Discrimination of Brettanomyces/Dekkera yeast isolates from wine by using various DNA finger-printing methods

Abstract

In the recent years Brettanomyces/Dekkera yeasts are posing an increasingly severe quality problem in the wine industry. The early and specific detection of these yeasts is therefore needed. Here, we describe the application of genetic techniques in addition to routinely performed physiological tests to identify yeasts isolated from Cabernet Sauvignon wines characterized by wine-makers panels as having 'betty' aromas. Brettanomyces/Dekkera reference strains from a type culture collection were included for comparison. A RAPD-PCR assay was developed for species- and strain-specific discrimination of these yeasts. These data were compared to the chromosomal patterns of uncleaved, Sfi 1-digested DNA, and to the physiological behavior of the yeasts. Karyotyping gave clear distinctions, but did not allow for relatedness studies due to the lack of pattern conservation among species and strains. Conversely, RAPD-PCR allowed for species discrimination within the genus Brettanomyces and strain discrimination within the species D. bruxellensis. All wine-isolated Brettanomyces/Dekkera yeasts belonged to the species B. bruxellensis. Populations derived from one single clone were found in the 1992 and 1994 vintage wines, but in contrast, the 1989 yeast population differed from these two vintages: two different strains were found instead of a homogenous population. In conclusion, we show that RAPD-PCR can be successfully applied to discriminate Brettanomyces/Dekkera yeasts on the species and strain level, representing an accurate alternative to conventional physiological tests.