Improvement of protocols for the screening of biological control agents against white-nose syndrome

White-nose syndrome, caused by the fungus Pseudogymnoascus destructans (Pd), has become increasingly prevalent in North America since 2006 and has caused mass mortality in many bat populations. This study focuses on standardizing protocols for efficient Pd cultivation, which includes harvesting Pd spores from agar plates, culturing Pd on both normal and modified culture media and finally, testing chemical agents and cave bacterial isolates against Pd for antimicrobial activities. Pd cultivation was performed on Sabouraud Dextrose Agar (SDA), Rose Bengal Agar (RBA) and modified RBA-bat tissue media. Only Pd grown on RBA supplemented with 0.25% bat tissue had a final colony diameter that was significantly different from Pd grown on SDA and could be attributed to the difference in the growth rates. Pd lawns grown using both pour- and spread-plate techniques were observed on SDA media. Although the former technique led to slower fungal lawn growth in comparison to the latter, the more uniform lawn produced with the pour-plate technique allowed the size of inhibitory zones to be determined quantitatively. Further, the cultured Pd lawns were tested against the chemical antimicrobial agents Nystatin, peroxigard and bleach (10%, 50%, 100%) and cave bacterial isolates using a Kirby Bauer and agar plug diffusion assay. Both bleach (10%) and peroxigard (1.5%) created zones of inhibition in Pd lawns, with diameters measuring 25.5 mm and 12.7 mm, respectively, after 24 days of incubation. No antimicrobial activities were observed for the cave bacteria tested against Pd. Our findings will allow more efficient and reliable screening of cave bacteria against Pd, elucidating biological control agents against white-nose syndrome.