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Of those, 918 isolates (352 from 2002 and 566 from 2003) had been unable to utilize lysine as a nitrogen source and had been hence identified as representatives in the Saccharomyces genus (in accordance with [21,22]). The S. cerevisiae strain 6167 along with the H. uvarum 1-03 strain had been utilised as controls.. S. cerevisiae and S. bayanus would be the most representative species located in late fermentation musts [1]; as a result the 918 Saccharomyces isolates were plated on vitamin-free media (Biolife-Italy), to identify S. bayanus yeasts (which develop on this medium; [22]). The S. bayanus 11719 as well as the S. cerevisiae 6167 strains have been utilised as controls. No S. bayanus isolate was discovered. Thus we provisionally assigned our 918 isolates for the S. cerevisiae species.on the reaction was digested with three U in the HaeIII restriction endonuclease. Upon digestion, all of the Mild and by no means reached significance (Supplementary Figure S2). To explore this amplicons developed four fragments of 320, 225, 180 e 145 bps, typical with the S. cerevisiae and S. paradoxus species. A S. cerevisiae-specific PCR reaction was then performed with the SC1 (59-AACGGTGAGAGATTTCTGTGC-39) and SC2 (59-AGCTGGCAGTATTCCCACAG-39) primers, as described in [28].Phenotypic characterizationFermentation vigor and sulfite tolerance jir.2010.0097 have been assessed based on [14].Samples from web page B (B2 and B3) and one particular sample from web page D (D1) were selected. From the 2003 harvest, eleven samples have been obtained: 3 from site A (A5 to A7), 3 from website B (B4 to B6), two from web-site D (D2 and D3) and one from each and every of web-sites E, F jir.2014.0227 and G (E1, F1 and G1). Musts samples from stone-concrete fermentation troughs were put in sterile containers, a 50 (v/v) must:glycerol mixture was obtained and swiftly stored at 280uC (for no longer than eight months) to preserve microorganism viability. Saccharomyces colonies had been isolated as follows. Musts had been sequentially diluted from 1:ten to 1:100,000 in 0.1 (w/v) sterile peptone. 0.2 ml of every dilution was spread on WL Nutrient Agar Oxoid. After 4 days in culture at 28uC, three colony morphologies had been detected: 1-colonies having a creamy to greenish colour and having a knob-like, opaque, smooth surface, typical on the Saccharomyces/Torulaspora genera [18]; 2-flat colonies of intense green color, smooth and opaque surface, standard of Hanseniaspora/Materials and Procedures Yeast strainsThe S. cerevisiae strain L404 and 6167 as well as the S. bayanus strain 11719 belong for the DIPROVAL collection with the University of Bologna (commercialized by Oliver-Ogar, Italy). The S. cerevisiae EC1118, ICV D254, QD145 and RC212 strains are commer-Figure 1. Analysis location (A) and location with the wineries (B) exactly where need to sampling was carried out (collection websites are indicated by capital letters). doi:ten.1371/journal.pone.0030428.gPLoS One | www.plosone.orgYeast Biodiversity Economic PotentialKloeckera genera [18]; 3-colonies using a dark intense green center, clear rim and domed surface, referred as Candida stellata [19] (and most most likely belonging towards the Candida zemplinina species [20]). Need to samples with morphology 1 inside a ratio of 20:1 for the other individuals, have been chosen for additional evaluation.