Isoamyl acetate

Pure cultures of B. circina, C. muscae, and M. ramosissimus were cultured on synthetic mucor agar (SMA; 40 g dextrose, 2 g asparagine, 0.5 g KH₂PO₄, 0.25 g MgSO₄·7H₂O, 0.5 g thiamine chloride, and 15 g agar in 1 L of deionized water). The plates were incubated at 10, 15, 20, 25, 30, 35, and 40 °C in the dark for 5 days. Fragments of mycelia were removed from the cultures, placed on microscope slides with lactophenol solution (Junsei Chemical Co. Ltd., Tokyo, Japan) and observed under a light microscope (Olympus, Tokyo, Japan). The fine structure of C. muscae was observed using scanning electron microscopy (Hitachi S4700; Hitachi, Tokyo, Japan). The isolates were fixed in 2.5% paraformaldehyde-glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 2 h and then washed with 0.05 M cacodylate buffer (Junsei Chemical Co. Ltd.). Cellular membranes were preserved by fixing the samples in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) diluted in 0.05 M cacodylate buffer for 1 h, washing again in 0.05 M cacodylate buffer, dehydrating in graded ethanol (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei Chemical Co. Ltd.), and drying in a fume hood. Finally, the samples were sputter-coated with gold and observed under a Hitachi S4700 field emission scanning electron microscope at the Korea Basic Science Institute (Gwangju, Korea).

To obtain samples for microscopic examination and growth rate determination, isolates CNUFC-1YSRS2-4 and CNUFC-GSNPF3-1 were cultured on each of the 3 media: PDA, malt extract agar (MEA; 33.6 g MEA in 1 L of deionized water; Becton, Dickinson and Co.), and oatmeal agar (OA; 1.5% oatmeal and 1.5% agar in 1 L of deionized water; Junsei, Tokyo, Japan). The plates were incubated at 25℃ in the dark for 7 days. Samples were mounted in a drop of distilled water on a glass slide and were examined under an Olympus BX51 microscope with DIC optics (Olympus, Tokyo, Japan). Fine structures of the fungi were analyzed by scanning electron microscopy (Hitachi S4700 field emission scanning electron microscope; Hitachi, Tokyo, Japan). Samples were fixed in 2.5% paraformaldehyde-glutaraldehyde buffer with 0.05M phosphate (pH 7.2) (Junsei) for 2 hr and washed in cacodylate buffer (Junsei). Cellular membranes were preserved by fixing the samples in 1% osmium tetroxide (diluted in cacodylate buffer; Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hr, by washing again in cacodylate buffer, dehydrating in a graded series of ethanol solutions (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei), and by drying in a fume hood. Finally, samples were covered with gold in a sputter coater and examined at Korea Basic Science Institute, Gwangju, Korea.

For microscopic examination and determination of the growth rate, EML-KWD01 isolate was grown on PDA, oatmeal agar (OA; oatmeal 30 g/L, agar 15 g/L), malt extract agar (MEA; Difco), or V-8 juice agar (V-8; Campbell, Camden, NJ, USA) at 18℃, 25℃, 32℃, and 37℃ in the dark for 7 days. Morphological characteristics of fungal structures were determined under a light microscope (DFC290; Leica, Wetzlar, Germany) after preparing lactophenol slide mounts. Fine structures of the fungus were observed using scanning electron microscopy. Fungal samples were cultured on PDA medium in the dark at 27℃ for 7 days. Samples were fixed in 2.5% paraformaldehyde-glutaraldehyde buffer with 0.05 M phosphate (pH 7.2; Junsei, Tokyo, Japan) for 2 hr and washed in carcodylate buffer (Junsei). Cellular membranes were preserved by fixing the samples in 1% osmium tetroxide (diluted in carcodylate buffer; Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hr, washed again in carcodylate buffer, dehydrated in graded ethanol (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei), and dried under a fume hood. Finally, these samples were covered with gold in a sputter coater and observed using a Hitachi S4700 field emission scanning electron microscope (Hitachi, Tokyo, Japan) at Korea Basic Science Institute, Gwangju, Korea.