Investigation of yeasts and yeast-like fungi associated with Australian wine grapes using cultural and molecular methods

摘要

This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation.The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries.PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures.Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice.A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.