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

Authors: WANG, XUEFEI - Washington State University; GLAWE, DEAN - Washington State University; Weller, David; Okubara, Patricia

Submitted to: Journal of Microbial Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2019
Publication Date: 11/23/2019
Citation: Wang, X., Glawe, D.A., Weller, D.M., Okubara, P.A. 2019. Real-time PCR assays for the quantification of native yeast DNA in grape berry and fermentation extracts. Journal of Microbial Methods. 168:105794. https://doi.org/10.1016/j.mimet.2019.105794.
DOI: https://doi.org/10.1016/j.mimet.2019.105794

Interpretive Summary: Native yeasts are distinct from starter yeasts that are introduced to finish the conversion of sugar to alcohol during wine fermentation. Native yeasts are not directly altered by the hand of man; they naturally colonize grape berries and other vine tissues and become a part of the fermentation upon crush. They also are known to have biocontrol activity against grape pathogens and to impart flavors, aromas and textures (some undesirable) to wines. We developed eight molecular diagnostic assays for selected native yeast species or species pairs, in order to provide vineyard managers and winemakers the tools for monitoring key yeasts that have the potential impact on vineyard health and wine quality. The advantages of these diagnostics are that they are rapid, sensitive and specific, and can be made available to members of the wine industry through a commercial service.

Technical Abstract: Native yeasts are part of the microbial community in grape vineyards and have roles in the alcoholic fermentation process and wine quality. Monitoring populations of native yeast in the vineyards and during the fermentation process is important for providing viticulturalists and oenologists with the microbiological information needed to suppress spoilage microbes or enhance desirable yeasts. This is especially the case in low-intervention winemaking methods that depend primarily on fermentation by native yeasts. In this study, we developed real-time polymerase chain reaction (qPCR) assays to rapidly detect and quantify eight grape yeast species or species combinations that occur in Washington State and in vineyards throughout the world. The assays targeted Candida californica, Curvibasidium pallidicorallinum, Hanseniaspora uvarum plus H. opuntiae, Metschnikowia pulcherrima plus Mt. chrysoperlae, Meyerozyma caribbica plus Me. guilliermondii, and Saccharomyces cerevisiae. We also developed assays for the spoilage yeast Brettanomyces bruxellensis, and the yeast-like fungus Aureobasidium pullulans. Primers were designed to sequences in the internal transcribed spacer (ITS) and large ribosome subunit (LSU) region of the 26S ribosomal RNA subunit. Known cell populations of the strains, added to grape juice, were significantly correlated to amounts of purified DNA (pg) for six of the yeasts; exceptions were A. pullulans and Cur. pallidicorallinum. The utility of the Metschnikowia, Meyerozyma and S. cerevisiae assays was further validated using fermentation samples from a yeast community profiling project, for which there was a strong correlation between the number of sequences in the selected fermentation samples and pg of DNA from qPCR. Overall, the assays will aid in species identification and monitoring of specific yeasts for various applications.