Detection of Dekkera/Brettanomyces bruxellensis in pressed Sangiovese grapes by real time PCR

Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) is a controversial yeast that is relevant for the wine industry. Although Dekkera/ Brettanomyces have been extensively studied, their ecological niche has not been well defined. In this study, a Real Time PCR-based assay was applied to the microbial DNA extracted directly from pressed Sangiovese grapes. Two different DNA extraction procedures were compared in an attempt to obtain a clean and QPCR-amplifiable DNA from grapes. Plasmidic DNA was used to evaluate both the extraction efficiency and the matrix effect. Such an effect was analysed in order to circumvent the difficulty of using filtersterilized pressed grapes and to obtain a calibration curve for quantifying D./ B. bruxellensis. Using specific primers for D./B. bruxellensis the number of these yeasts in Sangiovese grapes was in the range of 10 3-10 4 cells mL -1. This evidence supports the hypothesis that D./B. bruxellensis belongs to the native Sangiovese grape microflora, and that they can be detected by the method applied. Identifying the environmental reservoir of D./B. bruxellensis is of great interest to the wine industry. The method developed in this study, when applied to other vine species, could increase the knowledge about these controversial yeasts.