Since the yeast flora of Slovakian enology has not previously been investigated by culture-independent methods, this approach was applied to two most common cultivars Frankovka (red wine) and Veltlin (white wine), and complemented by cultivation. Model samples included grapes, initial must, middle fermenting must and must in the end-fermentation phase. The cultured isolates were characterized by length polymorphism of rDNA spacer two region using fluorescence PCR and capillary electrophoresis (f-ITS PCR), and some were identified by sequencing. The microbial DNA extracted directly from the samples without cultivation was analysed by f-ITS PCR, amplicons were cloned and sequenced. The use of universal fungal primers led to detection of both yeasts and filamentous fungi. The amplicon of highest intensity and present in all the samples corresponded to Hanseniaspora uvarum. Other species demonstrated by both approaches included Saccharomyces sp., Metschnikowia pulcherrima or M. chrysoperlae, Candida zemplinina, Cladosporium cladosporioides, Botryotinia fuckeliana, Pichia anomala, Candida railenensis, Cryptococcus magnus, Metschnikowia viticola or Candida kofuensis, Pichia kluyveri or Pichia fermentas, Pichia membranifaciens, Aureobasidium pullulans, Alternaria alternata, Erysiphe necator, Rhodotorula glutinis, Issatchenkia terricola and Debaryomyces hansenii. Endemism of Slovakian enological yeasts was suggested on the level of minor genetic variations of the known species and probably not accounting for novel species. The prevalence of H. uvarum over Saccharomyces sp. in the samples was indicated. This is the first culture-independent study of Slovakian enology and the first time f-ITS PCR profiling was used on wine-related microbial communities.
Aims: The investigation of yeast microflora during the must fermentation of two wine varieties (Frankovka modra – Blaufränkisch and Veltlinske zelene – Grüner Veltliner) from two consecutive vintages was performed using a three‐step approach.
Methods and Results: The investigation strategy consisted of the combination of yeast cultivation, selection of the isolated yeasts based on the amplification of internal transcribed spacer 2 using a fluorescence‐labelled primer (f‐ITS‐PCR) and a final identification step based on amplification and sequencing of the ITS1‐5.8S rDNA‐ITS2 region of the selected yeasts. By this three‐step approach, it was possible to screen 433 yeasts isolates that belonged to 13 different species.
Conclusions: The f‐ITS‐PCR allowed the unambiguous differentiation of all isolated yeast species that produced their typical f‐ITS‐PCR profile.
Significance and Impact of the Study: This is one of few reports that treat the yeast diversity in Slovakian wines and in two varieties largely cultivated in Central Europe. The three‐step approach permitted the rapid and reliable identification of isolated yeasts. The f‐ITS‐PCR with its good discrimination power can represent a suitable molecular tool for the selection of yeast members recovered from food or other environments.
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