Chestnut blight, incited by Cryphonectria parasitica (Murr.) Barr, devastated the American chestnut tree (Castanea dentata(Borkh.) Marsh) in the first half of the 20^thcentury, 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 (N_ei) of about 50 have been producing progeny since 2006. The N_eiis 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 N_ei 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.