Acta Hort. 1019:85-89. Double, M.L. and MacDonald, W.L., eds. 2014. Proceedings of the Fifth International Chestnut Symposium. 4-8 Sep 2012, Shepherdstown, WV, 270 pp. ISBN: 978-94-6261-006-4. Double, M.L. and Marshall, M.R. 2014. Strawberry amendment to potato dextrose agar to increase conidiation in Cryphonectria parasitica. Acta Hort. 1019:81-84. Double, M.L., Kolp, M.R., Jarosz, A.M., Davelos Baines, A., Fulbright, D.W. and MacDonald, W.L. 2014. Fungi associated with hypovirulent cankers of differing ages on American chestnut. Acta Hort. 1043:57-65. Double, M.L., MacDonald, W.L., Jarosz, A.M., Fulbright, D.W., Cummings Carlson, J. and Dahir, S. 2014. Recapping twenty years of biological control efforts in a stand of American chestnut in western Wisconsin. J. Amer. Chestnut Found. 27:19-23. Fang, G.C., Blackmon B.P., Staton M.E., Nelson C.D., Kubisiak T.L., Olukolu B.A., Henry D., Zhebentyayeva T., Saski C.A., Cheng C.H., Monsanto M., Ficklin S., Atkins M., Georgi L.L., Barakat A., Wheeler N., Carlson J.E., Sederoff R. and Abbott A.G. 2013. A physical map of the Chinese chestnut (Castanea mollissima) genome and its integration with the genetic map. Tree Genetics and Genomes 9:525-537. Fulbright, D.W. 2013. Internal kernal breakdown (IKB) of chestnut appears when European X Japanese hybrid cultivars are pollinized by Chinese chestnut. Annual Rept. Northern Nut Growers 103:3-11. Fulbright, D.W., Stadt, S., Medina-Mora, C., Mandujano, M., Serdar, Ü. and Donis-González, I.R. 2014. Kernel breakdown appears when hybrid Castanea Cultivars are pollinized by Castanea mollissima. Acta Hort. 1019:91-97. Galic, D., Dale, A. and Alward, M. 2014. Vegetative propagation of American chestnut. Acta Hort. 1019:99-103. Garziosi, I. and Rieski, L.K. 2014. Local spread of an exotic invader: using remote sensing and spatial analysis to document proliferation of the invasive Asian chestnut gall wasp. Acta Hort. 1019:113-118. Georgi, L.L., Hebard, F.V., Staton, M.E., Olukolu, B.A., Abbott, A.G. and Nelson, C.D. 2014. Adapting chestnut single nulceotide polymorphisms for use in breeding. Acta Hort. 1019:105-112. Gold, M.A. 2014. Agroforestry. Encyclopaedia Brittanica Online. Encyclopaedia Brittanica Inc. Graziosi, I., and Rieske, L.K. 2014. Local spread of an exotic invader: using remote sensing and spatial analysis to document proliferation of the invasive Asian chestnut gall wasp. Acta Hort. 1019:113-118. Graziosi, I., and Rieske, L.K. 2014. Potential fecundity of a highly invasive gall maker, Dryocosmus kuriphilus (Hymenoptera: Cynipidae). Environ. Entomol. (in press). Guyer, D.E., Donis-González, I.R., Burns, J. and De Kleine, M.E. 2014. Is internal quality of chestnuts influenced by harvest methods and physical stresses? Acta Hort. 1019:119-125. Hao, J.J., Donis-González, I.R., Jiang, H.H., Liu, H. and Fulbright, D.W. 2014. Antimicrobial activity in chestnut tissues corresponding with flavonol glycoside and terpenoid substances. Acta Hort. 1019:127-134. Harris, A.P. 2013. A vascular flora of Bendabout Farm, Bradley County, Tennessee and survey of native Castanea dentata (Marsh.) Borkh. (Fagaceae, American chestnut). M.S. Thesis, University of Tennessee, Chattanooga. Chattanooga, TN. Hebard, F.V., Fitzsimmons, S.F., Gurney, K.M. and Saielli, T.M. 2014. The breeding program of The American Chestnut Foundation. Acta Hort. 1019:135-139. Herr, J.R. and Carlson, J.E. 2013. Traditional breeding, genomics-assisted breeding and biotechnological modification of forest trees in short rotation woody crops. In: Jacobson, M. and D. Ciolkosz (eds.), Wood-Based Energy in the Northern Forests. New York: Springer, pp 79-99. ISBN: 978-1-4614-9477-5 (print); 978-1-4614-9478-2 (online). Jarosz, A.M., Springer, J.C., Fulbright, D.W., Double, M.L. and MacDonald, W.L. 2014. Hypovirus influence on survivorship and growth of American chestnuts at West Salem, Wisconsin, USA. Acta Hort. 1019-157-163. Jose, S. and Gold, M.A. 2014. Agroforestry. In Rowe, D. (Ed.) Achieving Sustainability: Visions, Principles, and Practices. 1st Edition. Macmillan Reference, MI, USA. Kenaley, S.C., Double, M.L. and MacDonald, W.L. 2014. Effect of spore concentration on the establishment of cytoplasmic hypovirulent (hv), transgenic hv, and virulent isolates of Cryphonectria parasitica, the chestnut blight fungus. Acta Hort. 1019-165-171. Kubisiak, T.L., Nelson, C.D., Staton, M.E., Zhebentyayeva, T., Smith, C., Olukolu, B.A., Fang, G.-C., Hebard, F.V., Anagnostakis, S., Wheeler, N., Sisco, P.H., Abbott, A.G. and Sederoff, R.R. 2013. A transcriptome-based genetic map of Chinese chestnut (Castanea mollissima) and identification of regions of segmental homology with peach (Prunus persica). Tree Genetics and Genomes 9:557-571. McCleary T.S., McAllister, M., Coggeshall, M. and Romero-Severson, J. 2013. EST-SSR markers reveal synonymies, homonymies and relationships inconsistent with putative pedigrees in chestnut cultivars. Genetic Resources and Crop Evolution. 60:1209-1222. Medina-Mora, C., Fulbright, D.W. and Jarosz, A.M. 2014. SSR genotyping of progeny from a chestnut orchard in Michigan. Acta Hort. 1019-173-178. Metaxas, A.M. 2013. Chestnut (Castanea spp.) cultivar evaluation for commercial chestnut production in Hamilton County, TN. M.S. Thesis, University of Tennessee, Chattanooga. Chattanooga, TN. Nelson, C.D., Merkle, S.A., Nairn, C.J., Kong, L., Staton, M.E., Abbott, A.G., Olukolu, B.A., Zhebentyayeva, T., Powell, W.A., Maynard, C.A., Baier, K.M., Newhouse, A., Carlson, J.E., Addo-Quaye, C., Hebard, F.V. and Georgi, L.L. 2014. The forest health initiative, American chestnut (Castanea dentata) as a model for forest tree restoration: biological research program. Acta Hort. 1019-179-189. Nelson, C.D., Powell, W.A., Merkle, S.A., Carlson, J.E., Hebard, F.V, Islam-Faridi, N., Staton, M.E. and Georgi, L.L. 2014. Chestnut. In: Ramawat, K., Merillon J.M., Ahuja M.R. (eds), Tree Biotechnology, Chapter 1. CRC Press, Boca Raton, Florida, USA, in press (accepted, August 2013). 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