Prevention of wood decay by fungi of the phylum Basidiomycota is an important issue of wood protection. The most destructive fungus in buildings in Central Europe is the dry rot fungus Serpula lacrymans, which needs more extensive restauration measures than all other wood decay fungi. Its close relative S. himantioides occurs more frequently in outdoor environment. The assessment and remediation of fungal damages require an identification of the infesting species, in particular the proof or exclusion of S. lacrymans. Morphological identification fails, if no fungal structures (fruit bodies, spores, mycelia and strands) with specific characteristics are available. In this case, DNA based methods can be used. For detection of S. lacrymans and S. himantioides a conventional PCR procedure using specific primers from the ITS region (Internal transcribed spacer of nucleus ribosomal DNA) is well established (Schmidt 2009, Jacobs et al. 2011). But, in the analysis of environmental samples, numerous problems arose in recent years. The most important difficulties are the interpretation of equivocal agarose gel findings and additional analysis efforts with small DNA amounts, degraded DNA, or high content of inhibitory substances in the sample material. To overcome these disadvantages of conventional PCR, new diagnostic assays for the detection of S. lacrymans and S. himantiodes were developed. The four different assays are based on real-time PCR, using partially specific primers and species specific TaqMan probes. The assays are targeting two genomic DNA regions, the multi-copy ITS and the single-copy beta-tubulin gene (BET). Thus, the diagnostic findings can be confirmed by two independent detection parameters. It was expected that the high specificity of the TaqMan probe technology prohibits the formation of unspecific byproducts and PCR artifacts. As a result of the work, primers and specific probes were generated from verified reference data of the two target regions. Basic performance parameters of the designed assays were optimized. The experimentally determined limit of detection (LOD) with the ITS targeting assays was 0.5 pg genomic DNA of S. lacrymans or S. himantioides, and with the BET targeting assays 1.0 pg. Out of twenty reference fungi, all six included Serpula strains could be clearly assigned to the appropriate fungus, and no false findings with other fungi were obtained. A first exploratory practice test was done with ten environmental samples, taken from damaged wood. These tests confirmed that the new diagnostic tool is a fast and reliable method for detection of the two Serpula-species. Non-specific reactions, side reactions and the production of PCR artifacts could be eliminated completely. A further considered issue of assay development was the DNA isolation procedure, which is time-consuming and susceptible to both, cross contaminations and loss of small samples. Therefore, direct PCR approaches were tested by processing whole cells from suspended fresh spores as well as milled dry mycelia directly in PCR reactions. It could be shown that pure culture material can be successfully processed in a direct PCR without any prior cell disruption and DNA isolation procedure. With environmental samples, the strategy worked in case of fresh mycelia and fruit bodies but findings were not reliable with decayed wood and aged fungal strands. Further studies are necessary to clarify terms and limits of practical application.

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