Date of Award

Summer 7-12-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Sidney A. Crow Jr. Ph.D.

Second Advisor

George E. Pierce Ph.D.

Abstract

The recently identified causative agent of White-Nose Syndrome (WNS), Geomyces destructans, has been responsible for the mortality of an estimated 5.7 million North American bats since its emergence in 2006. A primary focus of the National Response Plan, established by US Fish and Wildlife in 2011, was the identification of biological and chemical control options. In an effort to identify potential biological and chemical control options for WNS, six previously described bacterially produced volatile organic compounds (VOCs) and multiply induced Rhodococcus rhodochrous DAP96253 were screened for anti-Geomyces destructans activity. Geomyces destructans conidia and mycelial plugs were exposed to the VOCs and induced Rhodococcus in a closed air space at 15°C and 4°C and evaluated for inhibition of conidia germination and mycelial extension. Additionally, in situ application methods for induced Rhodococcus, such as fixed cell catalyst and fermentation cell paste in non-growth conditions, were screened with positive results. Rhodococcus was assayed for ex vivo activity via exposure to bat tissue ex-plants inoculated with G. destructans conidia. All VOCs inhibited radial growth of mycelial plugs and growth from conidia at both temperatures, with the greatest effect at low temperature (4°C). Induced Rhodococcus completely inhibited growth from conidia at 15°C and had a strong fungistatic effect at 4°C. Induced Rhodococcus inhibited Geomyces destructans growth from conidia when cultured in a shared air space with bat tissue explants inoculated with Geomyces destructans conidia. During the evaluation diffusible brown pigment was observed in G. destructans cultures exposed to induced Rhodococcus or select VOCs. The pigment was induced by light and oxidative challenge and hypothesized to be melanin. Traditional microbiological methods, as well as copper sulfide-silver staining and ultraviolet-visible spectroscopy, were utilized to confirm this hypothesis. This was a noteworthy result as melanin is a known virulence factor in other pathogenic fungi and may play a significant role in WNS. The identification of bacterially produced VOCs and inducible biological agents with anti-Geomyces destructans activity expands the pool of potential biological and chemical control options for WNS and provides wildlife management personnel with tools to combat this devastating disease.

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