Source: https://www.nimss.org/projects/view/mrp/outline/18408
Timestamp: 2019-04-21 08:21:08+00:00

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Chestnut blight, incited by Cryphonectria parasitica (Murr.) Barr, devastated the American chestnut tree (Castanea dentata(Borkh.) Marsh) in the first half of the 20thcentury, killing approximately 4 billion dominant and codominant trees in the hardwood forests of the eastern United States. Prior to blight, the tree had many uses, producing sawtimber, poles, posts, fence rails, cord wood for fuel, paper and tannin extraction, and nuts for humans, livestock and wildlife. It also can be characterized as a member of our charismatic megaflora; many people mourn its loss and participate in citizen-science projects to restore it. Restoration of the American chestnut would be a beacon of light and hope shown by science in the face of continuing environmental degradation due to the advent of industrial and now postindustrial economies and the accompanying influx of exotic pests.
The United States Department of Agriculture (USDA), in cooperation with state and private agencies, began work in the 1910s to restore the chestnut tree after recognition it would be destroyed by blight. As part of their work, exotic species of Castanea were introduced, which has resulted in a nascent orchard industry in numerous states from coast to coast in the US. Although the aggregate production of edible chestnuts is still too small to be tallied separately by the USDA, in 2015, the United States had 919 farms producing chestnuts on more than 3,700 acres. The states with the most chestnut acreage were Michigan, Florida, California, Oregon, Virginia, and Iowa.Most of those trees are not afflicted by blight, but are affected by other pests, which need management. Additionally, cultivation techniques for the trees are required, and infrastructure to process and market chestnuts needs further development. NE-1333 members have done research and obtained funding to address these needs and have formulated extension recommendations.
The NE-1333 project and its predecessors have been the central organization coordinating chestnut research since 1982. Members span numerous disciplines in plant sciences, and the annual meeting provides an opportunity for members to be exposed to this diversity. NE-1333 has provided a forum for new and established researchers to develop collaborative relationships and to share resources and expertise. NE-1333 meetings are well attended, and about 30 presentations are typically made by participants each year. International visitors and collaborators are often included in these presentations, and two international symposia have been organized and hosted by NE-1333. Numerous multi-state and international research efforts have been undertaken by NE-1333 members. The project was initiated to explore the diversity of hypoviruses and their efficacy for controlling blight on American chestnut at different locations in its natural range. That original goal persists, but range-wide studies additionally include breeding and evaluation of disease-resistant progeny as well as studies of orchard chestnuts for nut production. An additional activity requiring a multistate effort has been development of genomic tools for Castanea.
The NE-1833 project comprises three objectives: 1) develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome; 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; and 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.
Objective 1: Develop and evaluate disease-resistant chestnuts for food and fiber through traditional and molecular approaches that incorporate knowledge of the chestnut genome.
Two diseases of particular interest are chestnut blight and Phytophthora root rot. Resistance is being addressed on the one hand through breeding, supported by genetic mapping and development of genomic selection as well as metabolomic analysis to facilitate selection; and on the other hand, by transformation, including both trans- and cis-genetic approaches. Development of genomic tools, including an assembled sequence, supports both breeding and genetic engineering approaches.
Blight resistance has been backcrossed from oriental into American chestnut. Seed orchards with an aggregate inbreeding effective population size (Nei) of about 50 have been producing progeny since 2006. The Neiis predicted to increase to 300 as satellite breeding programs in 14 states progress. The satellite programs have progressed to planting seedling seed orchards. Selection for blight resistance within the two original seed orchards is still incomplete. It is expected to be complete in 5-10 years. Once selection is complete, blight resistance is predicted from analysis of orchard progeny tests to be midway between Chinese and American chestnut. Forest progeny tests were begun in 2009; they have not matured enough to evaluate the severity of naturally occurring blight. However, growth rates of backcross families overlapped with that of families of pure American chestnut, and were significantly better than growth rates of Chinese chestnut families.
Resistance to Phytophthora root rot (PRR), incited by P. cinnamomi, occurs in advanced (intercrosses of third backcrosses) lines derived from the Graves source of blight resistance. Heritability of resistance to PRR is high enough that it should be possible to fix it while retaining Nei and blight resistance.
Techniques were developed to regenerate chestnut plants from embryo cultures, which was a significant accomplishment for this difficult-to-root hardwood. A gene from wheat encoding oxalate oxidase conferred blight resistance on plantlets and greenhouse seedlings when transformed into American chestnut. Transformant events are being propagated to tree size for further characterization of blight resistance and for increase into American and backcross chestnut populations. The oxalate oxidase gene is heritable. Regulatory approval is being sought to release genetically transformed trees into the wild. A thorough pipeline has been developed for producing trees from embryonic cultures.
Integrated physical and genetic maps of Chinese chestnut were prepared and blight resistance mapped to three quantitative trait loci (QTL) in a small set of Chinese x American F2 progeny. BAC contigs covering the three blight resistance QTL were deeply sequenced, assembled into scaffolds and genes identified. Several candidate genes for bight resistance from the QTL have been transformed into American chestnut but no plantlets have been evaluated for blight resistance yet. Two QTL for PRR resistance have been detected using Restriction site-Associated DNA Sequencing (RAD-Seq). This accords with the single factor of high heritability predicted by classical analysis. The sequence variation responsible for blight resistance is also being characterized by RAD-Seq and sequence capture.
Version 2 of the Castanea genome sequence has 14,100 total scaffolds, of which the 5,745 largest were anchored to the integrated genetic-physical map, to produce aset of 12 pseudo-chromosome sequences representing the 12 linkage groups and providing 798 Mbp (98%) of genome coverage. Predicted gene positions are being transferred over to the pseudo-chromosomes, as well as the previously assembled blight-resistance-QTL sequences. Long-read PACBio sequences are being used for further validation of chromosome sequence order and for gap closing. To test the value of the chestnut reference genome for use in genetic variation studies and in Genome-Wide-Selection in breeding programs, 10X depth sequence data were produced for 21 Castanea genotypes. Genomic selection, including selection for major QTL for blight resistance, may be extremely helpful in speeding up selection in seed orchards, which currently requires progeny tests in addition to phenotypic testing. That selection may also be facilitated by more rapid screens for blight resistance and by metabolomic analyses.
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.
Chestnut blight appears to have been controlled by naturally occurring hypoviruses on C. sativain Europe but not on C. dentatain North America, except in specialized settings. Research by NE-1333 members and their European colleagues contributed to the view that control in North America was hampered by the much larger number of strains of C. parasiticain different vegetative compatibility groups than occurred in Europe. Other factors hampering control in North America versus Europe may have included greater competition from other hardwood species, greater susceptibility to blight in C. dentata and differing forest management practices.
The RNA sequence of an hypovirus was first determined by members of NE-1333, and a number of species of virus were found based on sequence analysis. Viruses in families other than the Hypoviridae, including mitochondrial plasmids, were found infecting C. parasitica,some associated with reduced virulence and biocontrol. Transformation of C. parasiticawith cDNA of Cryphonectria hypovirus 1 resulted in transmission of the DNA in ascospores and regeneration of RNA viruses in progeny. This completed Koch's Postulates for the hypovirus. Unfortunately, while transformed fungus strains could produce progeny that infected adjacent chestnut trees with hypovirus-containing C. parasitica,disease remission did not occur in the general populations.
Regarding the molecular basis of hypovirulence, NE-1333 researchers found fungal genes involved. Their protein products were components of complex signaling or response mechanisms critical for basic cellular functions, for example, G-protein signaling pathway components. Additionally, hypovirus-infected mycelium often failed to transition to lipid metabolism. A contributing factor to the failure might have been sequestration of lipids necessary for hypoviral replication.
Strain Ep155 of C. parasiticawas crossed with a European strain and six vegetative compatibility loci, known as Vic genes, were genetically mapped in the progeny. The DNA of strain Ep155 of C. parasiticawas sequenced and the European strain resequenced. The six Vic genes were identified and a "super donor" strain prepared with five inactivated Vic genes (four Vic genes were knocked out). The super donor strain should be able to transmit hypoviruses to strains with any combination of Vic genes. The strain is being tested in the forest for disease control.
Strain Ep155 has very high pathogenicity. It is used to screen chestnut trees for blight resistance in combination with a virulent strain of low pathogenicity known as SG2-3. The two strains were crossed and 96 progeny evaluated for pathogenicity. It is hoped that the progeny can be resequenced and QTL for pathogenicity identified by knockout. This should help lead to further understanding of mechanisms of pathogenicity in the fungus. The mechanisms of virulence reduction by hypoviruses in the fungus also remain an active area of investigation, as do other aspects of virus activity in the fungus. Reannotation of the fungus genome is almost complete, which should facilitate transcriptomics analyses and the above studies.
Blight cankers on chestnut are perennial and have been observed to persist more than 40 years. A rather complex community develops in cankers, especially as they age. NE-1333 members have documented numerous species of invertebrates and microorganisms in cankers. The blight fungus itself becomes a host for various viruses and similar entities, and multiple strains can be isolated from cankers. The development of these communities and association of their composition with canker longevity is a promising area for metagenomic investigation.
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.
In addition to the activities discussed under Objectives 1 and 2 above, research is ongoing on gall wasp, silvics, juvenile versusadult chestnut blight resistance, genetic variation in American chestnut, and integrating resistance with hypovirulence to control blight, inter alia.
It has been found that introduced and native parasites of the gall wasp control the pest after the first few years of infestation. Despite a plant quarantine, Michigan is now in the third year of gall-wasp infestation. While nut harvests are markedly decreased during those first few years of infestation, insecticidal treatments also would destroy the parasites, so the recommendation is to NOT spray insecticides to control gall wasp. Dispersal of parasites is recommended, but none are being produced currently. There have been efforts to bring parasite-infested boughs to new areas of gall-wasp infestation, to introduce parasites earlier than occurs naturally. There is variation between cultivars in their susceptibility to gall wasp.
In general, silvicultural evaluations of American chestnut in several states have found that it is a very rapid grower, frequently much faster than oak and walnut. Chestnut growth varies with site, like most hardwoods, so it is not faster than oak and walnut on all sites. Earlier research found that exotic chestnut species do not grow well in native forests, unlike American chestnut. This finding was part of the motivation leading to the proposal to backcross resistance from exotic into American chestnut.
The results of inoculating young seedlings of the three Chinese chestnut species do not match up with blight severity on mature specimens of the three species in China; this result needs more detailed experimental evaluation. Low levels of blight resistance occur in a few American chestnut trees. Intercrossing of these to enhance that resistance has been pursued for a long time. In combination with hypoviruses, impressive levels of blight control have been observed on some pure American chestnut with low levels of resistance. The hypothesis that hypoviruses coupled with resistance in backcross progenies will diminish blight severity is being evaluated.
Castanea species native to the U.S. (dentata, pumila var. pumila,and pumila var. ozarkensis), Castanea species imported from elsewhere (crenata, mollissima, henryi, seguinii, and sativa), and Castanea hybrids are maintained and studied by NE-1033 scientists and their citizen-scientist collaborators. These trees are in: Alabama, California, Connecticut, Delaware, Florida, Georgia, Indiana, Kansas, Kentucky, Louisiana, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, New Jersey, New Hampshire, New York, North Carolina, Ohio, Pennsylvania, Rhode Island, South Carolina, Tennessee, Vermont, Virginia, West Virginia, and Wisconsin. Strains of C. parasitica are shared by members of NE-1333, and important strains are deposited with the American Type Culture Collection. Strains with genetic markers are available, and information on the genetic determinants of vegetative incompatibility (vic genes) is available for use in population studies. Hypovirus types from France, Italy, MI, WV, KY, and China are studied and shared by NE-1033 members.
From January, 2013, to November, 2017, NE-1333 members collectively published 55 peer-reviewed technical articles, 5 Ph.D. Dissertations, 8 M.S. Theses, 1 undergraduate honors thesis, 4 book chapters, 1 proceedings volume, 39 proceedings articles, 9 articles in popular journals, 2 encyclopedia articles, and 16 abstracts. Members are renowned for their work on chestnut, Cryphonectria, and fungal viruses. Venues for presentations included the Plant and Animal Genome Conference, the American Phytopathological Society, the Mycological Society of America, the Ecological Society of America, the Society of American Foresters, various venues of the International Union of Forest Research Organizations, The American Chestnut Foundation (TACF), Encyclopedia Britannica, International Plant Protection Congress, the Entomological Society of America, and the American Society for Virology. The results of research have been extended to growers, especially in Pennsylvania, Michigan and Missouri, and to volunteer citizen scientists in 21 states guided by TACF and the American Chestnut Cooperators' Foundation (ACCF).
In recognition of these successes, NE-1333 received the ESS Excellence in Multistate Research award in 2010 (NE-1333 was then known as NE-1033). NE-1333 has met the milestones detailed in the project description and will continue to work on similar collaborative projects in the next 5 years. Data generated under the auspices of the NE-1333 project have been used by members to gain intramural and extramural funding for all aspects of chestnut biology and restoration.
In summary, the NE-1333 project is a productive group of collaborators that has provided new and meaningful information to all clients interested in chestnut biology and restoration, from the bench scientist to the professional orchardist and to the individual volunteer grower of chestnut for restoration. In the next 5 years, we will continue to pursue collaborative projects under our 3 stated objectives. This will lead to increased production of chestnuts in orchards and will further restoration of the iconic American chestnut. Great progress has been achieved toward restoration and continued research efforts are needed to guide it to completion.
The Connecticut Agricultural Experiment Station (CAES) has trees of all of the species and most of the potential hybrids of chestnut. These plantings are available to researchers (Anagnostakis 2012). Initial identification of the vegetative incompatibility system in C. parasitica was done at CAES, and tester strains of the pathogen have been made available to researchers throughout the US (Anagnostakis, 1977). C. ozarkensiswas included in the CAES breeding program, both for production of blight-resistant chinquapins and for use of the Asian chestnut gall wasp (ACGW) resistance of this species in timber and orchard breeding. Preliminary results with hybrids resulting from crosses of species resistant to ACGW indicate that this resistance is not simply inherited.
TACF has been backcrossing blight resistance fromC. mollissima into C. dentata since 1984. Steiner et al (2017) reviewed progress to date (and defines terminology for various chestnut crosses, such as B3). Currently, two B3F2 seedling seed orchards derived from the Graves and Clapper sources of blight resistance are about five years from completion of selection. Additional seed orchards are being developed at ten state chapters and include additional sources of blight resistance. The Nei of these progeny is expected to be 300. After selection, we estimate B3F3 progeny from the first two seed orchards will be equal in blight resistance to that of F1s between C. mollissima andC. dentata, rather than equal to that of C. mollissima.
Progeny testing is required to select B3F2 parents, even to detect individuals homozygous for major QTL for resistance, let al one minor QTL. This is a daunting task. TACF plans to expedite it using genomic selection.
Genotyping-by-sequencing of 480 Graves B3F2 progeny yielded 20,000 SNPs which were used to estimate genomic prediction accuracy of canker severity ratings. When seed orchard block, year of inoculation, and kinship relations were included in the model, median SNP predictive ability was 0.6 (Westbrook and Holliday, unpublished).
Genomic prediction was performed with 47 B3F2 mother trees whose heritable genetic blight resistance was estimated from canker severity after inoculation of 11 to 30 B3F3 with weak and strong strains of C. parasitica. Genomic predictive abilities were approximately 0.55 and 0.4 for the two strains.
For Phytophthora root rot (PRR) resistance, the predictive ability of genomic selection was 0.65 when roots were rated but only 0.45 for above-ground wilting. The predictive ability for PRR was encouragingly high despite the small training populations and is expected to increase when more progeny-tested trees are genotyped. Encouragingly, PRR resistance occurs in about a quarter of Graves B3F2 lines.
Clemson University. PRR was first noticed on C. dentata in 1856 (Crandall and Gravatt 1945, Rumbold 1911). Milburn and Gravatt (1932) noted that PRR was widespread in the southern states, and speculated that the “dying and recession” of southern C. dentata between 1830 and 1930 may have been due to PRR. Milburn and Gravatt isolated a species of Phytophthorafrom blackened roots of dying C. dentata. They tentatively but erroneously identified it as P. cambivora. It was subsequently identified as P. cinnamomi by Crandall et al (1945). Up until recently, P. cinnamomi was the only Phytophthora speciesreported to cause root rot on C. dentata. Currently, the widespread distribution of P. cinnamomi in southern soils is adversely affecting reintroduction of TACF backcross hybrids (Clark et al 2014, 2016; Pinchot 2017).
To map PRR resistance genes, Clemson is cooperating with the Forest Health Research and Extension Center (FHC) of theUniversity of Kentucky (UK) in the genotyping by sequencing of large TACF backcross families. Two resistance QTL map to one arm of linkage group E (Zhebentyayeva et al, in preparation).
During the past few years, the University of Georgia (UGA) has been working with Clemson to develop an in vitroscreen for P. cinnamomi resistance. Based on a recent report by Santos et al (2014) that used excised chestnut shoots for PRR resistance screening, we designed an in vitroapproach that employs micropropagated shoots from our chestnut cultures in water agar (actually water-Phytagel) inoculated with a plug of P. cinnamomi. Shoots of susceptible C. dentata clones became darkened and leaves wilted within a week of inoculation. While we lackedC. mollissima shoots to use as controls, we found it intriguing that transgenic C. dentata shoots engineered with a P. cinnamomi resistance candidate genes (RPH and NPR3/4), remained green and unwilted for several days after the wildtype shoots darkened. We recently obtained shoot cultures of C. mollissima from SUNY-ESF, and these will be added to the screens as resistant controls. Our next step is to apply the in vitroassay to B3F3 material that has inherited the PRR resistance of C. mollissima.
The University of Tennesseeat Chattanooga (UTC) will use grafting to increase the number of naturally occurring C. dentata in the ongoing TACF breeding program. Scions will be bench grafted, then grown under high temperature and photoperiod conditions in a greenhouse to expedite flowering, increasing the ease of pollen collection. Seeds also will be collected from clones transplanted outside in order to capture cytoplasmic genes. Target areas for collection are located at the southernmost fringe of the C. dentata range, with particular focus on disjunct populations in Alabama, Mississippi, and Middle and West Tennessee, which have been shown to harbor high levels of genetic diversity and possibly rare alleles not found in other parts of the species’ range (Gailing and Nelson 2017).
The SUNY College of Environmental Science & Forestry (SUNY-ESF) genetically engineered (GE) a blight-resistant C. dentata (Steiner et al 2016, Newhouse et al 2014b, Nelson et al 2014, Zang et al 2013, and Powell et al unpublished). This was accomplished by adding a gene from wheat encoding an oxalate oxidase enzyme. Seedlings are blight resistant when this enzyme is highly expressed in the stems of C. dentata. These GE C. dentata, once approved by Federal regulators, will augment the restoration of this species and may represent a template for using GE for the rescue of other threatened species.
The use of GE to rescue a “wild” plant species has introduced a new paradigm to federal regulators and conservation biologists. In all other GE species, containment of the transgene has been paramount. However for C. dentata, introgression of the transgene into wild populations may help rescue the remaining genetic diversity and possibly the species itself, returning it to its former role in the ecosystem.
Pennsylvania State University (PSU) developed three versions of the C. mollissima cv Vanuxem genome sequence during the past five years, called versions 1.1, 2.0 and 3.0.
For Version 1.1, in addition to shotgun sequencing, the Southern Institute of Forest Genetics (SIFG) led sequencing in great depth of BAC contigs spanning three blight resistance QTL. Atotal of 1,952 genes were predicted within the 3 QTL from which 15 candidate genes for blight resistance were selected, based on gene expression data, for transformation into somatic embryo cultures of' C. dentata (Nelson et al 2014). The version 1.1 sequence also was used as a reference to assess genetic variation among 18 genotypes of seven species from TACF and CAES. The University of Tennessee(UT) led the alignment of parental and B3genotype genome sequences to Version 1.1. Among other findings, the alignment revealed varying extents of transition of the B3genomes towards the C. dentata genome content from backcrossing (Staton et al 2015).
The scaffolds in Version 2.0 were distilled into 12 chromosome-scale sequences, with 95% genome coverage. The order of scaffolds in the “pseudo-chromosome” sequences was largely validated by comparison to the order of thousands of DNA-sequence–based loci in new medium and high-density genetic linkage maps developed by SIFG and TACF using SSRs developed by SIFG, SNPs developed by UT from the Version 2.0 sequence and SNPs developed by TACF and Virginia Tech (VT) from Restriction site Associated DNA Sequencing (RAD-Seq)(Carlson et al 2017). The Version 2.0 scaffold orders were also validated by fluorescent in situ hybridization (FISH) (Islam-Faridi et al 2015). Using FISH, SIFG and PSU also collaborated in documenting translocations in Castanea.
Version 3.0 is still under development. It was based on very long PACBio sequence reads. New pseudo-chromosome sequences were assembled using all 12,684 of the contigs (not scaffolds!) in the assembly, bringing the project closer to a whole genome reference tool for the community.
Hypovirus infection of C. parasitica often reduces canker expansion rates (Anagnostakis & Waggoner 1981). Heiniger and Rigling (1994) postulated that hypovirus infection led to recovery of many stands of C. sativa. Widespread recovery has not occurred in the U.S. on C. dentata despite 45 years of effort. Several factors have been proposed to explain this failure, including higher diversity of vegetative compatibility groups in the US (Anagnostakis et al 1986), the extreme susceptibility of C. dentata to blight, strong competition from other tree species in the US (MacDonald & Fulbright 1991; Heiniger & Rigling 1994), and different silvicultural practices (Mittempergher 1978; Griffin et al 2005).
Michigan State University (MSU) found that hypovirus infection of C. parasitica led to recovery of some C. dentata stands (Davelos & Jarosz 2004). However, further modeling suggested that equilibrium has not been attained (Davelos-Baines et al , 2014). Further, the frequency of hypovirus infection can change over time, leading to the decline of large trees (Springer et al , 2013).
Chestnut blight cankers are complex communities of interacting cohabitants (vertebrates, arthropods, microbes) that may modulate canker severity. Interactions can be further complicated by infection of C. parasitica by hypoviruses, other viruses and microbes, and by host resistance. We can predict the fate of a canker only at the extremes of these interactions.
Recent data suggest that microbial invaders of cankers may play an important role in canker severity. Invader frequency increases over time in cankers that do not kill the distal stem. In addition, the prevalenceof virulent C. parasitica declines steadily over time while hypovirulent C. parasitica remain at a moderate level (~25%) (Double et al 2018). We hypothesize that these invading microbes play an important role in canker severity and longevity.
Shenadoah University (SU) is comparing endophytic fungus populations in blight-free tissue of susceptible C. dentata to those in resistant C. mollissima and backcross hybrid chestnut. There have been two studies investigating the role of endophytes within Castanea sativa (Washington et al 1999 and Wilhelm et al 1998), but there has been no study of other Castanea spp.
Endophytic fungi are common in healthy trees (Bills 1991; Danti et al 2002), but are a poorly understood component of our ecosystem (Porras-Alfaro and Bayman 2011; Bacon and White 2000). Arnold et al (2003) found that endophytes may influence disease severity, and several endophytes secrete anti-microbial chemicals (Strobel et al 2001).
Studies in pines provide evidence that treatment of seeds and seedlings with endophytes increases resistance to pathogens (Brownbridgeet al 2012; Ganley et al 2008). Similar methods will be used to determine whether inoculation of C. dentata seeds or seedlings with endophytes will increase resistance to C. parasitica.
Rutgers and TACF have mapped LSAs in New Jersey, and identified 14 fungal and 24 bacterial species from cankers on LSAs in New Jersey, either after isolation into pure culture or total DNA extraction from cankers (metagenomics). Metagenomic data to date have not shown quantifiable differences between superficial and non-superficial cankers, so further cultivation and metagenomic analyses will be performed on this population. In related studies, C. parasitica isolatesco-infected with virus and the bacterium Lysobacter enzymogenesare being examined through transcriptome analysis using Illumina RNAseq. The effects of various mutations on the fungus and bacterium are also being studied.
Rutgers has identified viruses representing 7 families, 8 genera, and 12 species in C. parasitica. Most have a measurable effect on fungal growth and virulence (Hillman and Suzuki, 2004; Eusebio-Cope et al , 2015). Characterizations of two novel viruses are underway at Rutgers, strain RC1 from Michigan and strain JS13 from Japan.
Rutgers is examining interplay between C. parasitica transposons and viruses. Complete, active copes of a hAT-like element and several helitron copies, one predicted to be complete and possibly autonomous, are among numerous transposons found in C. parasitica.
West Virginia University. With few exceptions (Yu et al , 2013), mycoviruses have evolved exclusive intracellular lifestyles (Buck, 1986), limiting their transmission to intracellular mechanisms via conidia or anastomosis. Vegetative incompatibility (Vic) systems restrict mycovirus transmission (Boland, 2004; Caten, 1972; Hall et al , 2011; Biella et al , 2002) due to apoptosis triggered when vicincompatible individuals anastamose (Saupe, 2000; Jacobson et al , 1989; Glass et al , 2000).
Through a combination of systematic gene disruption and classical genetics, Zhang and Nuss (2016) developed the SD328/82 super donor (SD) strain, containing gene disruptions at vic1, vic3, vic6and vic7and both alleles of vic2. Under laboratory conditions, SD328/82 was able to transmit hypoviruses to uninfected strains heteroallelic at any viclocus. These strains will be tested for their efficacy in controlling blight onC. dentata in forest environments.
Using RNA-seq data in 2017, Mississippi State University (MissSU) prepared a new annotation of the C. parasitica genome sequence, called V3. One-third of predicted gene products differed from the previous annotation (Ren and Dawe, unpublished). PCR primers were designed to identify differences between potential mRNA products of the two annotations. V3 was correct on 21 occasions (< 80 %) and incorrect in one instance. The remaining four instances generated bands inconsistent with either prediction.
LysM-domain proteins have been shown to play important roles initiating defense cascades (de Jonge et al , 2010; Mentlak et al , 2012). One protein containing a LysM domain was LM12. LM12 functioned similarly to other LysM-containing effectors except that an LM12 knockout strain had a statistically significant increase in canker area compared to controls, opposite from prediction. This suggests that LysM effectors of necrotrophic fungal pathogens or woody stem pathogens, such as C. parasitica,may function very differently than those of biotrophs and hemibiotrophs.
TACF uses two strains of C. parasitica, Ep155 and SG2-3, to screen for blight resistance. They are near the top and bottom, respectively, of pathogenicity for virulent strains. The two strains were crossed and 96 progeny tested, revealing significant variation in pathogenicity. DNA has been extracted from these progeny in preparation for resequencing, identifying SNPs, mapping, and using GWAS to detect genes associated with pathogenic variation. We plan to knock out those genes to assess their function.
A putative orthologue of Neurospora crassa’s vib-1was found in C. parasitica. vib-1 ispart of the transcription cascade leading to apoptosis (Dementhon et al , 2006). We knocked out Cpvib-1to assess its role. The knockout did not change vegetative incompatibility in strain Ep155, but did in strain EU1. EU1 differs from Ep155 at vic4, yet the two strains with the two DCpvib-1knockouts were vegetatively compatible. We conclude that Cpvib-1plays a crucial role in the incompatibility reaction modulated by vic4.
USDA Forest Service, Delaware, OH. Reintroduction of C. dentata will require a tremendous and expensive effort. Studying biotic and abiotic factors affecting reintroduction will help guide decisions on site selection and silvicultural practices (Clark et al 2014, Jacobs et al 2013).
Historically, C. dentata was most abundant on well-drained, sub-xeric to mesic soils and more common on mid- to upper-slope positions (Russell 1987, Wang et al 2013). Its site preferences may have changed over time (Jacobs et al 2013). C. dentata growth after 2 years was greater on mesic than drier sites (Rhoades et al 2009), but longer study times are needed (Pinchot et al 2017). Competition from fast-growing hardwood species on higher quality sites has limited success of oak plantings (Dey et al 2008), and may similarly affect chestnut reintroduction. Available soil moisture also may affect blight severity (Gao and Shain 1995; Griffin 1991). A better understanding of the effects of site quality is necessary to guide site selection.
Several recent studies have tested the effects of various harvest treatments, including shelterwood, thinning, and midstory removal, on the response of subsequently planted chestnut (see Pinchot et al 2017). These studies found a positive relationship between chestnut growth and light availability, however chestnut planted in high-light treatments were out competed by shade-intolerant hardwoods. While chestnut growth is minimal in low light treatments, most studies found no detrimental impact of low light on chestnut survival. Additional testing is needed to develop region-specific guidelines for chestnut reintroduction.
UT will continue helping US Forest Service scientists evaluate cooperative research plantings of chestnut. The plantings were established between 2009 and 2017 and have yielded a number of publications (Case et al , 2016; Clark et al , 2010, 2012, 2014, 2016; Kapp et al , 2014; Pinchot et al , 2011, 2015, 2017). Notably, Clark et al (2016) found that most TACF B3F3 backcross hybrid families overlapped in mean growth rate with most C. dentata families.
There are 27 forest plantings and one field planting under active evaluation of: survival, growth, blight resistance, impact of P. cinnamomi and other pests, importance of seedling quality, competitive ability, breeding generation, effects of site quality, and/or response to different silvicultural treatments. Breeding materials were obtained from TACF, CAES, and ACCF.
UK is investigating the biology, ecology, and host-plant interactions of herbivores with chestnut, in particular the Asian chestnut gall wasp. We have found that gall wasp infestations can be ameliorated by both introduced and native parasitoids. These parasitoids also interact with one another, potentially forming antagonistic relationships (Cooper and Rieske, 2011, Graziosi and Rieske, 2013).
Gall wasps are obligate plant parasites and are affected by microorganisms associated with their host plant. A lesion-forming fungus was isolated that killed gall wasp larval but had no impact on native parasitoids within the gall, on reinoculation. The fungus was classified into a Colletotrichumspp. complex. That complex includes a member inciting blossom end rot in chestnut (Graziosi and Rieske, 2015).
Working with other scientists, CAES will continue the traditional breeding program using stock available in CT. In cooperation with Notre Dame, molecular markers will be sought to identify species of Castaneaand the fraction of each species in hybrids. These results will help identify critical regions of the genome associated with physical and physiological traits. Initial evaluation of nut cultivars will be for taste and nut size, followed by evaluation of the nutritional quality of nuts (Senter et al 1994). Seed and pollen of C. ozarkensisare being provided to CAES by the Ozark Chinquapin Foundation (AR), the Cherokee Nation (OK), and the Nature Conservancy’s Nickel Preserve (OK) to augment the CAES collection for breeding. Back-crossed hybrids of C. ozarkensis will be sent to all cooperators in OK and AR. Orchard and forest plantings will be established in the north eastern US [with C. Pinchot] to investigate optimal silvicultural parameters.
Increasing the speed and accuracy of selection for blight resistance: Currently, 1200 Clapper and 800 Graves trees remain in TACF seed orchards in Meadowview, VA, after initial culling of the most blight-susceptible trees. Many of these trees have not yet been progeny tested, but are being genotyped for genomic selection at Virginia Tech. Additional culling of inferior B3F2 trees will commence when genomic predictions of blight or PRR resistance are available in 2018.
Progeny testing will be accelerated by screening B3F3 seedlings for blight resistance in their first growing with small stem assays (Powell et al 2007). The resistance of trees that have not been progeny tested will be estimated with genomic prediction (Meuwissen et al 2001). Genomic prediction models for blight resistance are being developed separately for Clapper and Graves by genotyping training populations of 300 – 500 B3F2 mother trees from each source that have been progeny tested for blight resistance. Additional gains in blight resistance at B3F3 and beyond may be accelerated by developing high-throughput, non-destructive chemical fingerprinting approaches to predict the blight resistance of seedlings prior to out-planting, in cooperation with the FHC (Conrad et al 2014).
Diversifying transgenic blight-resistant populations:When permits are issued, the C. dentata clone (Ellis1) containing the oxalate oxidase gene will be outcrossed over three generations to 300 C. dentata and 200 backcross parents per generation. The backcross parents will be from the Graves, Clapper and Nanking sources of blight resistance. In the final generation of outcrossing, transgenic progeny will be bred with PRR-resistant Graves B3F2 selections to combine blight and root rot resistance. The 300 pure C. dentata breeding lines will be created by outcrossing transgenic trees to a diverse set of C. dentata sampled from across the species’ range by TACF’s volunteer network of 17 state chapters. Once outcrossing is complete, third generation transgenic outcross progeny will be planted in regional seed orchards where open pollination will be used to generate large quantities of seed for restoration.
Locating wild C. dentata will be expedited by using the TreeSnap cellphone app developed by the UT in cooperation with SIFG. TACF regional science coordinators are actively promoting it to citizen scientists.
At Clemson, over 200 backcross chestnut families were screened for PRR resistance Jeffers et al (2009 and 2012). Resistance has high heritability and is simply inherited in the advanced breeding lines derived from the Graves source of blight resistance.
R. Sharpe (2017 investigated the diversity of Phytophthora spp. associated with PRR on TACF seedlings that were planted in test plots in southeastern forests. Two-hundred, forty cultures of Phytophthora spp. were isolated from over 600 samples. P. cinnamomi (primarily), P. cambivora, P. heveae (infrequently), were recovered from plants and soil. P cryptogea was recovered from just plants at one site, and P quercetorum was recovered from soil at another site. Representative isolates were tested for pathogenicity on C. dentata seedlings. P. cinnamomi was most aggressive, but all spp were capable of inciting disease. Isolates were saved in a permanent culture collection.
In collaboration with TACF, soils have been assayed for Phytophthora spp., primarily in order to test sites as suitable for growing chestnut. Sites with soil positive for Phytophthora spp., especiallly P. cinnamomi and P. cambivora, are deemed unsuitable. Samples have come from 10 eastern states.
These methods will be applied in the future in support of the TACF breeding program.
At UGA, C. dentata and hybrid backcross B3F3 chestnut somatic seedlings will be produced from existing embryogenic culture lines using procedures described in Andrade and Merkle (2005). C. mollissima plantlets will be produced by harvesting shoots from existing proliferating shoot cultures of at least two cultivars and rooting them following the procedure modified from that described by Oakes et al (2013). Once the chestnut plantlets have developed root systems, they will be carefully removed from the germination medium under aseptic conditions and transferred to empty, sterile GA7 vessels that will immediately be filled with 100 ml of molten low-melting-point 1.5% water-agarose, held at 35º C in a water bath to keep it molten. Each plantlet will be held in place with forceps to keep it upright until the gel sets up. A day later, each GA7 will be inoculated with a 3 mm diameter plug of a P. cinnamomi isolate. Inoculated GA7 vessels with chestnut plantlets will be maintained in a lighted incubator at 22º C with 16 hour day-lengths and plantlets will be observed for PRR symptoms, including wilting and darkening of roots and stems. Data to be collected will include the number of days from P. cinnamomi inoculation to appearance of first symptoms and days from inoculation until plantlet death. After 6 weeks, all plantlets will be scored for severity of symptoms using the following scale: 0 = no symptoms, 1 = moderate root or stem lesions/darkening, 2 = extensive root or stem lesions/darkening, 3 = plantlet death. Symptoms will be photo-documented. Since B3F3plantlets are expected to display resistance levels between those of the two pure species, we will attempt to quantify the intermediate levels of resistance. Once we have documented the in vitro resistance/susceptibility of the clones, we will attempt to confirm these results using pot assays in the greenhouse.
At UTC, areas for scion wood collection will be divided into 4 regions – southeast Tennessee/northwest Georgia, south-central Tennessee/northern Alabama, northern-central Tennessee/southwestern Kentucky, and West Tennessee/northern Mississippi. Selection sites will be chosen using GIS prediction models based on bedrock composition, soil type, and dominant land use data. Under normal greenhouse conditions, scion wood collected from naturally occurring C. dentata will be grafted to container-grown C. mollissima, C. dentata, and hybrid rootstocks and grown ex situ under increased temperature (19 C in a heated greenhouse) and photoperiod (artificial lighting, 16 hr day) in order to expedite budburst and floral initiation.
At SUNY-ESF, two experiments are being pursued.
1) In order to increase the genetic diversity and regional adaptation of the GE C. dentata trees, transgenic pollen is being used to fertilize wild C. dentata from across its range, using a fast-breeding system we developed. Fast breeding is accomplished by treating tissue culture plantlets or seedlings from nuts to high doses of light (Baier et al , 2012). In approximately 30% of the trees, this induces pollen formation in less than on year. Half the offspring will inherit the OxO gene and be blight resistant. The first two leaves of seedlings are tested for oxalate oxidase activity with a simple leaf disk assay (Liang et al , 2002). A portion of the selections can be placed in the high-light growth chambers to start the next generation outcrossing. It takes 2 years to turnover generations. We are already producing T1s.
2) We aim to combine OxO blight resistance with PRR resistance and blight resistance genes from C. mollissima. TACF’s breeding program has captured both PRR and blight resistance in some of their Graves B3-F2 trees. GE C. dentata pollen will be used to fertilize the Graves B3-F2 PRR trees. Offspring will be tested for OxO expression and also PRR and blight resistance.
At PSU. the remaining work anticipated for developing the high-quality reference genome for the C. mollissima will involve validating the most recent assembly of chromosome-scale sequences by comparison to multiple high-quality, high-density chestnut genetic linkage maps, being prepared at Clemson University. The structure of the chromosome sequences will then be analyzed for repetitive DNA and gene content. To aid in identifying genes, new RNA sequence data was recently collected from tissues of the Vanuxem reference genotype, which will be mapped to the new pseudo-chromosomes and used with gene-finding bioinformatics programs to develop gene models supported by gene expression data. The genome assembly will also be validated by BUSCO analysis (Felipeet al 2015) which provides a quantitative measure of the assessment of genome assembly, gene identification, and transcriptome completeness, based on completeness of annotation of the known 330 single-copy gene orthologs shared among higher plants. Gene function predictions and classifications will be conducted to permit disease-resistance studies to focus on likely candidate genes. A stable genome assembly will be released as version 3.0, and published in a high-visibility journal.
Three projects are being pursued at MSU.
1) Long-term (20 years) annual monitoring of six C. dentata stands in Michigan is being continued using methods described by Davelos and Jarosz 2004. Edaphic and weather factors are now being investigated to determine correlates with spatial and temporal patterns. One unexpected result of our modeling work was the prediction that blight canker severity may increase over time (Davelos Baines et al 2014). In cooperation with Dr. Eric Eager, our future work will concentrate on analyzing these data using both simulation and mathematical modeling to confirm or refute the predictions of our initial simulation work. Sized-based projection matrices (Lefkovitch 1965) will be constructed for each year-to-year transition and used in the simulations. If these simulations also predict that recovery is not permanent, then sensitivity analyses will be used to evaluate how disease management should be applied (Caswell 2001). Sensitivities indicate which chestnut growth stages would produce the biggest change in population growth, if management protocols were implemented.
2) Spatio-temporal assessment of C. parasitica and hypovirus populations:The C. parasitica and hypovirus populations at six chestnut populations we have utilized for long-term monitoring were assessed in 1996 and 2009 (Springer et al 2013). We will resample all six sites again in 2018 with the aim of determining if further change has occurred. Springer et al (2103) found that some populations were largely invariant over time, while other sites changed in both v/c structure and hypovirus incidence. In particular our Frankfort site displayed an increase in v/c diversity and a decrease in hypovirus incidence in the C. parasitica population at the site. Associated with this change, dieback due to blight was more severe over the past decade than previously (Jarosz, Springer and Davelos Baines, unpublished data).
3) Microbial dynamics within cankers:We began annual isolation of microbes from individual cankers in 2012 to determine how changes in community composition influence the probability that a canker will girdle the infected branch (Kolp and Jarosz, unpublished data). Twenty-four samples, 12 along the canker margin and 12 from the interior of the canker, were collected annually from cankers in three chestnut populations in Michigan. Canker severity was rated on a 1-4 scale at each sampling date. In cooperation with Anita Davelos Baines and Eric Eager, a series of individual-based models (IBMs) models constructed in the summer of 2017 suggested that the amount and direction of changes in C. parasitica canker severity are influenced by variation in the species of invading microbes and the time of invasion. Canker severity was strongly influenced in some instances. We currently are analyzing the data to obtain parameter estimates for the competitive ability of individual microbial species to virulent and hypovirulent C. parasitica (Kolp, unpublished data). We estimate that model development and analyses will require two years of effort.
At SU, bark plugs will be collected from surface sterilized C. mollissima, dentata,and their backcross hybrids in breeding orchards of the Virginia Chapter of TACF, and placed in nutrient-free agar. Any fungi that grow from the bark plugs will be transferred to nutrient agar and maintained in culture. Each unique fungal culture will be identified based on DNA sequencing of fungal ITS regions. We will then evaluate the trees post-inoculation in order to determine the level of resistance each tree has against C. parasitica. The fungal microbiome of trees that demonstrate resistance to C. parasitica will be compared to trees that are not resistant to C. parasitica. If differences are found, we will use the list of fungi found on resistant trees in order to select those to test as a potential bio control against C. parasitica.
At Rutgers, Microbes are studied from cankers located both on LSAs and on C. dentata dying from blight. After subculture, the colony morphology of C. parasitica isolates will be classified as similar to that of virus-containing, hypovirulent colonies, or of virus-free, virulent colonies. All colonies will be saved and associated with the mapped trees from which they were isolated and dsRNA extracted and analyzed (Chung et al , 1994; Linder-Basso et al , 2005; Cai et al , 2013B). A subset of culturable bacteria and fungi that are identified as potentially of interest will be used in co-cultivation experiments with C. parasitica to examine interactions among these organisms in vitro(Kobayashi et al , 1995; Mathioni et al , 2013).
For metagenomics, four samples will be examined, two from superficial and two from non-superficial cankers. Genomic libraries will be made from DNA that has been amplified using universal DNA primers appropriate for bacteria or fungi (Cai et al 2013A; Cai and Hillman, manuscript in preparation). The abundant tools now available for metagenome analysis such as the Integrated Microbial Genomes tools (IMG; http://img.jgi.doe.gov/) and Metagenomics RAST server (http://metagenomics.anl.gov/) will be used to analyze the sequence data and begin to catalogue the organisms that are identified in these sequencing runs.
Fungal viruses. Rutgers will continue to characterize new viruses found in fungal isolates as they are identified. As stated previously, characterizations of two novel viruses, from Michigan strain RC1 and Japanese strain JS13, are underway in the Hillman lab.
Transposons. Rutgers will continue to do comparative analysis of the C. parasitica genome to examine the possible influence of its various transposons. To do this, we have sequenced and performed draft assembly of 12 C. parasitica genomes from North America, Asia, and Europe. In one European isolate, EU40, we found a complete helitron copy predicted to encode the genes permitting autonomous movement in the genome. Possible transposition is being examined first by crosses of EP155, which contains no autonomous helitron copies, with EU40, which contains one such complete copy. Analysis of single ascospore progeny will include mating type gene analysis and PCR examination of the site in which the complete helitron copy resides. Using RNAseq analysis, we will examine helitron-associated gene expression in C. parasitica isolates that contain or lack complete helitron copies.
WVU is deploying a hypovirus superdonor strain in the forest. Methods of application being tested include: (1) the traditional punch method; (2) scratch methods using bee combs; and (3) paint method without wounding. These plots will be followed to determine: (1) best method for introducing the SD328/82 formulation; (2) dissemination of hypovirus to non-treated cankers on treated trees and nearby non-treated trees; and, (3) if the SD328/82 formulation can arrest cankers with multiple applications.
B3-F3s being developed by TACF are predicted to have more resistance than C. dentata but less than C. mollissima.WVU hopes to test a superdonor strain with B3-F3s planted in the forest.
further identify potential genes with products involved in pathogenicity by leveraging the available genome sequence and recent annotation.
We will identify CpVIB-1-targeted DNA sequences by using CHIP-Seq technology and the recently re-annotated genome to determine which genes are targets for expression control by VIB-1 in response to incompatibilty.
For all projects, the basic methods to be employed will be similar. Gene knockouts will be made in the EP155 strain. The deletion constructs prepared using the method of Colot et al (2006)in which the flanking regions were assembled with the Hygrmarker using a yeast-based recombination system. Transformants will then be single-spored for nuclear homogeneity and verified by Southern blot. Analysis of the secretion characteristics of the LysM proteins will be accomplished by fusing epitope tags to the gene sequences and verifying secretion by western blot. Transcriptome data will be prepared by using Illumina sequencing of RNA from multiple samples, to control for variation. CHIP-Seq analysis will be conducted using an available tagged-VIB-1 protein.
A study was established in spring, 2017 to evaluate the long-term (10+ year) survival, growth, and competitive ability of chestnut seed and seedlings planted in each of the three stages of the three-stage shelterwood harvest system commonly used to regenerate oak on the Allegheny plateau region. In April, 2017, 1374 B3F3chestnut seeds and seedlings (from CAES and TACF) were planted on a 12’ x 12’ grid, planted in each of three replicates of each of the three silvicultural treatments. All chestnuts are protected from browsing using five foot-tall tree shelters. During the first, fifth, and tenth growing seasons, hemispherical photos will be taken adjacent to a sub-sample of living chestnut seedling to evaluate light availability. At the end of the first ten growing seasons, height and diameter of each seedling will be recorded, as well as height and species of the tallest competing seedling.
As resources permit, UT annually will evaluate plantations listed in the previous section.
UK will continue to elucidate interactions among hymenopteran parasitoids of the chestnut gall wasp, and evaluate the host range and entomopathogenic properties of the member of the Colletotrichumspp. complex infecting chestnut galls and their inhabitants. The methods used will be similar to those used for the publications cited in the previous section.
•	Continue elucidating chestnut gall wasp-parasitoid-Colletotricum dynamics.
•	Public release of backcross hybrid chestnut trees will start restoring the benefits of wild American chestnut to wildlife and humans. One benefit will be greater and more consistent amounts of mast for animal and human consumption; for wildlife, this results in larger populations (Diamond et al 2000, Lutts 2004). Another benefit will be a substantial increase (up to 100%) in rates of timber growth and faster harvest rotations on currently unproductive mountain land (Jaynes and Graves 1963; Kuhlman 1978; Smith 2000). Finally, starting the restoration of American chestnut would be a beacon of light and hope shown by science in the face of continuing environmental degradation (Youngs 2000).
•	Incorporation of PRR resistance will broaden the niche of blight-resistant trees to include areas where PRR eliminated American chestnut prior to blight.
•	Development of a screen for PRR resistance in tissue cultures will speed up testing of genetically modified chestnut and enable clonal testing. Likewise as above for Ozark chinkapin.
•	Genetic diversity is crucial for this highly heterozygous, obligately outcrossing genus. The transgenic chestnut ideally is a single clone to facilitate true breeding for the transgene (from trees homozygous for the transgene). Using a single clone also lessens the chance for gene silencing due to combination in single trees of transgenic events at different loci. Thus increasing effective population size is crucial.
•	Combining OxO blight resistance with PRR and blight resistance from Chinese chestnut may lead to less disease severity. Decreased disease severity may prove crucial to long-term growth.
•	Solid DNA sequences of C. dentata and C. mollissima will be crucial to further progress with genomic selection and most any other genetic analysis or intervention.
•	Genomic selection augmented for major QTL should allow completion of selection in the first seed orchards for production of blight-resistant backcross hybrids within the next five years.
•	Estimation of effective population size in breeding populations will be very helpful in optimizing genetic diversity.
•	Prediction of future blight severity in recovering stands of chestnut will identify stands where intervention is indicated.
•	Identification and culture of biota in chestnut may provide materials to help control blight.
•	Comparison of individual-based models (IBMs) and stands with differing predictions of future recovery may help identify promising biocontrol agents.
•	Correlations of biota present in cankers differing in severity may identify promising biocontrol agents.
•	The foregoing studies of canker biota may lead to deliberate creation of more LSAs.
•	Superdonor strains of C. parasitica, that can anastamose with strains with any combination of the 5 known vic genes, hold promise for effecting biocontrol of blight on American chestnut similar to that observed on European chestnut because the barrier of multiple vc groups to spread of hypoviruses will have been minimized.
•	Superdonors plus blight-resistant stock may yield better blight control than either alone and may help elucidate the relative effect of resistance and vc group on control.
•	Transposons in C. parasitica may be important components of their evolution and may be helpful to biocontrol efforts.
•	Characterization of LysM will better our understanding of the role of LysM genes in fungal virulence.
•	Fine mapping of QTL for pathogenicity in C. parasitica should identify genes for pathogenicity that can be tested by gene knockout. Knowledge of genes for pathogenicity and resistance in the pathogen and host, respectively may help elucidate their roles and lead to better disease control through resistance and pathogenicity modification.
•	Characterization of VIB-1 and its DNA targets will lead to improved understanding of the vegetative incompatibility process, in particular the molecular signals that lead to apoptosis and interrupt hypovirus transfer.
•	Comparison of data from different forest plantings will uncover commonalities and patterns across studies, e.g., linking performance of planted seedlings under similar silvicultural treatments in different regions. Collectively, these plantings should lead to improved guidance for optimal seedling quality and planting conditions, pre- and post-planting management, and performance of hybrid chestnut generations. Tracking regional sources of American parents will help determine possible seed zones and the impacts of adaptation.
•	Chestnut gall wasp can effectively eliminate nut production in commercial orchards for up to 4 years. Furthermore, gall wasp infestations can wax and wane. Continued elucidation of chestnut gall wasp-parasitoid-Colletotricum dynamics may suggest measures to stabilize nut production in commercial orchards.
The greatest outcome of the NE-1333 project and its predecessors has been to provide an annual meeting for scientists studying C. dentata and its pests and pathogens. Numerous collaborations have been struck up over the years since 1983, the first meeting of NE-140. These deeply interwoven, long-term collaborations between stations should be evident throughout this proposal and in the following, which describes the rationale for the experiments at the core of this project.
Breeding. The ultimate goal of the traditional and GE breeding programs is to release genetically diverse, disease-resistant breeding populations adapted to the range of environments encountered between Maine and Georgia. That genetic diversity should enable the trees to adapt to changes in their environment, such as global warming. The first release of B3-F3s is expected within 5 years. These trees will be adapted to Meadowview, Virginia and environs.
Trees are selected for blight resistance during breeding by measuring canker size or rating canker severity. Measurements are more accurate than rating in the hands of amateur volunteers and technicians. Cankers are induced by direct inoculation using the cork-borer, agar-disk method (Hebard 2006) or variants (Powell et al 2007). Selection can be based on parental phenotype or the collective phenotypes of offspring. Early in backcrossing, there are sufficient genetic differences between straight backcrosses for direct selection to be successful. However, three major QTL are involved in resistance (Kubisiak et al 2013; Nelson, Abbott, Fan, Georgi and Hebard, unpublished) and their individual heritability is too low to permit its direct selection in straight or backcross F2s homozygous for all three. That homozygosity has to be detected by markers, progeny testing, or waiting 20 years for highly resistant phenotypes to manifest in the face of numerous infections under a fairly full sampling of environmental conditions (Hebard unpublished).
The earliest screen for blight resistance in transgenic plantlets that SUNY-ESF has been able to devise is a detached leaf assay performed on leaves of potted plantlets (Newhouse et al 2014b). This assay is too laborious for mass screening of thousands to tens of thousands of seedlings.
Two virulent strains of C. parasitica are used for selection, Ep155 and SG2-3. They are from the two extremes of the distribution of pathogenicity (canker size) for virulent strains of C. parasitica. Ep155 from the high end is useful for quantifying high levels of resistance and SG2-3 from the low end, low levels of resistance. SG2-3 is also useful in screening trees for resistance without killing them. In general, seedlings at least 2 years old with Chinese levels of blight resistance can be screened without killing them, using SG2-3 (Hebard 2012).
Chestnut blight canker length or width approximates a sigmoid curve when plotted against host resistance, fungal pathogenicity, or both, and is not linear, although variances of length or width are constant, except for cankers showing no expansion whatsoever. This non-linearity can lead to significant host-parasite interactions in ANOVAs, but such interactions do not indicate race specificity without more careful analysis; none have been found yet in chestnut. Likewise one strain or another will showa significant QTL, but that is likely a reflection of the non-linearity rather than any specificity of a QTL for one strain (Hebard 2012).
Ep155 was originally isolated by CAES and is in common use throughout NE-1333. SG2-3 was isolated by TACF and is used by SUNY-ESF. Their pathogenicity was determined by Hebard (2012). Powell et al's (2007) small-stem assay is being tested extensively by TACF to accelerate progeny testing of B3-F2s from three years to one. This early testing is expected to kill the inoculated trees.
P. cinnamomi presents a different set of challenges. It is only known to reproduce clonally (Zentmeyer 1980) and is not found above 40 degrees north latitude, approximately (Balci et al 2007). It can be eliminated from soil by cold weather in winter, perhaps because it does not produce oospores (Griffin et al 2009). It spreads rather slowly in soil, but can persist indefinitely in warm climes. Infested soil can be a major source of movement, as well as infected plant materials, such as seedlings from southern forest nurseries. P. cambivora can infest nurseries further north, but infection of susceptible plants doesn't always result in death and is more easily controlled with fumigation, unlike P. cinnamomi. Unfortunately, P. cinnamomi can infect seedlings in forest nurseries at very low frequency, making improvement in control methods very difficult, yet allowing infestation of areas previously free of P. cinnamomi (https://content.ces.ncsu.edu/management-of-phytophthora-root-rot-in-fraser-fir-christmas-trees). In areas with general infestation of soil by P. cinnamomi, C. dentata in plantations die at frequencies approaching 100% whereas C. mollissima and its hybrids can thrive.
Screens for PRR resistance are quick and easy. Unfortunately, the pathogen is quite destructive and many promising plants die after a few seasons, but not all (James unpublished). Therefore, the method devised by Jeffers at Clemson, cited above in Methods, was used for progeny testing; another reason for progeny testing was that propagation of chestnut by nuts is easy, unlike various methods for rooting cuttings. Classical analysis over several generations of crossing indicated simple inheritance of rPRR resistance (Hebard and Georgi unpublished), in line with previously reported heritabilities obtained on clonal chestnut in Spain exceeding 0.8 (Fernandez-Lopez et al 2001). Molecular analysis also indicated PRR resistance is conferred by one gene or two closely linked genes (Zhebentyayeva et al, in preparation).
Fortuitously, the Graves tree, a B1, and its progeny inherited PRR resistance as expected for a single gene, out to B3-F3. The Graves tree, located at CAES, is one of three major sources of blight resistance in the TACF program, along with the Clapper B1, also at CAES, and C. mollissima cv Nanking, at VT. PRR resistance is incompletely dominant and some Graves B3-F2s show high resistance, compatible with frequencies of trees homozygous for a single gene expected under the Hardy-Weinberg model (Hebard and Georgi unpublished). Thus it should be relatively straightforward to fix the resistance in all Graves lines and simutaneously to introgress it into populations from other sources of blight resistance, including GE chestnut. Genetic markers could greatly simplify and accelerate this process, all but replacing progeny testing.
An in vitro screen for PRR resistance could be used on GE chestnut cultures, including, hopefully, some that will be identified in C. mollissima in the next year. The gene identification would be done by the collaborative efforts of PSU, TACF, UT, VT, UK and Clemson and genes inserted into chestnut and identified by UGA. Especially if genes in founder populations could be edited with a CRISPER/Cas9 system, great strides could be made quickly.
Meg Staton of UT developed the TreeSnap app for cellphones to enable quick cataloguing of tree locations and diagnostic photos for identification. It is being used by TACF to augment C. dentata populations from throughout the range for crossing with the OxO GE tree from SUNY-ESF.
Genomics. The ultimate goals for the genomics component of the USDA multi-state research project have been: 1) to develop high quality, chromosome-scale reference genomes for C. mollissima and C. dentata; and 2) to apply \ the high-quality genomes to accelerating the breeding of blight-resistant C. dentata.
Jason Holiday at Virginia Tech and Jared Westbrook of TACF are in the process of developing a Genomic Selection model for use in accelerating the TACF backcross breeding program, with funding from a USDA NIFA grant. The Staton group at The University of Tennessee has produced a set of 714,039 SNPs from the mapping of genome sequence data for three American genotypes to the Vanuxem genome, which will be used in developing the Genome-Wide-Selection model(s). Staton is also maintaining the sequence database at http://hardwoodgenomics.org; she developed the database. The Carlson lab will use the new chromatin-interaction technology to prepare chromosome assemblies for additional genotypes that are important in the TACF breeding program, including the C. mollissima blight-resistance donor genotypes “Mahogany” and “Nanking” and early F1hybrid “Clapper.” In addition, a reference genome for an C. dentata genotype is being prepared for TACF at the Hudson-Alpha Institute, which is expected to be available by late 2018.
Tatyana Zhebentyayeva at Clemson, working with Bert Abbott at UK and Dana Nelson at SIFG, has developed SNP markers for mapping PRR resistance QTL. Her work indicated two genes for PRR resistance on the long arm of linkage group E, as mentioned above in the breeding portion of this Outreach Plan plus in Related, Current and Previous Work (Zhebentyayeva et al, in preparation). Abbott, Nelson, Fan, Georgi (TACF) and Hebard (TACF) are remapping blight resistance QTL in F2, B1, and B2populations. Jared Westbrook and Jason Holliday are mapping blight resistance QTL in B3-F2progeny using GWAS of SNPs derived from RAD-Seq data and from Meg Staton at UK, mentioned above. Markers flanking QTL derived from these data should augment genomic selection models (Spindel et al 2015). (Whether one uses traditional QTL analysis or GWAS depends primarily on population/pedigree structure and how one accounts for linkage disequilibrium; the most glaring difference might be whether the x axis of Manhattan plots is centiMorgans or base pairs).
Upon validation of the C. mollissima and C. dentata genomes, in-depth comparative genomics studies will be conducted to identify regions that differ in structure as well as specific sequence variations related to resistance and susceptibility to chestnut blight and Phytophthora. Comparative studies will then be extended to genomes of other Fagaceae species, as well as other, more distantly related hardwood forest tree species, to determine the extent to which genome organization and gene sequences have been conserved or diverged among trees.
Hypovirulence. In the Previous Work section above, four reasons were given why hypoviruses have resulted in widespread remission of blight in Europe but not North America: 1) fewer vc groups; 2) more blight susceptibility; 3) stronger competition from other tree species; 4) differences in forest management. The superdonor strain being deployed by WVU solves the vc issue, but there are some additional reasons and considerations that are important to the success of field deployments of hypoviruses and to further observation and collection in the field. One additional reason given in a recent paper still in print (Double et al 2018) is proper choice of hypovirus.
Absence of resistance in C. dentata is the null hypothesis.
Survival of LSAs was associated with reduced virulence.
Resistance was also detected in most LSAs studied by Griffin et al (1983).
For LSAs without resistance, survival was most likely due to reduced virulence.
Hebard (1991) reported that cankers on small stems in the crowns of LSAs with resistance were superficial, but not those in the crowns of LSAs without resistance; this should be noted during further field studies of LSAs.
Chestnut blight is endemic on understory sprouts in mature forest but becomes epidemic on sprouts in clearcuts, leading to almost complete mortality 10 years after cutting (Hebard, 1982). Hypovirus deployment in clearcuts needs to begin 2-3 years after cutting before the disease becomes epidemic; otherwise inoculum of virulent strains becomes overwhelming. Remission in clearcuts may not be possible during the first epidemic in parallel to the situation in Italy. There, blight started in one coppice cycle but remission associated with hypovirulence did not begin until the next coppice cycle (Mittempergher, 1978).
Ascospore concentrations in air should be sampled. No reports subsequent to 1915 have occurred in the US (Guerin et al 2001).
Small C. dentata surviving blight are termed SSAs. When progeny of SSAs from clearcuts were tested from resistance, no significant family differences were detected. SSAs in clearcuts may have survived longer when competing vegetation was removed.
The frequency of SSAs in clearcuts is about 1 per cut. Cuts in the Jefferson National Forest usually contain between 100 and 1000 sprout clumps of C. dentata.
Some isolates from SSAs in clearcuts were virulent but had reduced pathogenicity, including SG2-3.
Unlike LSAs, which are mostly single trees or in pairs, the trees in Michigan are in stands. This suggests that hypoviruses are important to recovery but not resistance, since so many trees are recovering; one would expect resistance alleles to be rare in C. dentata due to the absence of selection prior to blight (Burnham, personal communication). Rarity of random alleles for blight resistance also would argue for their rarity in founders of stand populations of C. dentata outside its native range.
Anecdotally, Jeff Springer's experience in Michigan is that blight can be controlled by treatment of cankers with hypoviruses on Castanea spp and hybrids possessing intermediate levels of blight resistance but is difficult on C. dentata and unnecessary on C. millissima.
Large (> 25 cm dbh) mollissima in China are more severely cankered than C. henryi orC. sequinii in forested situations, yet seedlings of C. mollissima test as more resistant. Progeny tests of the three species should be established in forest in their native habitat in China, such as at Dalaoling National Forest Park.
The metagenomic analyses planned by Brad Hillman at Rutgers hopefully will lead to detection of viruses and other parasites of C. parasitica in cankers,including those found by Andy Jarosz at MSU and Laurel Rodgers at SU. One also can envision Brad's metagenomic subjects being extended to Michigan and West Salem to look for co-occurrences with recovery and to follow biocontrol over time in all of its subjects. Hillman's continued characterization of viruses that infect C. parasitica, including transposonsshould provide additional reference sequence to search for in the metagenomic data. One wonders also about the host range of these viruses.
Angus Dawe's fundamental studies of C. parasitica at MissSU do not have any immediate translational benefit as a goal, but neither did Don Nuss' when he started to sequence hypoviruses in 1985. Nuss' brilliant work eventually led to the superdonor strains. Comparable benefits could arise from further characterization of the molecular basis of vegetative incompatibility, identification of fungal genes associated with pathogenicity, and the molecular basis of pathogenicity.
Outplantings. Jacobs et al (1913) developed a theoretical framework for restoration. Schlarbaum et al(1994) found that C. mollissima does not persist in most forested settings if competing tree species are allowed to grow; it gets overtopped and dies, apparently due to increased blight severity (Miller et al 2014). Miller et al (2014) also studied population dynamics of C. mollissima, and found it could produce viable offspring on a site with low competition.
If bred trees are not competitive in the forest, one can anticipate that blight severity will increase. In turn, blight reduces the competitive ability of chestnut (Uchida, 1977), which then precipitates a negative feedback loop leading to death. Because of the poor competitive ability of C. mollissima, we do not know whether or not it has sufficient blight resistance to persist in unmanaged forests except on sites with little competition. Such sites are poor.
The amount of blight resistance necessary for chestnut to be a dominant forest tree is unknown, and will only be determined empirically if we are fortunate enough to be able to increase it should current levels be inadequate. We are fortunate enough to know that most families of TACF's current backcross hybrids, at least when young, grow (compete) as fast as most C. dentata families. This occurred in studies installed by Stacy Clark of the USDA Forest Service and Scott Schlarbaum of UT, mentioned above in the Previous Work section. Blight incidence should be starting to increase in these plantings. Measuring it and mortality will give an excellent first look at the performance of B3-F2s in the forest in the face of competition and naturally occurring blight.
The Clark/Schlarbaum studies of B3F2s were installed beginning in 2009, and illustrate the challenges encountered with hardwood plantations in the forest, along with numerous studies they installed previously using pure C. dentata and backcross hybrids from earlier generations. In one B3-F2study, the trees were decimated by a cicada outbreak, allowing competing Liriodendron tulipferato overtop them (Hebard unpublished). Another was hampered by deer predation (Hebard unpublished). An earlier planting was invaded by clear-wing moth (Pinchot et al 2011). There are myriad factors that can negatively impact a particular planting. Devastation of hardwood forest plantings approaches being a stochastic process. It is best to install numerous plantings, which then implies minimal data collection per planting (quantitative data nevertheless!) but perhaps multiple visits throughout the year. The intensity of data collection should be varied depending on results and observation.
In addition to Clark, Nelson and Pinchot, USDA Forest Service scientists helping install chestnut plantings in the forest include Paul Barrang, Barb Crane, Paul Schaberg, and numerous other scientists and district personnel working with them (Clark et al 2014). University personnel installing plantings include Kim Steiner (PSU), Brian McCarthy (Ohio University), Carolyn Keiffer (Miami University of Ohio) Hill Craddock (UTC), Doug Jacobs (Purdue), Harmony Dalgleish (William and Mary), Michael Saunders (Purdue), Marty Cippolini (Berry College), Sunshine Brosi (Frostburg State University), and Brian Roth (University of Maine). TACF personnel include Jared Westbrook, Fred Hebard, Tom Saielli, Sara Fitzsimmons, Kendra Collins, Welles Thurber, and Michael French.
Gall Wasp. Lynne Rieske-Kinney continues to investigate parasitoids and fungi associated with the waxing and waning of Asian chestnut gall wasp. During peaks of gall wasp infestation, nut production in orchards can be reduced 10 or 100 fold for 2-3 years. This is financially ruinous for chestnut orchardists. It is only through continued study that more effective means of control can be devised than just waiting for an infestation to subside.
Publicity. Regarding publicity, which the title of this section suggests it is about, TACF survives due to publicity and will always publicize developments. Members of the project also take advantage of the public outreach specialists at land-grant and non land-grant institutions, especially extension personnel, of whom we have a participant from PSU. Technical publications also are a form of outreach. Interested members of the public, aka stakeholders, are permitted to and have attended NE-1333 meetings; however meetings are not publicized in advance.
The organization of the regional research project was established in accordance with the format suggested in the "Manual for Cooperative Regional Research". One person at each participating agency is designated, with approval of the agency director, as the voting member of the Technical Committee. Other agency individuals and interested parties are encouraged to participate as non-voting members of the committee. Each year, members elect a Chair-elect, whose duties begin the following year as Chair.
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