Multiplex PCR for species discrimination of Sclerotiniaceae by novel laccase introns

Common PCR-based targets for the identification of filamentous fungi and yeasts are the sequences of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Within the Sclerotiniaceae the ITS-region is homogenous and the identification is almost impossible. Furthermore, the lack of IGS-data (intergenic spacer region) requires new specific marker genes for a rapid identification of phytopathogenic Sclerotiniaceae. We sequenced and analyzed new laccase2 ( lcc2) genes from the phylogenetically related Sclerotinia sclerotiorum (Lib.) de Bary, Sclerotinia minor Jagger, and Monilinia fructigena Honey. Comparative analysis revealed remarkable differences in length and sequence compared to the well-known lcc2 gene of Botrytis cinerea caused by a different number of intron sequences. These results gave us the possibility to develop a primer set for a rapid multiplex PCR-identification of different species in environmental samples, e.g. wine, fruit, or soil. Therefore, the application of this technique allows the simultaneous detection of different phytopathogenic Sclerotiniaceae in complex microbiota like decomposed herbal material. In the present study prevailed problems in the field of a general identification of fungal specimen are highlighted.