Date of Graduation

2016

Document Type

Thesis

Degree Type

MS

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

William L MacDonald

Committee Co-Chair

Matthew T Kasson

Committee Member

Fredrick V Hebard

Committee Member

Daniel G Panaccione

Abstract

Chestnut blight is a devastating canker disease of numerous Castanea spp. resulting from the infection of wounds by Cryphonectria parasitica. The restoration of American chestnut has been pursued through the generation of blight resistant American x Chinese hybrids and employment of virulence attenuating hypoviruses of C. parasitica. The goals of this study were: 1) to conduct comparisons of host resistance among American (C. dentata), European (C. sativa), Chinese (C. mollisima), and three American x Chinese hybrid generations (B2F2, B2F3, B3F2) produced by The American Chestnut Foundation (TACF) to isogenic virulent and hypovirulent (CHV1) strains of C. parasitica; 2) to validate the high-throughput use and reproducibility of the chestnut leaf susceptibility assay against the same fungal strains on representative leaves sampled across the various host backgrounds; and 3) to conduct comparisons among hypovirulent strains of C. parasitica using living stem inoculation, excised-leaf, and apple assays. The comparison of host resistance was conducted on populations of trees growing at the West Virginia University agronomy farm. Infections were initiated on live-stems with a virulent strain designated 'Weekly' and an isogenic, hypovirulent 'Weekly-CHV1' strain (Euro7-CHV1). Subsequent canker measurements and fungal stromata counts were performed every two months for a year to assess host resistance. For virulent Weekly inoculations, Chinese chestnuts were often significantly more resistant, but few significant differences existed among the other hosts. Average linear growth measurements taken one year post-inoculation revealed B3F2 chestnuts to have the most linear growth of 20.02 cm and Chinese chestnuts to have the least linear growth of 13.92 cm. Stromata density estimates were highly variable within each host background with the highest density on B3F2 at an average of 0.65 stromata/cm 2 and the least on Chinese chestnuts at an average of 0.25 stromata/cm 2. Additional measurements taken for virulent Weekly inoculations hinted at a higher level of resistance within the B2F2 hybrid generation while all other hosts aside from Chinese were often similar to American chestnut. Weekly-CHV1 cankers expanded during the first two months of the study, but no subsequent growth was observed on any host despite the recovery of these isolates nine months post-inoculation. An excised-leaf assay was conducted using leaves from a subset of trees included in the living stem assay. Weekly and Weekly-CHV1 were used to inoculate the midvein of leaves from all previously mentioned host backgrounds. There were no significant differences for the Weekly isolate inoculations, but the average lesion area for American chestnut (78.5 mm2) was largest and Chinese chestnut (33.1 mm 2) was smallest among the tree species and breeding lines tested. For Weekly-CHV1 inoculations, Chinese chestnut (42.7 mm2) had significantly smaller lesion areas while all other hosts had average lesion areas similar to each other with the exception of B2F2 (63.72 mm2). Weekly (58.1 mm2) produced a significantly smaller average leaf-lesion area across all hosts than Weekly-CHV1 (86.4 mm 2). Further, the incidence of infection was significantly lower for Weekly (39.5%) than Weekly-CHV1 (79.1%). The virulence of selected hypovirulent isolates also was examined through a living branch assay that employed a clonal clump of wild American chestnut sprouts, an excised-leaf assay using leaves from the same clonal clump, and an apple assay. Weekly-CHV1 once again produced significantly smaller cankers in the living branch assay than Weekly. Interestingly, Weekly-CHV1 produced larger lesions than Weekly in the leaf and apple assays while all other virulent strains produced larger lesions than their hypovirulent counterparts. Host resistance and pathogen virulence are the balancing point for the survival of an infected chestnut host. Here, a selection of American, European, TACF hybrid chestnuts were shown to be equally susceptible to stem infections of C. parasitica. The excised-leaf assay produced similar results with regards to host response, but hypovirulent Weekly-CHV1 unexpectedly produced larger lesions than virulent Weekly. This same observation was made for an apple assay and a second excised-leaf assay. These findings provide evidence for unique interactions between C. parasitica and hypoviruses not previously observed.

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