Yeast Populations Residing on Healthy orBotrytis-Infected Grapes from a Vineyard in Attica, Greece

Abstract

The yeast flora associated with healthy and Botrytis-infected grapes was assessed. Molecular identification methods assigned isolates to six genera and nine species. For the first time Hanseniaspora opuntiae was encountered as an inhabitant of the grape ecosystem. By using DraI, an informative restriction fragment length polymorphism pattern was generated to distinguish H. opuntiae from the closely related organism Hanseniaspora guilliermondii. Botrytis infection resulted in a larger population and greater diversity of yeasts enriched with fermentative or spoilage species.Grape berries, especially the interface between soluble nutrients and the septic world, are common niches for yeasts. From a biotechnological point of view, grapes are the primordial source of the microorganisms necessary for alcoholic fermentation to occur, providing must with both beneficial and potentially spoilage species. Nevertheless, the yeast flora of grapes is surprisingly poorly documented (13, 19). As determined so far, the physiognomy of the grape microflora may change in response to factors such as the climate, grape variety, and geographical region (4,18,21). Biological invasion is a critical concern for widespread changes in the community. Botrytis is among the most important pathogens that cause grape damage (gray rot) or drying (noble rot), yet its role in yeast ecology has not been studied previously. We assessed and compared yeasts present on Botrytis-infected and healthy grapes. Different molecular methods were used for species identification, and the robustness of these methods is discussed below.Grape samples were collected at the time of harvest (2005 vintage) from the experimental vineyard of the Agricultural University of Athens (37 58Ј⌵, 23 32Ј⌭; 30 m above sea level). The grapevines were treated with ground sulfur rock during the spring, and no other chemicals were applied after this. 'Mavroliatis' and 'Sefka', two red Vitis vinifera varieties, were included in the analysis. Vines of each variety were cultivated in parallel single rows that were 20 m long and 40 m apart. Healthy and rotten bunches of each variety were randomly and aseptically collected from throughout the rows. The platetrapped antigen enzyme-linked immunosorbent assay was used to confirm Botrytis infection of rotten grapes, using monoclonal antibody BC-12.CA4 (14) as described previously (5). One hundred grams of randomly collected berries from each sample was aseptically crushed with a Stomacher (Lab Blender 400), and the pH of the juice was recorded. D-Glucose/Dfructose and ethanol contents were determined by using appropriate enzymatic kits (Boehringer Mannheim/R-Biopharm, Germany). Decimal dilutions (10 Ϫ1 to 10 Ϫ6 ) in Ringer's solution were prepared, and 100-l portions were spread on different culture media. For enumeration and isolation of total yeasts, non-Saccharomyces yeasts, Saccharomyces, and Dekkera/ Brettanomyces spp., samples were spread in triplicate on Wallerstein laboratory nutrient agar (Oxoid Ltd.), lysine medium agar (Oxoid Ltd.)...

Association between Grape Yeast Communities and the Vineyard Ecosystems

“…On the other hand, H . opuntiae has been less frequently reported as inhabitant of grape ecosystem [9,66–68], but in our study this species corresponded to 3.5% of the total isolates from freshly crushed grapes, despite being found only in 2 samples. Interestingly, in both samples H .…”

Yeast ecology of vineyards within Marsala wine area (western Sicily) in two consecutive vintages and selection of autochthonous Saccharomyces cerevisiae strains

“…Interestingly, this species was found when H. uvarum was absent and its presence was more frequent in the vintage 2009. H. opuntiae has been reported to be a member of the grape ecosystem (38) and to dominate the first stages of alcoholic fermentation (39), but no information is available in literature on its presence at the late phases of the process. In this work H. opuntiae was detected at approximately 10 7 CFU/mL at 3/5 sugar consumption.…”

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

“…Restriction fragment length polymorphism (RFLP) can be used in conjunction with ITS-PCR to differentiate closely-related species such as those of the Saccharomyces sensustricto complex 18 . PCR amplification of rDNA and restriction digestion of the amplicon has not been used extensively for the identification of yeasts isolated during brewing or from beer 27,53 , but has seen wide application in other fermentation industries, particularly for the identification of yeasts occurring during oenological fermentations 13,14,17,18,21,22,36,43,44,48,62 . The technique has also been used to identify yeasts occurring as contaminants or natural, spontaneous fermenters of cider 15,39 and rice wine 28 as well as yeast associated with non-alcoholic citrus juice beverages 3,32 .…”