ABSTRACT
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.
Subject(s)
Ascomycota/isolation & purification , Botrytis/isolation & purification , Candida/isolation & purification , Laccase/genetics , Polymerase Chain Reaction/methods , Amino Acid Sequence , Ascomycota/classification , Base Sequence , Botrytis/classification , Candida/classification , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal Spacer/chemistry , DNA, Ribosomal Spacer/genetics , Food Microbiology , Genes, Fungal/genetics , Genetic Markers , Introns , Molecular Sequence Data , Mycological Typing Techniques , Phylogeny , Sensitivity and Specificity , Sequence Analysis, DNA , Species SpecificityABSTRACT
To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-labeled specific oligonucleotides for fluorescence in situ hybridization (FISH). These probes are partial complementary to each other. This feature promotes the accessibility to the target sequence within the ribosome and enhances the fluorescence signal. For the rapid identification of Oenococci both the 5S rRNA gene and the ITS-2 region are useful targets.
