Abstract

OBJECTIVES Scopulariopsis species and their teleomorphs of the genus Microascus are commonly isolated from soil, decaying plant material and indoor environments. Moreover, certain Scopulariopsis and Microascus species are recognised as opportunistic human pathogens. Although most species can be identified by detailed morphological study, phenotypic characters appear to overlap in several cases and morphology seems to be insufficient for establishing species limits in these fungi. Molecular typing has improved understanding of species concepts in many fungal groups, but such a study has not been performed for Microascus and Scopulariopsis genera. The aim of this study was to investigate the relationships between Scopulariopsis and Microascus isolates and evaluate the differentiation potential of six RAPD assays towards fungi of Scopulariopsis and Microascus genera. METHODS The study included DNA samples of 38 Scopulariopsis and Microascus isolates representing 24 species, isolated with Brillowska-Dąbrowska method (J Clin Microbiol 2007; 45(4):1200-4). The strains were obtained from international culture collections (CBS; BCCM/IHEM; DSMZ) and Molecular Biotechnology and Microbiology Department collection of fungi (Gdańsk University of Technology, Gdańsk, Poland). Six primers: LIAN2004-4, LIAN2004-7, CgAP50-1, (GACA)4, CgRAPD-EDP4, CgRAPD-EDP8 were evaluated in RAPD assays. RAPD mixtures, of 20 µ each, consisted of 10 µl of 2x PCR Master Mix Plus High GC (A&A Biotechnology, Gdynia, Poland), 0,2 µl of primer at 100 µM, and 2 µl of DNA. RAPD time-temperature profile included initial denaturation for 3 min at 94°C followed by 40 cycles of 45 s at 94°C, 45 s at 45°C, and 90 s at 72°C. The presence of amplicons was examined electrophoretically on 2% agarose gels, stained with ethidium bromide. RESULTS Application of RAPD analysis of DNA extracted from Scopulariopsis and Microascus fungi revealed differences between species and even single isolates among species. We were able to recognise between 20 and 24 genotypes, depending on the primer used. CONCLUSION In this study we found, that RAPD is sufficient for typing of Scopulariopsis and Microascus fungi. Obtained results support recent redefinition of Scopulariopsis, Microascus and allied genera by Sandoval-Denis et al. (Persoonia 2016; 36:1-36).

Authors (3)