Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA

Four different primer pairs were designed for the preferential PCR amplification of fungal SSU rDNA directly from environmental samples. Most of the amplification products obtained from a reference collection of 46 wood-decomposing fungi could be separated by denaturing gradient gel electrophoresis (DGGE) using 1650 bp rDNA fragments produced by primer pair FR1 + NS1. A relatively high level of resolution was also achieved using 390 bp products amplified with primer pair FRI + FF390. In contrast, the separation of amplification products obtained by the remaining two primer pairs (FR1 + FF700 and FR1 + FF1100; 700 bp and 1100 bp, respectively) was inadequate when applied to our fungal collection. Differentiation between all the species tested was achieved by combined analysis of the rDNA fragments produced by primer pairs FR1 + NS1 and FRI + FF390. The DGGE analysis of environmental samples collected from Norway spruce stumps showed that the analysis of fungal DNA extracted directly from wood was usually in accordance with the investigation of mycelial cultures isolated from the same decay column. In some cases, however, disagreement was observed, which suggests that these two fundamentally different techniques present different views about fungal diversity. The investigation of fungal species profiles directly from environmental samples using DGGE analysis of PCR-amplified SSU rDNA molecules can be used to improve the detection of fungal groups that are difficult to cultivate.