Identification of Brettanomyces/Dekkera Species Based on Polymorphism in the rRNA Internal Transcribed Spacer Region

Abstract

Brettanomyces/Dekkera yeasts grow in wine mainly during barrel aging. Their presence is often associated with formation of off-flavors. This potential spoilage generates a strong demand for a sensitive, rapid, and reliable identification procedure. Ribosomal DNA restriction fragment length polymorphism and comparative sequence analysis of the two internal transcribed spacer (ITS) regions located between the ribosomal RNA genes was carried out using Brettanomyces/Dekkera yeast reference strains and wine isolates. ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. Specific oligonucleotides were designed for each Brettanomyces/Dekkera species and evaluated for specificity and reliability. No cross-reaction products were detected when the specific primers were assayed in a PCR reaction with Brettanomyces/Dekkera, strains of different species or other wine-related non-Brettanomyces/Dekkera yeasts. Thus, PCR using a combination of all four specific primers gave a specific and reproducible detection assay for the genus Brettanomyces/Dekkera. Use of these specific primers allowed for species-specific discrimination. Brettanomyces/Dekkera. yeast isolates from wine were shown to uniquely belong to the species B. bruxellensis.