Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

Abstract

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera <i>Cladosporium</i>, <i>Geomyces</i>, <i>Fusarium</i>, and <i>Mortierella</i> could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. <i>Absidia</i>, <i>Mucor</i>, <i>Trichoderma</i>, and <i>Penicillium</i> isolates had noticeably lower PCR success.