A comparison of microbiota isolation methods reveals habitat preferences for fermentative yeasts and plant pathogenic fungi in the grape berry

Abstract

The methodologies for profiling the grape berry microbiota have exponentially evolved in the past 25 years. Recently, concerns arose regarding the homogeneity in the protocols of grape harvesting, sequencing and bioinformatic analyses, but the bias introduced by the microbiota isolation method is still unexplored. This study followed a simple approach of comparing two most used methods of microbiota collection from grape berries (washing vs crushing), hypothesizing a significant impact in the outcome of the microbiota profiles analyzed by NGS metabarcoding. Experiments conducted in fruits of three cultivars of the Douro wine region showed that only 52 % of OTUs were common to both surface and juice microbiota, suggesting specific microbial niches. Thirteen fungal genera were abundantly detected in the fruit surface, including Alternaria, Aureobasidium, Cladosporium, Didymella and Bipolaris. Fermentative yeasts including Meyerozyma and Saccharomyces cerevisiae were exclusively detected in the juice, together with several Penicillium species. Distinct habitat preferences of species within the genera Alternaria, Sporobolomyces and Rhodotorula were also revealed. The study showed that the microbiota isolation method is crucial in the detection of certain plant pathogenic/saprophytic fungi and yeasts with biotechnological and oenological interest, adding novelty to the globally accepted assumption that S. cerevisiae in musts originates primarily from the cellar.