The suspensions obtained from each soil samples were seeded onto nutritive plates, and incubated in triplicate over a range of temperatures (4, 10, 15 and 22°C). After 30–90 days of incubation, approximately 30 to 60 yeast-like colonies developed on each plate. In contrast, no colonies or low colony numbers (4 to 8) appeared on plates from water samples. Because large numbers of
isolates were obtained, isolates were grouped according to their isolation growth temperature and colony characteristics such as pigmentation, texture, elevation and size. Among the 64 groups, several differed only by isolation growth temperature. These isolates were buy I-BET-762 grown at different temperatures and re-grouped according to macromorphological characteristics at their optimal growth temperature. In this way, 35 groups were ultimately generated. Several isolates from each group (at least one isolate per sampling site; a total of 78 isolates) were selected for molecular and biochemical analyses. Molecular identification of yeasts The chromosomal DNA was purified from cultures of each yeast isolate and the D1/D2 region of 26S rDNA and the ITS1-5.8S- ITS2 (hereafter designated the ITS region for simplicity) regions of the rDNA were amplified OSI-027 chemical structure by PCR. The amplicons obtained were purified from gels and sequenced on both strands. Isolates showing 100% identity in both rDNA sequences
were grouped and their DNA sequences were submitted to GenBank under the accession numbers Anlotinib ic50 listed in Table 1. Species identification was performed
by comparison with the GenBank references, using as criterion the Blast-hits with ≤ 0.5% difference with the query [14]. In 84% of the isolates the closest Blast-hits obtained for both rDNA sequences were coincident. When this NADPH-cytochrome-c2 reductase was not the case, the D1/D2 results were used for identification because they yielded higher identity percentages than did the ITS (see Additional file 1). 76% of the isolates could be identified to species level by this molecular analysis. 22 species belonging to12 genera were identified, of which 80 and 20% were Basidiomycetes and Ascomycetes, respectively. The genera containing the highest number of species were Mrakia (5 species) and Cryptococcus (4 species). However, the species Sporidiobolus salmonicolor was the most abundant, being identified in 24 isolates from 13 different sampling sites. Mrakia gelida was the only yeast species present in both water and soil samples. Of the three isolates identified as Leuconeurospora sp., two of them (T11Cd2 and T27Cd2) possessed identical D1/D2 and ITS sequences, both of which differed from the third (T17Cd1) by 0.7%. However, the macromorphological characteristics of the three isolates, including pigmentation, differed markedly under identical culture conditions (see Additional file 2). Because of these discrepancies, the molecular and morphological analyses were repeated several times, but the results were highly consistent.