Sixty-two natural cankers on branches and main stems of three 16-year-old grafted American chestnut trees at the Lesesne State Forest were sampled for Cryphonectria parasitica. Cankers were sampled in 1996 and 1997 at various distances from the main stem zone on the grafts (ground to 183 cm) that was inoculated in 1982 and 1983 with a mixture of dsRNA-containing white and pigmented hypovirulent strains. Grafted trees exhibited a high level of blight control, and all bark cores extracted from cankers on the grafted trees showed superficial necrosis. Bark cores extracted from these cankers yielded 156 isolates of C. parasitica. Fifty-three of these isolates were white, and 103 were pigmented. The farthest canker containing a white isolate was located 564 cm from the zone inoculated with hypovirulent strains (H-inoculated zone). The number of white isolates recovered per canker on the grafted trees near the H-inoculated zone (< 0.5 maximum sampling distance) was significantly greater (P=0.0039) than the number of white isolates recovered per canker on the grafted trees far from the H-inoculated zone (>0.5 maximum sampling distance). Lloyd's index of patchiness value for the frequency of white isolates in cankers was 1.36, indicating that white isolates were slightly aggregated in cankers. White isolates of C. parasitica were found in two of seven artificially established cankers 5 months after inoculation with a pigmented virulent strain (WK). Thirteen of 14 pigmented isolates collected from these cankers after 5 months were compatible with WK in vegetative compatibility (VC) tests. Eight of 25 white isolates recovered 5, 11, and 50 months after WK inoculation converted the pigmented WK strain to the white hypovirulent phenotype in vitro. Sixty-five pigmented isolates collected from natural cankers were paired in VC assays, revealing 28 VC groups. All 11 white isolates of C. parasitica assayed contained a 12.7 kb dsRNA in high concentrations. None of 48 pigmented isolates assayed contained dsRNA. All white isolates tested in virulence trials on American chestnut stems in a forest clearcut were hypovirulent, based on low canker severity indices. Little or no dissemination of white strains to cankers on the American chestnut stump sprout clusters, which surround the grafted trees, was found. In the future, to maximize spread of white hypovirulent strains on American chestnut trees, it may be beneficial to re-inoculate trees with hypovirulent strains farther up the main stem after substantial tree growth has occurred. / Master of Science

Intracellular sterol redistribution is an important step in the lipid homeostasis of organisms, a process directly linked to the organizational arrangement in the plasma membrane (PM) of cells. Previous studies in the budding yeast Saccharomyces cerevisiae have demonstrated that the ARV1 (ACAT-related enzyme-2 required for viability 1) protein is a major regulator of sterol transport from the endoplasmic reticulum to the plasma membrane, contributing to the structural organization of the PM, rendering it resistant to anti-fungal compounds as well as maintaining ER integrity. This study assessed the significance of ARV1 in the plant pathogenic fungus Cryphonectria parasitica (Cparv1) and investigated its role in the pathogenesis and virulence of the fungus. C. parasitica is the causative agent of Chestnut blight, which has wreaked havoc on the American chestnut species. Genomic analysis revealed that the Cparv1 gene is very closely linked to another gene that putatively encodes a cyanamide hydratase (Cpcah). An initial gene deletion event resulted in the elimination of both genes and a highly deformed phenotype in C. parasitica that was fully recoverable by complementation. PCR-based expression analysis determined that the lack of Cparv1 was responsible for the debilitated phenotype of the double mutant, with no transcript detectable from Cpcah. Subsequent complementation of the Cparv1 gene was also observed to restore the wildtype phenotype. Mass spectrometry-based (MS) results indicated a decrease in sterol content of the DCparv1 mutant strain compared to wildtype EP155 thus confirming a role for Cparv1 in sterol homeostasis. It has been shown that infection of C. parasitica with virulence-attenuating hypoviruses altered intracellular lipid content and protein secretion. Ultrastructure studies conducted on the Cparv1 strain showed disrupted organelle integrity and the presence of cytoplasmic double membrane stretches. Decreased sterol content in C. parasitica infected with CHV1-EP713 was observed similar to DCparv1 suggesting a connection between the hypovirus-infected phenotype and Cparv1. Furthermore, a non-targeted metabolomic study on all three strains identified 324 metabolites. Through the subsequent pathway analysis, we have investigated the pleiotropic effects in the C. parasitica strains and established a mechanistic linkage between this the activity of the ARV-1 protein and the hypovirus-infected phenotype.