Brief Summary of Minutes of Annual Meeting

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

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

Objectives of NE1833:

Objective 1: Develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome.

Objective 2: Evaluate biological approaches for controlling chestnut blight from the ecological to the molecular level by utilizing knowledge of the fungal and hypovirus genomes to investigate the mechanisms that regulate virulence and hypovirulence in C. parasitica.

Objective 3: Investigate chestnut reestablishment in orchard and forest settings with special consideration of the current and historical knowledge of the species and its interaction with other pests and pathogens.

Reports:

Sandra L. Anagnostakis, The Connecticut Agricultural Experiment Station

Topic: Breeding Chestnut Trees for Gall Wasp Resistance (all Objective 1)

Summary:

• Dryocosmus kuriphilus has distribution across range of chestnut with new detection in CT in 2011
• Breeding efforts are underway using C. sativa, C. ozarkensis, C. pumila, and C. henryi
• First crosses, commercial trees x C. henryi, 2011; Second crosses, commercial trees x C. ozarkensis, 2013
• Crosses showing promise (starting to see growth beyond the galls)

Fred V. Hebard, TACF

Topic: The “what’s left from Chinese” question: can enumerating what remains from Chinese chestnut in the genome of selected B3-F2s help characterize genes for blight resistance? (all Objective 1)

Summary:

• Current Clapper BC₃F₂ selections range from 0% to 39% Chinese. Median 13% Chinese
• It is only this year that chromosome-level DNA sequences became available for chestnut. The data were voluminous, millions of data. The markers were distilled to about 5000 loci, depicted here.
• To explore the usefulness of testing the “What’s left from Chinese” hypothesis on the full dataset
  • Only seven of 163 B3-F2s had more than one locus homozygous Chinese.
  • Forty-seven of 163 selections were homozygous Chinese on at least one locus among the five gut chromosomes.
  • Ranking by the mean frequency of American alleles across the most Chinese locus on each chromosome spotlighted five putative loci for blight resistance that had shown signal in previous QTL searches.
• The comparison of expected to observed genotype frequencies yielded suggestions of dominant and recessive gene action.
• The great virtue of this approach is that it depends solely on data intrinsic to DNA sequence. It does not depend on measurements of blight resistance
• Backcrossing was key to removing irrelevant loci.

Brad Hillman, Rutgers University

Topic: Viruses and transposons of Cryphonectria parasitica, the chestnut blight fungus (all Objective 2)

Summary:

• At least three independent horizontal transmission events were responsible for the four known hypovirus species of C. parasitica
• Infection of the reovirus MyRV2 from West Virginia C. parasitica strain C-18 is stabilized by coinfection with the hypovirus CHV-4
• The C. parasitica mitochondrial virus CpMV1 can be introduced into different fungal strains, species, genera and families by protoplast fusion, is stable in many but not all
• Through RNAseq and PCR, we examined expression of the 9.2 kb C. parasitica helitron transposable element, and found that only the 5’-terminal 0.8 kb of the element appears to be expressed in culture

Linda McGuigan and Kristen Stewart, SUNY ESF

Topic: Transgenic American chestnut Update (all Objective 1)

Summary:

• Laccase-like gene from Chinese chestnut (cisgene); Laccase-like gene expression much higher in resistant Chinese chestnut vs. the susceptible American chestnut
• Phytophthora Resistance Genes?
• Quick Leaf Assay Survey of T2 offspring
• Initiating Ozark Chinquapin Embryos in Tissue Culture
• New Ozark chinquapin embryo lines have been established in tissue culture for future transformations
• First transformation with embryos from OK is complete
• Optimizing multiplication, rooting, and acclimatization of Ozark chinquapin in anticipation of a transgenic OC
• Use genetic engineering to develop American elms with resistance/tolerance to:
  • Dutch elm disease
  • Elm yellows
  • Phytophthora
  • Other vascular wilt diseases

Steve Jeffers, Clemson

Topic: NE1833 Chestnut Research at Clemson University

Summary:

• Focus at Clemson: Phytophthora root rot (PRR)
  • PRR is the other major disease that kills American chestnut (Objective 2)
• Collaborating with TACF to screen backcross hybrid chestnut seedlings for resistance to Phytophthora cinnamomi (Objective 1)
• Evaluating the virulence of different populations of P. cinnamomi to hybrid chestnut seedlings (Objective 1)
• Excised twig assay to identify resistance in hybrid chestnut trees
• Detection of Phytophthora spp. in soils where chestnut are growing or might be planted (Objective 2)
• Efficacy of oomycete-specific fungicides for Phytophthora root rot (PRR) on American chestnut seedlings (Objective 2)

Angus Dawe, Mississippi State University

Topic: Mississippi Report

Summary: (all Objective 2)

• Description of predicted LysM proteins that play a role in regulation of growth and development in C. parasitica
• Genome sequencing of 90+ progeny from crosses of two standard C. parasitica strains, SG2-3 x EP155

Amy Metheny and Matt Kasson, WVU

Topic: WVU Report: Super Donor 2.0 (all Objective 2)

Summary:

• Application method and hypovirus both impact biocontrol efficacy using field deployed super donor strains in a forest setting

Paul Sisco, Carolinas Chapter - TACF

Topic: Results of Using F1’s as controls in the Carolinas Chapter Seed Orchard

Summary:

• We would like Restoration Chestnut trees to be as least as blight resistant as F1’s (Objective 3)
• Male-sterile F1’s will not contaminate seed orchard (Objective 3)
• New sources of resistance can be introduced by collecting seed of F1 x B3F2 trees selected for blight resistance “Better B1 trees” (Objective 2)
• Phytophthora resistance can be added by collecting seed of F1 x B3F2 selected for blight resistance “Better B1 trees” (Objective 2)

Tom Saielli, TACF

Topic: Engaging Citizen Scientists to help find surviving American chestnut

Summary:

• The more trees that are found, the more trees that can be used in the (3BUR) breeding program (Objective 1)
• To preserve native germplasm (in GCO’s) (Objective 1)
• Harvest open-pollinated American chestnut seeds (Objective 3)
• Cultural significance, educational opportunities, fun and interesting (Objective 3)

Jared Westbrook, TACF

Topic: Genome-wide ancestry inference in American chestnut backcross hybrids
With application for mapping loci associated with resistance to Phytophthora cinnamomic

Summary:

Discover regions of BC₃F₂ mother trees’ genomes associated with variation in mortality of their BC₃F₃ after infection with P. cinnamomic (Objective 1)

Rita Costa, INIAV

Topic: Understanding the Interaction of Phytophthora cinnamomi Rands with Castanea spp.

Summary: (all Objective 1)

• A breeding program for resistance of chestnut to Phytophthora cinnamomi was initiated in 2006, based on controlled crosses, using the Asian resistant species, C. crenata and C. mollissima, as donors of resistance and C. sativa as female parent.
• The progenies were root phenotyped through inoculation with the pathogen.
• Under the scope of the research program, candidate genes and QTLs were identified.
• The comprehension of the host-pathogen system in Castanea spp using histopathology is being performed and the functional validation through genetic transformation is being implemented
• The ultimate goal is to select molecular markers linked with resistance genes to expedite selection of genotypes with improved resistance to the pathogen

Tetyana Zhebentyayeva, John Carlson, Penn State

Topic: Update on Genetics of Resistance to Phytophthora cinnamomi in Chestnut: an Integrated Genetic and Genomic Approach for Candidate Gene Discovery Within QTL Interval

Summary:

• Examination of quantitative trait loci for resistance to Phytophthora cinnamomi (Objective 1)
• Update on genome sequencing of Castanea mollissima and Castanea dentata (Objective 1)

Dana Nelson, University of Kentucky

Topic: QTL Mapping Resistance to Cryphonectria parasitica in Chinese × American Chestnut Hybrid Families

Summary:

• Description of recent progress with mapping of quantitative trait loci for resistance against Cryphonectria parasitica in Castanea mollissima X Castanea dentata progeny (Objective 2)

Andrew Jarosz, Michigan State University

Topic: Michigan report

Summary:

• Perspective from the Midwest Chestnut Producers Council (Objective 3)
• Broad/Preliminary look at hypovirulence treatments from 2013 to 2019 at two large commercial orchards in Michigan.  (Objective 2)
• Utilizing tree ring analyses to determine the age of chestnut blight cankers. (Objective 2)

Westbrook, J.W., James, J.B., Sisco, P.H., Frampton, J., Lucas, S. and Jeffers, S.N., 2019. Resistance to Phytophthora cinnamomi in American chestnut (Castanea dentata) backcross populations that descended from two Chinese chestnut (Castanea mollissima) sources of resistance. Plant disease, 103(7), pp.1631-1641.

Zhebentyayeva, T.N., Sisco, P.H., Georgi, L.L., Jeffers, S.N., Perkins, M.T., James, J.B., Hebard, F.V., Saski, C., Nelson, C.D. and Abbott, A.G., 2019. Dissecting resistance to Phytophthora cinnamomi in interspecific hybrid chestnut crosses using sequence-based genotyping and QTL mapping. Phytopathology, 109(9), pp.1594-1604.

Perkins, M.T., Robinson, A.C., Cipollini, M.L. and Craddock, J.H., 2019. Identifying host resistance to Phytophthora cinnamomi in hybrid progeny of Castanea dentata and Castanea mollissima. HortScience, 54(2), pp.221-225.

Stauder, C.M., Nuss, D.L., Zhang, D.X., Double, M.L., MacDonald, W.L., Metheny, A.M. and Kasson, M.T., 2019. Enhanced hypovirus transmission by engineered super donor strains of the chestnut blight fungus, Cryphonectria parasitica, into a natural population of strains exhibiting diverse vegetative compatibility genotypes. Virology, 528, pp.1-6.

Staton, M., Addo-Quaye, C., Cannon, N., Sun, Y., Zhebentyayeva, T., Huff, M., Fan, S., Bellis, E., Islam-Faridi, N., Yu, J. and Henry, N., 2019. The Chinese chestnut genome: a reference for species restoration. bioRxiv, p.615047.

Perkins, M.T., Zhebentyayeva, T., Sisco, P.H. and Craddock, J.H., 2019. Genome-wide sequence-based genotyping supports a nonhybrid origin of Castanea alabamensis. BioRxiv, p.680371.

Sato, Y., Miyazaki, N., Kanematsu, S., Xie, J., Ghabrial, S.A., Hillman, B.I. and Suzuki, N., 2019. ICTV Virus Taxonomy profile: megabirnaviridae. Journal of General Virology, 100(9), pp.1269-1270.

Aulia, A., Andika, I.B., Kondo, H., Hillman, B.I. and Suzuki, N., 2019. A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing. Virology, 533, pp.99-107.

Shahi, S., Eusebio-Cope, A., Kondo, H., Hillman, B.I. and Suzuki, N., 2019. Investigation of host range of and host defense against a mitochondrially replicating mitovirus. Journal of Virology, 93(6).

Crouch, J.A., Dawe, A., Aerts, A., Barry, K., Churchill, A.C., Grimwood, J., Hillman, B.I., Milgroom, M.G., Pangilinan, J., Smith, M. and Salamov, A., 2020. Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen. Phytopathology, pp.PHYTO-12.

Mcguigan, L., Chartrand, A., Northern, L., Russell, K., Powell, W. and Maynard, C., 2019, August. Use of a Temporary Immersion Bioreactor System to Transform American Chestnut Somatic Embryos. In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT (Vol. 55, No. 4, pp. 483-484). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.

Pilkey, H.C., McGuigan, L.D. and Powell, W.A., 2019, August. Genetic Transformation of the Ozark Chinquapin (Castanea ozarkensis). In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT (Vol. 55, No. 4, pp. 487-488). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.

Brown, A.J., Newhouse, A.E., Powell, W.A. and Parry, D., 2019. Comparative efficacy of gypsy moth (Lepidoptera: Erebidae) entomopathogens on transgenic blight‐tolerant and wild‐type American, Chinese, and hybrid chestnuts (Fagales: Fagaceae). Insect Science.

Powell, W.A., Newhouse, A.E. and Coffey, V., 2019. Developing blight-tolerant American chestnut trees. Cold Spring Harbor Perspectives in Biology, 11(7), p.a034587.

Oakes, A.D., Pilkey, H.C. and Powell, W.A., 2019, June. Improving Ex Vitro Rooting and Acclimatization Techniques for Micropropagated American Chestnut. In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL (Vol. 55, pp. S71-S71). 233 SPRING ST, NEW YORK, NY 10013 USA: SPRINGER.

Goldspiel, H.B., Newhouse, A.E., Powell, W.A. and Gibbs, J.P., 2019. Effects of transgenic American chestnut leaf litter on growth and survival of wood frog larvae. Restoration Ecology, 27(2), pp.371-378.

Cipollini, M., Wessel, N., Moss, J.P. and Bailey, N., 2019. Seed and seedling characteristics of hybrid chestnuts (Castanea spp.) derived from a backcross blight-resistance breeding program. New Forests, pp.1-19.