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. 2012 Feb;78(3):735-43.
doi: 10.1128/AEM.06518-11. Epub 2011 Nov 18.

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Matilde Maqueda  1 , Emiliano Zamora, María L Álvarez, Manuel Ramírez
Affiliations

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Matilde Maqueda et al. Appl Environ Microbiol. 2012 Feb.

Abstract

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.

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Figures

Fig 1
Fig 1
Ecological distribution of the Saccharomyces sensu stricto killer yeasts among the five wine-producing subareas (A) and the six vintages (B) analyzed. White, nonkiller yeasts; black, killer K2 yeasts; gray, killer Klus yeasts. T, mean daily temperature during the ripening/harvest season; P, rainfall during the ripening/harvest season.
Fig 2
Fig 2
Genetic determinants of Klus and K2 toxins. (A and B) Presence of L-A and M dsRNA molecules in K2 (A) and Klus (B) strains. Nucleic acids were obtained from reference killer yeasts K1 (F166), K2 (EX73), K28 (F182), Klus strains (Mlus-1 to Mlus-4), and K2 strains (M2-1 to M2-4) containing different virus isotypes. Samples were separated by agarose gel electrophoresis. The ethidium bromide staining of the gel is shown. The size markers correspond to a 1- to 15-kb molecular ruler (Bio-Rad).
Fig 3
Fig 3
Frequencies of yeast isolates containing each virus isotype with each killer phenotype intensity. (A) Yeasts containing M2 virus isotypes. (B) Yeasts containing Mlus virus isotypes. The thicknesses of the growth inhibition halos (killer phenotype intensities) were as follows: white, less than 1 mm; gray, 1 to 2 mm; black, more than 2 mm.
Fig 4
Fig 4
Representative examples of the three different killer wild yeast population dynamics found among the spontaneous must fermentations. (A) The dominant yeasts were killer K2. (B) The dominant yeasts were killer Klus. (C) The killer K2 and Klus yeasts codominated at the end of must fermentation. White, nonkiller yeasts sensitive to K2 toxin; light gray, nonkiller yeasts resistant to K2 toxin; dark gray, killer Klus yeasts; and black, killer K2 yeasts. Dots and solid line, CFU of yeast/ml.
Fig 5
Fig 5
Representative vinification types according to the presence/absence of killer yeasts and the effect of the killer phenotype on the killer-sensitive yeast population. (A) VK-0. (B) VK-L. (C) VK-H. White, nonkiller yeasts sensitive to K2 toxin; gray, nonkiller yeasts resistant to K2 toxin; and black, killer K2 yeasts. Dots and solid lines, CFU of yeast/ml.
Fig 6
Fig 6
Average changes in the proportion of killer yeasts during the VK-L and VK-H fermentations. White, nonkiller yeasts sensitive to K2 toxin; grey, nonkiller yeasts resistant to K2 toxin; and black, killer K2 and Klus yeasts. Dots and solid lines, CFU of yeast/ml. The data are the mean values of 17 VK-L and 8 VK-H fermentations. The error bars indicate standard errors of the mean.
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