Date of Award

2002

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Botany and Plant Pathology

Advisor

Joyce Longcore

Second Committee Member

Seanna Annis

Third Committee Member

David Lambert

Abstract

Balrachochytrium dendrobatidis is a pathogen of amphibians that has caused severe population declines on several continents, and little is known about the conditions that favor epidemics. The zoospore activity, temperature, and pH requirements of B. dendrobutidis were investigated to help understand the ecology and transmission of this pathogen. Over 95% of the chytrid's zoospores stop moving in less than 24 hours, and the zoospores swam less than 2 cm before encysting on tryptone agar. B. dendrobutidis zoospores were not attracted to tryptone, gelatin hydrolysate, casamino acids, keratin, gelatin, glucose, or lactose. The chytrid grew and reproduced at temperatures ranging from 4 to 25 OC, and grew best from 17 to 25 OC; it survived and reproduced for more than 6 months at 4 "C. Exposure of cultures to 30 OC for 8 days killed 50% of the cultures. Different isolates of B, dendrobutidis did not differ in their temperature optima. The chytrid grew best at a pHs ranging from 6 to 7, but live zoospores were present after two weeks of incubation at pHs ranging from 4 to 8. The zoospore activity and physiological parameters could determine the transmission and persistence of B. dendrobutidis in the environment. The nutritional requirements of Butrachochytriunz dendrobatidis were studied to help determine if the chytrid could live on saprophytic substrates outside its host, and to aid in designing an optimal culture medium for the fungus. No synthetic medium tested supported growth. B. dendrobutidis cultures grew densest with tryptone or peptonized milk as a nitrogen source. The chytrid did not require additional sugars when grown in tryptone; and grew densest in a liquid medium with 0.5 % tryptone alone. Liquid media with glucose concentrations greater than 1.8% or tryptone concentrations greater than 2% hindered growth. The chytrid grew on autoclaved snakeskin in water or on 1 % keratin agar. B. dendrobutidis produced extracellular proteases that degraded casein and gelatin, but had no measurable activity against keratin azure. The proteases were most active against casein at temperatures from 23 to 30 OC at pH 8.0; however, they were active at temperatures from 6 to 37 OC, and in a pH range from 6 to 8. SDS-PAGE analysis of the culture supernatant yielded no visible protein bands when stained with Coomassie blue or copper chloride; but activity gels with 0.5% skim milk revealed two distinct clearings

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