Thesis (M.S.)--Mississippi State University. Department of Entomology and Plant Pathology. / Title from title screen. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains vii, 93 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 75-83).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vii, 79 p. : ill. (some col.), col. map. Includes abstract. Includes bibliographical references (p. 68-75).

The American chestnut tree (Castanea dentata) is a deciduous tree that largely exists in the eastern United States along the Appalachian Mountain range. Approximately 100 years ago, a fungal pathogen (Cryphonectria parasitica) decimated chestnut populations, resulting in the loss of billions of trees. Disease-resistant American chestnut populations have been developed, but the introgression of wild adaptive diversity into these breeding populations will be necessary to develop locally adapted and disease resistant chestnut trees for reintroduction. In this dissertation, I presented our findings which addressed previous gaps in knowledge regarding the population genomics of wild and backcross American chestnut populations. I 1) estimated the genomic diversity, population structure, and demographic history of remnant wild American chestnut populations; 2) revealed the genomic basis of local climate adaptation in American chestnut, developed a novel method to make tree sampling estimates for germplasm conservation, and defined unique seed zones for American chestnut based on climate and genotype, and 3) determined the amount of wild adaptive diversity captured by the backcross breeding program and made recommendations for their replanting region. These results will inform the development of a breeding plan for the introgression of adaptive diversity into backcross and transgenic chestnut populations. / Doctor of Philosophy / The American chestnut tree (Castanea dentata) is a deciduous tree that largely exists in the eastern United States along the Appalachian Mountain range. Approximately 100 years, a fungal disease (Cryphonectria parasitica) decimated chestnut populations, resulting in the loss of billions of trees. The American Chestnut Foundation developed disease-resistant American chestnut backcross trees by breeding American chestnut trees with Chinese chestnut trees (Castanea mollissima). These trees will need additional breeding with wild American chestnut trees so that their offspring will have both the disease-resistant traits and the adaptations to the local environment where they will be replanted. This is important, because trees that are both disease-resistant and locally adapted will be most likely to survive and thrive in their replanting location. However, a comprehensive evaluation of the genomic basis for local adaptation in American chestnut populations is lacking. In this dissertation, I presented our findings which addressed previous gaps in knowledge regarding the population genomics of wild and backcross American chestnut populations. I 1) estimated the genomic diversity, number of unique populations, and population size changes over time in wild American chestnut; 2) revealed the genes related to local adaptation in American chestnut, developed a novel method to make tree sampling estimates for conserving wild American chestnut diversity, and defined unique seed zones (areas within the species range that have unique adaptations to environment) for American chestnut based on climate (ie, precipitation and temperature values) and genotype (DNA), and 3) determined the amount of wild genomic diversity related to local adaption captured by the backcross breeding program and made recommendations for their replanting region. These results will inform the development of a breeding plan of wild American chestnut with backcross and transgenic chestnut populations to create locally adapted and disease-resistant chestnut populations for reintroduction.

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Cryphonectria parasitica is the causal agent of chestnut blight, which devastated the American Chestnut tree population in the early 20th century. The discovery of hypoviruses that reduce the severity of the chestnut blight infection offers the potential for biological control. However, the spread of the hypoviruses is hampered by a diverse genetically controlled nonself-recognition system, vegetative incompatibility (vic). CPVIB-1 was identified as a transcription regulator playing an important role in the programmed cell death response to this stimulus. In this study, we have found that CPVIB-1 is ubiquitin-decorated which might lead to its degradation in the proteasome pathway. RNA-Seq and ChIP-Seq were used to further explore the downstream targets of CPVIB-1 that mediate the various metabolic changes that lead to the altered phenotype of the Δcpvib-1 mutant. Due to inaccuracies in the prior annotation, we performed a genome re-annotation to improve the accuracy using a MAKER2-two-pass pipeline. To validate the improvement a second pipeline, PEPA, was developed to compare quality metrics between the old and new annotations. Approximately 1/3 of the original annotations from 2009 were found to be inaccurate. Experimental confirmation by testing 27 predicted genes using a diagnostic PCR protocol to differentiate between prior and new transcript structures showed that over 80 % of tested genome locations supported for the new annotation. Using rapamycin treatment to mimic stimulation of the vic response and applying the RNA-seq and ChIP-seq data to this new information, we found that CPVIB-1 is related to TOR signaling pathways, promoting autophagy and the proteasome pathway, but repressing carbon metabolism, protein and lipid biosynthesis. In depth analysis of CPVIB-1-bound DNA targets showed that this protein is a member of the GAGA regulator family, a group of multifaceted transcription factors with diverse roles in gene activation and repression, maintenance of mitosis, and cell development. Following treatment with rapamycin the recognition sequence bound by CPBVIB-1 was altered leading to the regulation of different suite of genes with diverse metabolic functions. Ultimately, we have developed a revised model of TOR signaling pathway where TORC1 and TORC2 signaling pathways are connected by the action of CPVIB1.

<u>Endothia parasitica</u> (Murr.) P. J. & H. E. Anderson (syn: <u>Cryphonectria parasitica</u>(Murr.) Barr), the causal agent of chestnut blight, was able to grow in total aqueous and tannin extracts from blight-susceptible American chestnut as well as in blight-resistant Chinese chestnut bark extracts, from winter and summer bark. Differences in the amount of conidial germination and growth in extracts of the two species were small. The, <u>E. parasitica</u> tannase was more abundant intracellularly than extracellularly. Total tannase activities from cultures in American chestnut aqueous and tannin extracts were greater than in the Chinese chestnut extracts, for both winter and summer bark. The tannase was isolated from the mycelium of E. parasitica and purified 142-fold with a 10% yield by anion exchange chromatography and gel filtration. The estimated molecular weight was 240 kD and the molecule may be a tetramer composed of four subunits with a molecular weight of 58 kD. The pH optimum of the purified tannase was 5.5 and the temperature optimum for activity was 30 C. The enzyme was separated into six bands in the pH range of 4.6 to 5.1 which may represent isoenzymes or post-translational modifications. / Ph. D.

Results of inoculations of grafts, seedlings, excised stems, and intact trees of large, surviving American chestnut indicated that some have heritable blight resistance. Some degree of resistance in American chestnut possibly would assist spread of hypovirulence (H) factors. The pathogenicity of the H isolate but not the V (virulent) isolate appeared to influence the size and sporulating characteristics of biocontrolled H+V cankers. The histopathology of canker development on blight-resistant Chinese and blight-resistant and blight-susceptible American chestnut was examined after inoculation with V and H E-parasitica. Rapid formation and continual growth of mycelial fans distinguished chestnut blight cankers incited by V E-parasitica from those incited by H E-parasitica, and distinguished cankers on susceptible chestnut from cankers on resistant chestnut. Differences among such cankers in wound periderm and other pathological alterations of bark and wood anatomy probably were results of differences in rates of fan formation and growth. Wound periderm formation began at the deepest point of a wound or canker and progressed outward to the bark surface. Superficial cankers arose in resistant trees or with hypovirulent fungus when slow-forming mycelial fans expanded through areas where wound periderm had not formed. Rapid epidemics of chestnut blight occur in recently clearcut areas but not in areas with large trees of species other than American chestnut, most probably because chestnut sprouts released after cutting become larger than they do when overtopped by large forest trees. The size of chestnut sprouts appeared to influence the rate of blight progress by setting a maximum limit on canker size and thus canker sporulation. It may be possible to foster spread of H factors in the Appalachians if blight epidemics in clearcuts can be prolonged. This might be done by restricting inoculum production. Cutting and removing some blighted sprouts, cutting some large unblighted sprouts, or increasing blight resistance in the chestnut population are three potential means of restricting inoculum production. / Ph. D.

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