Molecular typing of the yeast species Dekkera bruxellensis and Pichia guilliermondii recovered from wine related sources Patricia Martorell a , Andre ´ Barata b , Manuel Malfeito-Ferreira b , M. Teresa Ferna ´ndez-Espinar a , Virgilio Loureiro b , Amparo Querol a, * a Departamento de Biotecnologı ´a, Instituto de Agroquı ´mica y Tecnologı ´a de Alimentos (CSIC), P.O. Box 73, E-46100 Burjassot, Vale `ncia, Spain b Laborato ´rio de Microbiologia, Departamento de Bota ˆ nica e Engenharia Biolo ´gica, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal Received 21 November 2004; received in revised form 19 May 2005; accepted 30 May 2005 Abstract A total of 63 strains of Dekkera bruxellensis and 32 strains of Pichia guilliermondii isolated from wine related environments were identified by restriction analysis of the 5.8S-ITS region of the rDNA. These strains were subjected to intraspecific discrimination using mtDNA restriction and RAPD-PCR analysis. The isolates identified as D. bruxellensis yielded 3 different molecular patterns of mtDNA restriction using the endonuclease HinfI. The pattern A was the most frequent (58 strains) among strains from different sources, regions and countries. Pattern B (4 strains) and C (one strain) were determined in isolates from Portuguese wines. The discrimination among the pattern A strains was achieved by a RAPD-PCR assay with 3 primers (OPA-2, OPA-3 and OPA-9). Atotal of 12 haplotypes were obtained with the combination of the patterns provided by the 3 OPAs. The pattern 2 was the most frequent and extensively distributed being found in strains from different countries and from different sources like wine, barrique wood and insects. The strains of P. guilliermondii were characterized with restriction of mtDNA using the endonuclease Hin fI yielding 7 different restriction patterns. These patterns were associated with different efficiencies of 4-ethylphenol production. Patterns A to D corresponded to 19 strains producing low levels of 4-ethylphenol (<1 mg/l) while patterns F and G grouped 13 strains producing high levels of 4-ethylphenol (>50 mg/l), when grown in synthetic media supplemented with 100 mg/l of p-coumaric acid. The high degree of polymorphism observed shows that intraspecific typing is essential for accurate yeast dissemination studies in wine related environments. D 2005 Elsevier B.V. All rights reserved. Keywords: Dekkera bruxellensis ; Pichia guilliermondii ; Molecular typing; 4-Ethylphenol; Wine 1. Introduction In wine industry, although lactic and acid bacteria have been described as important spoilers (Sponholz, 1992; Ribe ´reau-Gayon et al., 2000), yeasts are now regarded as the most feared contaminants in wine. Their spoilage effects are film formation in stored wine, cloudiness, sediments and gas production in bottled wines, and off-odors and off-tastes during wine production and bottled wines (Loureiro and Malfeito-Ferreira, 2003). The yeasts of the genus Dekkera (Brettanomyces , imperfect form) are described as the most serious spoilage yeasts in red wines, because of their ability to produce high amounts of volatile phenols (4-ethylphenol and 4-ethylguaiacol) imparting off-flavors to red wines (Chatonnet et al., 1992, 1995, 1997). Among the species of this genus, Dekkera bruxellensis is the most representative in wines (Mitrakul et al., 1999; Rodrigues et al., 2001). In addition, it has been found that other species are capable of producing volatile phenols (Dias et al., 2003b). Among these, Pichia guilliermondii has the ability to produce 4-ethylphenol with efficiencies as high as those observed in D. bruxellensis (Dias et al., 2003b). The risk of wine contamina- tion by these yeasts thus justifies the effort to develop rapid identification techniques. Several molecular-based methodolo- gies have been described for a rapid detection and identifica- tion of Dekkera/Brettanomyces , such as nested PCR, AFLP, RT-PCR, PCR-RFLP, and fluorescence in situ hybridization 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.05.014 * Corresponding author. Tel.: +34 96390 0022x2306; fax: +34 96363 6301. E-mail address: aquerol@iata.csic.es (A. Querol). International Journal of Food Microbiology 106 (2006) 79 – 84 www.elsevier.com/locate/ijfoodmicro