Semester

Summer

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Not Listed

Committee Chair

Matt Kasson

Committee Co-Chair

Dan Panaccione

Committee Member

Jim Kotcon

Committee Member

Brad Hillman

Abstract

American chestnut (Castanea dentata) and various oak species (Quercus spp.) are impacted by several economically important canker pathogens in the fungal order Diaporthales, namely Cryphonectria parasitica and Endothia gyrosa (Cryphonectriaceae). Cytospora canker caused by several closely related fungi in the fungal genera Cytospora and Valsa (Cytosporaceae) have also been implicated in causing canker diseases of Quercus and Castanea in Asia, Europe and North America. The impacts of C. parasitica on American chestnut have been exhaustively studied and it’s impacts on various oak hosts have also been broadly investigated and reported. Endothia gyrosa has also been thoroughly investigated on oak, but to a lesser extent on chestnut species. Cytospora canker has been reported from numerous woody hosts, but formal pathogenicity studies on native oaks and chestnut in the United States have not been undertaken. Direct comparisons between these phytopathogens on single hosts have also not been formally investigated.

From 2021-2025, we conducted a series of lab and field experiments to compare the pathogenicity of lab strains of C. parasitica, E. gyrosa and a field recovered Cytospora ceratosperma strain to better understand the relative risks posed by each fungus. Lab studies were conducted on cut stems of northern red oak (Quercus rubra; red oak family) in 2021 and 2024, while field investigations were conducted in 2023 and 2024 on both red oak and chestnut oak trees (Quercus montana; white oak family). Overall, experiments showed E. gyrosa was more aggressive than C. parasitica on cut stem assays, whereas field studies revealed only C. parasitica produced significant cankers on northern red oak after six months. However, none of the fungi tested caused significantly larger cankers on chestnut oak compared to the negative control, yet all treatments produced cankers. Recovery of naturally occurring Cytospora ceratosperma from 2023 and 2024 inoculation sites across all treatments helped in part to explain the presence of some cankers in negative control treatments as well as clarify the pervasiveness of this fungus in the forest environment. Temperature assays between C. parasitica and E. gyrosa indicated that C. parasitica grew better than E. gyrosa across a range of tested temperatures that these fungi typically experience under field conditions but neither was subjected to lethal temperatures during our studies. These results indicate that C. parasitica, E. gyrosa, and C. ceratosperma (in the case of northern red oak) are weak pathogens on the two oak species tested and likely only infect or cause significant disease on these hosts opportunistically as trees age or experience other environmental stressors. Follow-up studies looking at timing of inoculation and combinations of these fungi in enhancing disease are needed to better understand disease dynamics in this pathosystem.

To further our understanding of C. parasitica and E. gyrosa, a series of additional experiments comparing virulent (V) and transfected strains of each on cut-stem and apples were undertaken. Temperature assays were also conducted to determine the effect of hypovirus, if any, on mycelial growth under different temperatures. As part of these investigations, we successfully transfected two strains of E. gyrosa with CHV1 hypoviruses via anastomosis with engineered super donor (SD) strains of C. parasitica, which to our knowledge represents the first successful transfection through anastomosis. Cut stem assays conducted on northern red oak and apple assays indicated that though putative transfected E. gyrosa strain phenotypes had reduced pigmentation as is expected with CHV1 hypovirus infection, they behaved as if they had enhanced virulence exhibiting superior growth across a range of temperatures and increased virulence on apples and cut red oak stems compared to V counterparts. Transfected C. parasitica strains exhibited more typical behavior with diminished growth compared to the V isogenic counterparts. In temperature assays, differences in growth were more pronounced between V and transfected C. parasitica strains at 10 °C and 15 °C and between V and transfected strains of E. gyrosa at 30 °C. Finally, historically vic incompatible virulent European strains of C. parasitica(EU65-EU73) were paired with SD strains to help determine vic barriers that prevented successful anastomosis with EU tester strains and subsequent transfection. All of the EU65-EU73 strains were successfully transfected using SD strains, but transmission rates for certain strains, namely EU65 and EU70, never exceeded 10% or 21%, respectively, for either hypovirus strain tested. These data support the possibility of additional vic alleles or vic genes in atypical European C. parasitica strains from France and Switzerland. Together these findings advance our understanding of hypovirus-fungus interactions and pave the way for transformative studies that may serve to better control devastating plant pathogenic fungi.

Finally, in 2016-2017, SD strains of C. parasitica were deployed at two field sites on the Savage River State Forest in Western Maryland to assess the biocontrol efficacy of SD strains using different hypoviruses and application methods against stands with naturally occurring chestnut blight. Though both experiments were initially successful with most cankers diminishing and transfection occurring in most isolates one-year post-application, without additional treatment, a majority of trees succumbed to disease and died within 2-3 years’ time. In 2022, SD treated trees were evaluated, sampled and harvested to allow for comprehensive vic genotyping of cankers on surviving and chestnut blight killed American chestnut trees. A total of 296 C. parasitica strains representing five unique phenotypes were recovered from across the 69 previously treated American chestnut trees including ten trees that were still alive at the time of resampling. Vic genotyping was performed on 110 recovered C. parasitica strains to compare with previous vic genotype diversity at both sites. Overall, vic genotype diversity increased from 22 vic genotypes to 45 vic genotypes between 2017 and 2022 at SD site 1.0 and from 36 to 44 between 2018 and 2022 at SD site 2.0, the latter of which appears to be near the upper limit of vic genotypic diversity reported from a single site and the highest reported recovery at a single site to this date. However, no C. parasitica phenotypes or dominant vic genotypes were specifically associated with the surviving trees or treatments. In vitro hypovirus transfection pairing assays performed on atypical putative transfected phenotypes confirmed successful transmission though transfection rates varied across phenotypes and replicates. SD strains still offer great promise in controlling chestnut blight but will require reapplication to previously treated cankers and newly formed cankers on previously treated trees to counteract the ever-increasing vic diversity of virulent strains in the environment. Together these findings advance our understanding of hypovirus-fungus interactions and pave the way for transformative studies that may serve to better control devastating plant pathogenic fungi.

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