NE1333: Biological Improvement of Chestnut through Technologies that Address Management of the Species, its Pathogens and Pests
(Multistate Research Project)
Status: Inactive/Terminating
Date of Annual Report: 12/06/2013
Report Information
Annual Meeting Dates: 09/05/2013
- 09/07/2013
Period the Report Covers: 10/01/2008 - 09/01/2013
Period the Report Covers: 10/01/2008 - 09/01/2013
Participants
Anagnostakis, Sandra - Connecticut Agricultural Experiment Station;Baines, Anita - University of Wisconsin La Crosse;
Brosi, Sunshine - Frostburg State University;
Burhans, Brian - The American Chestnut Foundation;
Clark, Stacy - USDA Forest Service;
Craddock, Hill - University of Tennessee Chattanooga;
Dawe, Angus - New Mexico State University;
Double, Mark - West Virginia University;
Fulbright, Dennis - Michigan State University;
Graziosi, Ignazio - University of Kentucky;
Hebard, Fred - The American Chestnut Foundation;
Jarosz, Andy - Michigan State University;
Kazmierczak, Pam - University of California Davis;
Kolp, Matt - Michigan State University;
MacDonald, William - West Virginia University;
McCampbell, Michael - Frostburg State University;
McGuigan, Linda Polin - State University of New York;
Micsky, Gary - Penn State Extension;
Moore, Claire - Michigan State University;
Nuss, Don - Institute for Bioscience and Biotechnology Research, Univ of Maryland, Shady Grove Campus;
Regnery, Russell;
Rieske-Kinney, Lynne - University of Kentucky;
Romero-Severson, Jeanne University of Notre Dame;
Sisco, Paul - The American Chestnut Foundation (ret);
Springer, Josh - Michigan State University;
Stewart, Kristen - State University of New York;
Van Alfen, Neal - University of California Davis;
Brief Summary of Minutes
Accomplishments
Accomplishments<br /> <br /> 2009 Milestones Accomplished:<br /> " Genetic map of Chinese and American chestnut <br /> " Genetic analysis of regions near chromosome-specific markers<br /> " Refined genetic linkage and genome sequence maps for map-based cloning of fungal vic and pathogenicity genes.<br /> " Assembly and community manual annotation of the C. parasitica genome sequence<br /> " C. parasitica genome sequence used to develop new microarray chip and proteomics platforms for analysis of global gene expression in the blight fungus when challenged by viral pathogens <br /> " Secondary structural motifs within the hypovirus genome identified<br /> " Anatomical studies of Chinese chestnut stems to determine relationship between protophloem fibers and grafting failure<br /> " Genetic fingerprints of tested cultivars through the use of microsatellite markers<br /> " Cultures of new embryogenic clones of known parentage established<br /> " Multiple transclones with 3 to 6 first-generation anti-fungal gene constructs regenerated<br /> " Tissue from chestnut families segregating for resistance to the blight collected<br /> " Differential responses of American and Chinese chestnut to foliar application of jasmonic acid characterized<br /> <br /> 2010 Milestones Accomplished:<br /> " Region of Chinese chestnut genome responsible for resistance to the blight pathogen delineated<br /> " Common secondary structural motifs shared with other hypoviruses identified<br /> " Fungal proteins altered in response to infection with hypovirus identified<br /> " Robust genetic linkage map for C. parasitica based on genomic sequence generated<br /> " C. parasitica antiviral RNA silencing pathways characterized<br /> " Genetic and physical causes of graft union failure in newly budded trees elucidated<br /> " Nitrogen recommendations for chestnut orchards finalized<br /> " Interaction between Asian chestnut gall wasp stem galls and chestnut foliage characterized<br /> " Influence of gall structure on ecological associations of the Asian chestnut gall wasp characterized<br /> " Chestnut volatiles as potential attractants for adult chestnut weevils characterized<br /> " DNA marker technology further developed<br /> " Engineer altered secondary structures and assess impact on hypovirus viability and transmission<br /> " Resistance of embryogenic clones in greenhouse tested<br /> " Field tests using embryogenic clones established<br /> " Contribution of saprophytic growth of C. parasitica to hypovirulent inoculum production assessed <br /> <br /> 2011 Milestones Accomplished:<br /> " Gains provided by release of transgenic strains of C. parasitica in the field assessed<br /> " Summary of early performance of 'Eaton' and 'Auburn Super' scions grafted onto 'Little Giant' interstem and 'Cropper' seedling rootstock completed (5 yr summary)<br /> " Genetic and physical causes of delayed incompatibility on bearing trees determined<br /> " Determine the effects of different pruning methods on nut yield and quality<br /> " Determine the attractiveness of chestnut volatiles to adult chestnut weevil<br /> " Make F2 crosses with Canadian chestnut and collect and store fruit<br /> " Sequence regions of the Chinese chestnut genome responsible for resistance to the blight pathogen and identify candidate resistance genes<br /> " C. parasitica vic genes identified, cloned and disrupted<br /> " C. parasitica secreted proteins and plant cell degrading enzymes and hypovirus-mediated modulations identified<br /> <br /> 2012 Milestones Accomplished:<br /> " F2 Canadian chestnut seedlings planted in field<br /> " Initiated functional tests by genetic transformation of candidate genes for resistance to the blight pathogen identified in the Chinese chestnut genome<br /> " Evaluated preliminary results from field tests using embryogenic clones<br /> " Identified fungal genes responsible for differences in C. parasitica virulence levels through genetic and genomic analyses <br /> " C. parasitica genes required for hypovirus replication and symptom expression through genetic and genomic analyses identified<br /> " Role of jasmonic acid in development of galls produced by Asian chestnut gall wasp elucidated<br /> " Molecular characterization of the Asian chestnut gall wasps introduced parasitoid, Torymus sinensis, and a native parasitoid, Ormyrus labotus, and their interactions characterized<br /> <br /> 2013 Milestones Accomplished:<br /> " Genes associated with vegetative incompatibility identified and characterized (vic1 and vic3)<br /> " Generation of a complete set of allele-specific PCR primers for all vic loci generated, available to interested researchers.<br /> " Survey of vic-genotypes for C. parasitica field isolate populations completed<br /> " Identified and sequence analysis of vic gene homologs in other Cryphonectria species. <br /> " F2 Canadian chestnut seedlings for screened for blight resistance<br /> " First test plants of chestnut trees with improved blight resistance deployed<br /> " Initiated development of dwarf chestnut trees of potential value to growers with limited space<br /> " Guidelines for P. cinnamomi control in southern forest nurseries established<br /> " Historical data to determine most suitable sites for chestnut reestablishment synthesized<br /> " Elucidated role of abiotic forces and landscape factors in localized spread of the Asian chestnut gall wasp <br /> " Role of olfactory and visual cues in host location by Torymus sinensis, the introduced parasitoid of the Asian chestnut gall wasp elucidated<br /> " Determined the role of a Colletotrichum/ Glomerella sp. fungus colonizing Asian chestnut gall wasp galls; the fungus causes high gall wasp mortality and no parasitoid mortality. <br />Publications
Impact Statements
- "Establishment of breeding orchards to generate large numbers of backcross generations for forest and orchard testing for pest resistance and regional adaptability;
- "Evaluation of Castanea genomic data to identify genes that confer desirable traits and enable rapid screening for those traits;
- "Development of in vitro mass propagation systems for Castanea spp. so that elite genotypes can be clonally propagated for reforestation;
- "Evaluation of the blight fungus genome to further our understanding of the genetic basis for pathogenesis and hypovirus regulation;
- "Development and deployment of genetically engineered virus for enhanced biocontrol;
- "Utilization of biological control agents to reduce the impacts of blight and other pests and pathogens; and,
- "In the longer-term, the project will lead to the return of an important timber species, mast species for wildlife, and commercial nut crop.
Date of Annual Report: 12/09/2015
Report Information
Annual Meeting Dates: 09/05/2014
- 09/06/2014
Period the Report Covers: 09/01/2013 - 09/01/2014
Period the Report Covers: 09/01/2013 - 09/01/2014
Participants
Attendance:Canada: Myron Smith (Carleton University, Ottawa)
Connecticut: Sandra Anagnostakis (Connecticut Agricultural Experiment Station)
Kentucky: Lynne Rieske Kinney, Ignazio Graziosi (University of Kentucky)
Maryland: Donald Nuss (University of Maryland Institute of Bioscience and Biotechnology Research, Shady Grove)
Michigan: Dennis Fulbright, Matt Kolp (Michigan State University)
New Jersey: Brad Hillman, Administrative Advisor (Rutgers University)
New Mexico: Angus Dawe (New Mexico State University)
New York: Kathleen Baier, Linda McGuigan (SUNY-ESF); Steve Jakobi (Alfred Station)
North Carolina: Jared Westbrook (TACF®, Asheville)
Pennsylvania: Sara Fitzsimmons (TACF®, University Park); Nathaniel Cannon (Penn State)
South Carolina: Steve Jeffers (Clemson University)
Tennessee: Hill Craddock, Taylor Perkins (UT Chattanooga)
Vermont: Kendra Gurney (TACF®, South Burlington)
Virginia: Fred Hebard—chair-elect, Laura Georgi (TACF®, Meadowview)
West Virginia: William MacDonald, Mark Double, Cameron Stauder (West Virginia University)
Wisconsin: Anita Davelos Baines—chair, Eric Eager, Dustin Stevens, Brandon Potter, Rebekah Guthman (University of Wisconsin-La Crosse)
Brief Summary of Minutes
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 genomeSUNY-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.
Accomplishments
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).<br /> <br /> 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.)<br /> <br /> 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. <br /> <br /> 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.<br /> <br /> 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. <br /> <br /> 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. <br /> <br /> 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. <br /> <br /> 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. <br /> <br /> Cultivars ‘Nevada ‘and ‘Okei', planted as pollinizers for ‘Colossal’ in Michigan are no longer recommended, as they are too sensitive to cold temperatures. <br /> <br /> 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. <br /> <br /> For the chestnut pest, Asian Chestnut Gall Wasp, it was found that insect weight and metosomal width can predict egg load.<br /> <br /> It was found that phosphite-based fungicides may impede root colonization by ectomycorrhizal fungi.<br />Publications
NE-1333 PUBLICATIONS 2013-2014<br /> <br /> 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.<br /> Anagnostakis, S.L. and Pinchot, C.C. 2014. Restoration of chestnuts as a timber crop in Connecticut. Acta Hort. 1019:17-19.<br /> 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.<br /> 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.<br /> 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.<br /> Craddock, J.H. 2014. A tree crop archetype. Acta Hort. 1019:45-47<br /> Dale, A. and Galic, D. 2014. Breeding blight resistant American chestnut for Canada. Acta Hort. 1019:49-53. <br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> Galic, D., Dale, A. and Alward, M. 2014. Vegetative propagation of American chestnut. Acta Hort. 1019:99-103.<br /> 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.<br /> 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.<br /> Gold, M.A. 2014. Agroforestry. Encyclopaedia Brittanica Online. Encyclopaedia Brittanica Inc.<br /> 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.<br /> Graziosi, I., and Rieske, L.K. 2014. Potential fecundity of a highly invasive gall maker, Dryocosmus kuriphilus (Hymenoptera: Cynipidae). Environ. Entomol. (in press).<br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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).<br /> 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.<br /> Jose, S. and Gold, M.A. 2014. Agroforestry. In Rowe, D. (Ed.) Achieving Sustainability: Visions, Principles, and Practices. 1st Edition. Macmillan Reference, MI, USA.<br /> 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.<br /> 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. <br /> 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.<br /> 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.<br /> 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.<br /> 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.<br /> 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).<br /> 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 (in press) http://www.sciencedirect.com/science/article/pii/S016894521400079X.<br /> Newhouse, A.E., Spitzer J.E., Maynard, C.A. and Powell, W.A. 2014. Leaf Inoculation Assay as a Rapid Predictor of Chestnut Blight Susceptibility. Plant Disease 98:4-9.<br /> Oakes, A.D., Powell, W.A. and Maynard, C.A. 2013. Doubling acclimatization survival of micropropagated American chestnuts with darkness and shortened rooting induction time. J. Environ. Hort. 31:77–83.<br /> Petit, R.J., Carlson, J., Curtu, J., Loustau, M.L., Plomion, C., González-Rodriguez, A., Sork, V. and Ducousso, A. 2013. Fagaceae trees as models to integrate ecology, evolution and genomics. New Phytologist 197:369-371.<br /> Pinchot, C.C., Clark, S.L., Saxton, A.M., Schlarbaum, S.E., Schweitzer, C.J. and Hebard, F.V. 2014. Impact of silvicultural treatment on chestnut seedling growth and survival. Acta Hort. 1019: 191-197.<br /> Rieske, L.K. 2014. Accumulation of natural enemies by the Asian chestnut gall wasp in North America. Acta Hor. 1019:205-209.<br /> Sisco, P.H. Hebard, F.V., Neel, T.C., 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. 1019:215-222.<br /> Springer, J.C., Davelos Baines, A., Fulbright, D.W. Chansler, M.T. and Jarosz, A.M. 2013. Hyperparasites influence population structure of the chestnut blight pathogen, Cryphonectria parasitica. Phytopathology, doi: 10-12-02730-R.<br /> Stevens, D.J., Soltau, P. and Davelos Baines, A., 2014. American chestnut sprout dynamics. Acta Hort. 1019: 223-227.<br /> Warmund, M.R. 2104. Anatomical features of chestnut galls induced by Dryocosmus kuriphilus. Acta Hort. 1019.239-242.<br /> Warmund, M.R. 2014. Disinfestation of Dryocosmus kuriphilus Yasumatsu in Zhangscion wood. Acta Hort. 1019:243-250.<br /> Zhang, D.-X., Lu, H.-L., Liao, X., St. Leger, R.J. and Nuss, D.L. 2013. Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis. Fungal Genetics and Biology 61:1-8.<br /> Zhang B., Oakes, A.D., Newhouse, A.E., Baier, K.M., Maynard, C.A. and Powell, W.A. 2013. A threshold level of oxalate oxidase transgene expression reduces Cryphonectria parasitica - induced necrosis in a transgenic American chestnut (Castanea dentata) leaf bioassay. Transgenic Research 22:973-982.<br /> Zhang, D.X., Spiering, M.J., Choi, G.H. and Nuss, D.L. 2014. Genes associated with Cryphonectria parasitica vegetative incompatibility loci. Acta Hort. 1019:257-261.<br /> Zhang, D.-X., Spiering, M.J., Dawe, A.L. and Nuss, D.L. 2014. Vegetative incompatibility loci with dedicated roles in allorecognition restrict mycovirus transmission in chestnut blight fungus. Genetics 197:701-714.<br /> Zhebentyayeva, T., Chandra, A., Abbott, A., Olukolu, B.A., Jeffers, S.N., James, J.B., Staton, M.E., Hebard, F.V., Georgi, L.L., Sisco, P.H. and Nelson, C.D., 2014. Genetic and genomic resources for mapping resistance to Phytophthora cinnamomi in chestnut. Acta Hort. 1019:263-270.<br /> <br /> <br /> <br />Impact Statements
Date of Annual Report: 12/08/2015
Report Information
Annual Meeting Dates: 09/10/2015
- 09/12/2015
Period the Report Covers: 09/01/2014 - 09/01/2015
Period the Report Covers: 09/01/2014 - 09/01/2015
Participants
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)
Brief Summary of Minutes
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.
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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)
Accomplishments
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. <br /> <br /> <br /> 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.<br /> <br /> 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.<br /> <br /> 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.<br /> <br /> 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.<br /> <br /> 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.<br /> <br /> The University of TN upgraded the hardwoodgenomcis.org website fo the genome and QTL assemblies to JBrowse format.<br /> <br /> One thousand new SNPs were added to the Chinese X Chinese chestnut genetic map by The Southern Institute of Forest Genetics.<br /> <br /> 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.<br /> <br /> Hypovirus strain GH2 was found to efficacious when applied to cankers containing the five most common vc types in Michigan orchards.<br /> <br /> The OTC drug, naproxen, was found to inhibitory to the growth and development of C. parasitica in culture.<br /> <br />Publications
NE-1333 PUBLICATIONS 2014-2015<br /> <br /> 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. <br /> 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.<br /> 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. <br /> ?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.<br /> 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.<br /> 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.<br /> 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.<br /> Fulbright, D.W. and Serdar, Ü. 2015. Turkey: Chestnut blight cankers. J. Amer. Chestnut Found. 29:28-33. <br /> 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.<br /> Gold, M.A. Agroforestry. 2014. Encyclopædia Brittanica Online. Encyclopædia Brittanica Inc.<br /> 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.<br /> 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.<br /> Jose, S. and Gold, M.A. 2014. Agroforestry. In Rowe, D. (Ed.) Achieving Sustainability: Visions, Principles, and Practices. 1st Edition. Macmillan Reference, MI, USA.<br /> 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.<br /> 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.<br /> 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.<br /> Newhouse, A.N. Where there be mountains, there be chestnuts. Ozark Chinquapin Foundation, 14 Mar 2015, Rogers, AR.<br /> 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.<br /> 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.<br /> 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.<br /> Powell, W.A. Where there be mountains, there be chestnuts. Biotechnology Literacy Project, 31 May-4 Jun 2015, Davis, CA.<br /> 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.<br /> 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.<br /> Warmund, M. 2014. Nitrogen fertilization of young Chinese chestnut trees. American Society for Horticultural Science. 28-31 July 2014, Orlando, FL. <br /> 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.<br />Impact Statements
Date of Annual Report: 11/30/2016
Report Information
Annual Meeting Dates: 09/30/2016
- 10/01/2016
Period the Report Covers: 10/01/2015 - 09/30/2016
Period the Report Covers: 10/01/2015 - 09/30/2016
Participants
Brief Summary of Minutes
Accomplishments
Publications
Impact Statements
Date of Annual Report: 10/31/2017
Report Information
Annual Meeting Dates: 09/07/2017
- 09/10/2017
Period the Report Covers: 09/30/2016 - 09/01/2017
Period the Report Covers: 09/30/2016 - 09/01/2017
Participants
Alabama: David and Judi Morris (AL Chapter, TACF)Kentucky: Albert Abbott (University of Kentucky)
Mississippi: Angus Dawe, Didi Ren (Mississippi State University)
New York: Linda McGuigan, Dakota Matthews (SUNY-ESF)
North Carolina: Paul Sisco, Ben Jarrett, Jarrod Westbrook (TACF®, Asheville)
Pennsylvania: John Carlson (chair-elect) (Pennsylvania State University)
Portugal: Rita Costa (INIAV, Oeiras)
South Carolina: Steve Jeffers, Tatyana Zhebentyayeva (Clemson University)
Tennessee: Hill Craddock (Chair), Kirsten Hein, Taylor Perkins (UT Chattanooga)
Virginia: Fred Hebard (TACF®, Meadowview), Laurel Rodgers (Shenandoah University)
West Virginia: William MacDonald, Mark Double, Cameron Stauder (West Virginia University)