Fundação para a Ciência e a Tecnologia (FCT) - POCI 2010; POCTI/AGR/56102/2004.FEDER.AGRO (ENOSAFE, Nº 762)

Casal, Margarida

Dellinger, B.

Graça, Magda Maria Couto da Silva

Schuller, Dorit Elisabeth

Universidade do Minho: RepositoriUM

IDENTIFICATION OF WINE RELATED YEAST SPECIES BY CAPILLARY ELECTROPHORESIS SINGLE- STRAND CONFORMATION POLYMORPHISM B. DELLINGER, M. SILVA GRAÇA, M. CASAL, D. SCHULLER* Departamento/Centro de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal; *For correspondence: Dorit Schuller, e-mail: dschuller@bio.uminho.pt  Introduction The yeast species Dekkera bruxellensis is the major cause of red wine spoilage worldwide due to the production of high amounts of volatile phenols (4-ethylphenol and 4-ethylguaiacol), [1]. Predominating spoilage yeasts of high-alcohol beverages are also Saccharomyces spp., Zygosaccharomyces bailii and Saccharomycodes ludwigii [2]. In recent years, rapid and very sensitive identification techniques, mostly PCR-based, have been developed and applied for spoilage yeast detection in wine, such as nested PCR, AFLP, RT-PCR, FISH, PCR-RFLP and TGGE [3]. Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) is a powerful analysis technique that separates heat denaturated DNA fragments of the same length according to their sequence. This technique presents very high sensitivity, detecting differences up to one base pair [4]. CE-SSCP has been applied in studies such as profiling of bacteria and yeast communities in raw milk Salers cheeses [5], but has not been used so far for wine yeast analysis. The aim of the present study was to develop and validate a new identification method, capable to distinguish between various wine yeasts, including the most relevant spoilage yeast species.   2. Materials and Methods The 66 yeast strains (24 species) used in the present study are shown in Table 1. DNA isolation was performed using a previously described method [6]. From multiple sequence alignments of the D1/D2 domains of the 24 species a 164 bp region was chosen and amplified by primer pair NL5 (5’CGAGTTGTAATTTGGAGA-3’)-NL6 (5´TACCACCCACTTAGAGCT3’). The 5’ ends of NL5 and NL6 were labeled with the fluorescent dye HEX and 6-FAM, respectively. PCR reactions contained 20 ng template DNA, 0.4 mM of each primer, 2 mM MgCl2, 0.2 mM of each dNTP, 1x Taq polymerase buffer (MBI Fermentas) and 0.5 U Taq polymerase (MBI Fermentas). PCR conditions were as follows: initial denaturation at 94 ºC for 4 min, followed by 36 cycles of 30 sec at 94 ºC, 30 sec at 50ºC and 30 sec at 72 ºC, with a final extension at 72 ºC for 10 min. PCR products were diluted (1:40) and 1 μl was combined with 11.25 μl deionized formamide, 0.25 μl DNA molecular weight standard Genescan-500 (PE, Applied Biosystems) and 0.5 μl 0.3 N NaOH. Samples denaturated at 95ºC for 5 min and CE-SSCP analysis was performed at 25ºC on an ABI Prism 310 genetic analyzer [7]  using Gene Scan Analysis Software.   3. Results and Discussion  CE-SSCP analyses were applied to a collection of 21 mostly wine-related yeast species, including also taxonomically close species. As summarized in Table 1, it was possible to separate 19 of 21 species based on their combined mobility values. Sequencing of the 164 bp fragment revealed between one and 67 base pair differences among clearly distinguishable species (data not shown).  CE-SSCP analyses clearly distinguished the closely related Saccharomyces sensu stricto species S. bayanus, S. cerevisiae and S. paradoxus, that showed mobility values of 163/195, 168/194 and 167/195 in the HEX/6-FAM labelled strands, respectively. Two pairs of species (D. anomala and Candida vini; Hanseniaspora uvarum and Saccharomyces ludwigii) presented very similar/identical mobility values (161/194 and 161/193; 184/193 and 184/193 respectively, in the HEX/6-FAM labelled strands) and cannot be distinguished by CE-SSCP. With the exception of one Z. bailii strain, no base pair differences were found among multiple strains of a species. Candida famata and Candida stellata showed a more complex pattern consisting of multiple peaks, possibly due to the formation of several stable single strand conformations for both strands. With the exception of Metschnikowia pulcherrima, the HEX labelled strand showed lower mobility values than the 6-FAM labelled strand. The mobility values of the spoilage yeasts D. bruxellensis and Z. bailii can be clearly distinguished from S. cerevisiae (162/188, 162/182, and 168/194, in the HEX/6-FAM labelled strands, respectively). Intraspecific variation showed standard deviations of less than one mobility value between multiple strains of the same species. Standard deviation decreased with the temperature and showed values of 0.05 – 0.30 at 25 ºC, compared to 0.15 – 1.14 at 35ºC, which is in agreement with data from literature [7]. Variations derived from PCR amplifications, CE-SSCP sample preparations and repeated injections were between 0.1 and 0.7 mobility values (data not shown), corresponding to the values determined for inter-strain variation and indicating the high reproducibility of the method. Our results show that CE-SSCP can be successfully applied to analyze wine related yeast species, leading to highly reproducible results.  References [1] Chatonnet P, Viala C, Dubourdieu D (1997) Am J Enol Vitic 48:443-448 [2] du Toit M, Pretorius IS (2000) S Afr J Enol Viticult 21:74-96 [3] Fernández-Espinar MT, Martorell P, de Llanos R, Querol A (2006) in: Querol A and Fleet GH (Eds.) Yeasts in Food and Beverages. Springer Verlag Germany, pp. 55-82 [4] Tebbe CC, Schmalenberger A, Peters S, Schweiger F (2001) in: Rochelle, P. A. (Ed.), Environmental Molecular Microbiology: Products and Applications. Horizon Scientific Press, Wymondham, UK, pp 161-175 [5] Callon C, Delbes C, Duthoit F, Montel M-C (2006) Syst Appl Microbiol 29:172-180 [6] Lopez V, Querol A, Ramon D, Fernandez-Espinar MT. 2001. Int J Food Microbiol 68: 75-81 [7] Duthoit F, Godon JJ, Montel M-C (2003) Appl Environ Microbiol 69:384-3848 Acknowledgements  This study was financially supported by the programs POCI 2010 (FEDER/FCT, POCI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762). Table 1 - CE-SSCP mobility values for HEX/6-FAM labelled DNA strands of 60 yeast strains belonging to 21 species. Yeast strains were obtained from American Type Culture Collection (ATCC); Portuguese Yeast Culture Collection (PYCC) and Instituto Superior de Agronomia, Portugal (ISA).  Species Strain HEX 6-FAM  Species Strain HEX 6-FAMCandida cantarelli PYCC3073 166.21 190.06  Pichia guilliermondii ISA2126 174.64 197.08Candida famata PYCC3056 161.00 189.34  ISA2145 174.64 197.07 170.59   ISA2286 174.65 197.09Candida stellata CBS157 161.45 192.63  Saccharomyces bayanus PYCC4456T 163.26 195.48 177.52 197.55  PYCC4565 163.46 195.63Candida veronae PYCC3664 160.81 189.15  PYCC4569 163.37 195.48Candida vini ISA1007 161.42 192.61  PYCC4568 163.55 195.94PYCC2597 161.56 192.61  Saccharomyces cerevisiae PYCC4455T 168.07 194.29Dekkera anomala ISA1652T 161.16 193.86  PYCC2608 167.80 193.86PYCC5133 161.22 193.59  PYCC3983 167.95 194.00Dekkera bruxellensis ISA1600 161.68 188.30  PYCC3931 168.09 194.00ISA1649 162.00 187.96  L169 167.86 194.39ISA1700 162.13 187.82  L170 168.07 194.28ISA1703 162.08 187.70  Saccharomyces ludwigii ISA1089 183.92 193.05ISA2117 162.09 187.71  ISA1088 184.10 193.18Hanseniaspora uvarum ISA1189 184.40 193.32  Saccharomyces paradoxus PYCC4570T  166.63 194.88MT1/BG/10 184.40 193.32  PYCC4576 166.54 195.04Kluyveromyces marxianus PYCC3886T 169.40 186.37  PYCC4578 166.59 194.88CBS6432 169.47 186.63  PYCC4656 166.63 194.89PYCC3286 169.41 186.65  Saccharomycodes ludwigii ISA1083 155.84 190.44PYCC2902 169.35 186.53  Zygosaccharomyces  bailii PYCC5167T 162.16 182.71Lodderomyces elongisporus ISA 1421 186.37 202.23  ISA1022 162.66 182.19ISA1308 185.26 202.09  PYCC4806 162.35 182.90Metschnikowia pulcherrima PYCC5625 160.95 158.72  CBS2856 162.79 182.32PYCC4384 160.88 157.57  ISA1265 162.38 182.39Pichia anomala PYCC4121T 156.05 190.69  PYCC4531 162.36 182.48PYCC2495 156.02 190.51  Zygosaccharomyces bisporus PYCC5335T 162.26 182.75PYCC3294 155.91 190.49  PYCC5336 162.38 182.79PYCC4380 156.04 190.64  PYCC5337 162.21 182.78Pichia guilliermondii ISA 2105 174.63 197.09   PYCC5381 162.37 182.78   

Identification of wine related yeast species by capillary electrophoresis single- strand conformation polymorphism

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