Real-time PCR assays for the quantification of native yeast DNA in grape berry and fermentation extracts

Native yeasts comprise part of the microbial community in grape vineyards and play roles in alcoholic fermentation and wine quality. Monitoring populations of native yeast in vineyards, during fermentation and after bottling will provide viticulturalists and oenologists with information needed to help control spoilage and to enhance desirable wine properties. This is especially crucial for low-intervention winemaking, in which fermentation is driven by native rather than starter microbes. In this study, we report real-time polymerase chain reaction (qPCR) assays for rapid quantification of seven grape yeast species or species combinations that occur in vineyards of Washington State and throughout the world. The assays targeted Candida californica, Curvibasidium pallidicorallinum, Metschnikowia spp., Meyerozyma caribbica/Me. guilliermondii, and Saccharomyces cerevisiae/S. bayanus. We also developed assays for the spoilage yeast Brettanomyces bruxellensis, and the yeast-like fungus Aureobasidium pullulans. Primers were designed for sequences in the internal transcribed spacer (ITS) and large ribosome subunit (LSU) gene. Known populations of yeast cells, added to fermentation extract, were significantly correlated to amounts of purified DNA in picograms (pg) for most of the yeasts; exceptions were A. pullulans and Cu. pallidicorallinum. The utility of the Metschnikowia, Meyerozyma and Saccharomyces assays was further validated by good correlations (R² = 0.75-0.83) between the number of target sequences and pg of DNA from qPCR for selected vineyard and fermentation samples. Overall, the assays will aid in species identification and monitoring of specific yeasts from cultures, vineyards and fermentation samples. Topics: Food Microbiology, Microbiological Method.