Brief Summary of Minutes of Annual Meeting

Milestone Accomplishments

• A thorough analysis of the four parasitica RNA-dependent RNA polymerase (rdr) genes was completed during this reporting period. Disruption mutants were made for each of the rdr genes independently.  Double (rdr1/rdr3 and rdr2/rdr3) and triple rdr1/rdr2/rdr3 mutants were also made to overcome potential problems of functional redundancy (a quadruple mutant was not prepared because rdr4 appears to be a pseudo-gene).  None of the rdr disruption mutants displayed any growth or morphology phenotypes that differed from the wild-type strain either with or without hypovirus infection.  Deletion of the rdr genes also failed to result in detectable changes in transposon expression or hypovirus recombination activity.  We conclude that rdr genes in C. parasitica do not have significant roles in RNA silencing as part of defense responses against mycoviruses or transposons or have a significant role in viral RNA recombination has we have shown previously for dcl2 and agl2.
• A simple and efficient system was developed by adapting the Cre-loxP recombination system for unlimited recycling of the limited number of available selectable marker genes (SMGs). The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi.
• Mutational analyses of the infectious CHV-1/EP713 infectious cDNA clone defined the requirements for autocatalytic cleavage of papain-like leader proteases p29 and p48 and the functional importance of autoproteolysis in the context of hypovirus replication. The studies also exposed an alternative p48 processing pathway independent of the encoded papain-like protease activities.
• In order to effectively determine the vegetative incompatibility genetic structure of parasitica field populations, we designed PCR primer sets 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 isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement 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.
• The identification of vic genes and adaptation of the Cre-loxP recombination system in previous years allowed us to systematicly disrupt multilocus vic genes and excize exogenic genes to generate strains of the chestnut blight fungus able to transmit hypovirulence to strains with genotypic differences at any or all of the defined vic The results demonstrate the feasibility of modulating fungal allorecognition to promote transmission of virulence-attenuating mycoviruese for enhanced biocontrol potential. These “Super Donor” strains are currently being tested in USDA permitted field trials near Grantsville, MD.  Results from a 1-year study involving naturally occurring cankers challenged with super donor strains indicate significantly less canker expansion compared to cytoplasmic hypovirulent isolates (without gene knockouts) and water agar controls.
• A BC₃F₃ orchard was established at the Univeristy Forest in Preston County, WV as a demonstration orchard with public access.
• Over 60,000 American chestnut backcross trees have been planted in TACF’s Meadowview seed orchards since 2002. After inoculation and culling, 5000 trees remain from which to make the final selections of 500 of the most disease resistant trees.
• Version 1.1 of the Chinese chestnut genome is available to the public at the website created and curated by Dr. Margaret Staton at the University of Tennessee-Knoxville. The version 1.1 genome assembly (for TACF cv. Vanuxem) consists of 4 Mb in 41,270 scaffolds, averaging app. 40,000 bp in length.  A total of 36,146 gene models and 38,146 peptide sequences were predicted in the genome.  BAC contigs spanning the 3 blight resistance QTL (identified in the early F2 QTL mapping population) were also sequenced and assembled into 395 scaffolds.
• Leaves from SUNY-ESF’s transgenic trees were used as a foodsource for tadpoles to discern if TG leaves were deleterious to leg development. For all leaf sources (chestnut and non-chestnut), In fact, tadpoles developed fastest on American chestnut leaves, both transgenic and non-transgenic. 

Publications 2016-2017

Cannon, N., Staton, M.E., Addo-Quaye, C., Islam-Faridi, N. Tomsho, L.P., Ficklin, S., Saski, C., Burhans, R., Drautz, D., Zembower, N., Schuster, S.C., Abbott, A.G. Nelson, C.D., Hebard, F.V., Carlson, J.E.  2017. The physical and genetic structure of the Chinese chestnut (Castanea Mollissima) genome. Plant and Animal Genome XXV Conference, San Diego, CA, Jan. 14-18, 2017, abstract.

Case, A., Clark, S.L., and Schlarbaum, S.E. 2016. Threats to American chestnut, butternut, and black walnut and attempts to conservation and resistance. North Carolina Arboretum, 4 Nov 2016, Asheville, NC.

Case, A.E., Mayfield III, A.E., Clark, S.L., Schlarbaum, S.E. and Reynolds, B.C.  2016.  Frequency and abundance of Asiatic oak weevil (Cyrtepistomus castaneus) on American, Chinese, and hybrid chestnut (Castanea) seedlings.  Journal of Insect Science. 16:1–8. DOI 10.1093/jisesa/iew012.

Clark, S.L. and Schlarbaum, S.E. 2017. Reintroduction of the American chestnut: Impacts of traditional breeding and forest management in restoration of an extirpated species. ARS, Society for Range Management, 20 Jan 2017, St. George Utah.

Ćurković-Perica, M., Ježić, M., Karin, V., Idžojtić, M., Sotivorski, K., Risteski, M., Rigling, D., Prospero, S., Kolp, M., and Double, M. 2017.  Biocontrol of chestnut blight: (in)stable infection of the chestnut blight fungus by Cryphonectria hypovirus 1.  7^th Congress of European Microbiologists, Federation of European Microbiological Societies, 9-13 July 2017, Valencia, Spain.  Abstract 713.

Double, M.L., Nuss, D.L., Rittenour, W.R., Holásková, I., Short, D.P.G., Kasson M.T., and MacDonald, W.L.  2017.  Long-term field study of transgenic hypovirulent strains of Cryphonectria parasitica in a forest setting.  Forest Pathology. doi.org 10.1111.efp12367.

Gold, M.A.  2016.  Agroforestry.  Encyclopaedia Brittanica Online.  Encyclopaedia Brittanica, Inc. 

Islam-Faridi N., Majid M.A., Zhebentyayeva T., Georgi L.L., Fan S., Hebard V., Sisco P.H., Westbrook J., Carlson J.E., Abbott A.G., Nelson C.D. 2016. FISH Confirmation of a Reciprocal Translocation in Chestnut. In Cytogenetic And Genome Research 2016 Jan 1, Vol. 148, No. 2-3, pp. 144-144, Allschwilerstrasse 10, Ch-4009 Basel, Switzerland: Karger.

Kolp, M., Double, M., Fulbright, D.W., MacDonald, W., and Jarosz, A.M. 2017.  Fungal community structure in chestnut blight cankers on American chestnut in Michigan and Wisconsin.  Ecological Society of America. 6-11 Aug. 2017, Portland, OR.

Lovell, S.T., Dupraz, C., Gold, M.A., Jose, S., Revord, R., Stanek, E., and Wolz. K. 2017. Temperate Agroforestry Research – Considering Multifunctional Woody Polycultures and the Design of Long-Term Field Trials. Agroforestry Systems doi:10.1007/s10457-017-0087-4.

Nelson, C., Powell, W., Merkle, S., Carlson, J., Staton, M., Nairn, C., Holliday, J., Westbrook, J., Georgi, L., Hebard, F., Zhebentyayeva, T., Jeffers, S., Sisco, P., James, J., and Abbott, A. 2017.  Shovel-ready trees: Forest Health Initiative a model for rapid development and deployment of disease resistant trees. Phytopathology 106 (12):170 (abstract). 

Perkins, M.T., Harris, J., Shaw, J. and Craddock, J.H. 2017.  Chloroplast DNA phylogenetics of the North American Castanea. Southeastern Biology. (In press).

Pinchot, L.C., Schlarbaum, S.E., Clark, S.L., Saxton, A.E., Sharp, A.M., Schweitzer, C.J., and Hebard, F.V.  2017.  Growth, survival, and competitive ability of chestnut (Castanea Mill.) seedlings planted across a gradient of light levels. New Forests. DOI 10.1007/s11056-017-9577-5.

Rhodes, T.K., Aguilar, F.X., Jose, S., and Gold, M.A. 2016.  Factors influencing the adoption of riparian forest buffers in the Tuttle Creek Reservoir watershed of Kansas, USA.  Agroforest Syst.  doi:10.1007/s10457-016-0045-6.

Robinson, A.C. 2016. Measuring Phytophthora resistance phenotypes in segregating testcross families of hybrid American chestnut trees. Honors Thesis, Univesity of Tennessee Chattanooga, Chattanooga, TN.

Steiner, K.C., Westbrook, J.W., Hebard, F.V., Georgi, L.L., Powell, W.A. and Fitzsimmons, S.F. 2017. Rescue of American chestnut with extraspecific genes following its destruction by a naturalized pathogen. New Forests 48:317–336.

Santos, C., Nelson, C.D., Zhebentyayeva, T., Machado, H., Gomes-Laranjo, J., Costa, R.L.  2017. First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi. PLoSONE 12(9): e0184381. https://doi.org/10.1371/journal.pone.018438.

Santos, C., Duarte, S., Tedesco, S., Fevereiro, P. and Costa, R.L.  2017. Expression profiling of Castanea genes during resistant and susceptible interactions with the oomycete pathogen Phytophthora cinnamomi reveal possible mechanisms of immunity. Frontiers in Plant Science, doi: 10.3389/fpls.2017.00515.