Methods for Yeast Identification

The specificity of nucleic acid sequences has prompted the development of several methods for rapid species identification. The comparison of RNA (rRNA) and its template ribosomal DNA (rDNA) has been used extensively in recent years to assess both close and distant relationships among many kinds of organisms as a result of the existence of conserved sequences within this region, and the concerted evolution among units (Kurtzman and Robnett 1998). Using the information from this region, different methods have been developed for yeast species identification.

3.1.1 Ribosomal RNA Gene Sequencing

The determination and comparison of nucleotide sequences for the purpose of recognizing similarities or differences between organisms is at the heart of any molecular approach. Therefore, choosing the appropriate gene is paramount to the success or failure of any such sequence analysis between species. The 26S, 18S, 5.8S, and 5S ribosomal genes have undergone a relatively slow evolution, allowing for their use in the comparison of distantly related organisms. Work has

Table 1 Main enzymatic activities described in non-Saccharomyces wine yeasts

Enzymatic activity

Yeasts

References

Protease

Candida, Kloeckera, Pichia

Charoenchai et al. 1997

ß-Glucosidase

Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera,

Grossmann, et al. 1987; Rosi et al. 1994;

Kluyveromyces, Metschnikowia, Pichia, Saccharomycodes,

Manzanares et al. 2000

Schizosaccharomyces, Zygosaccharomyces

Esterase

Brettanomyces, Debaryomyces, Rhodotorula

Besancon et al. 1995

Pectinase

Candida, Cryptococcus, Kluyveromyces, Rhodotorula

McKay 1990

Lipase

Candida

Charoenchai et al. 1997

Table 2 Studies about wine yeast identification using molecular biology techniques

Methodology

Genus

References

AFLP 8 elements Intron splice site Karyotype

Microsatellite

Nested-PCR

Plasmids

RAPDs

RFLP-karyotype

RFLP-mtDNA

RFLP-ITS/5.8S rRNA gene

Saccharomyces Saccharomyces Saccharomyces Saccharomyces

Hanseniaspora

Zygosaccharomyces

Saccharomyces

Brettanomyces

Saccharomyces

Zygosaccharomyces

Saccharomyces

Metschnikowia

Rhodotorula

Zygosaccharomyces

Candida

Picchia

Torulaspora

Hansenula

Candida

Kloeckera

Schizosaccharomyces Saccharomyces

Kluyveromyces

Zygosaccharomyces

Brettanomyces

Candida

Hanseniaspora

Saccharomyces

25 different genera de Barros Lopes de et al. (1999)

de Barros Lopes de et al. (1999)

Cardinali and Martini, 1994; Guillamon et al (1996);

Ibeas et al. (1997); Martinez et al. (1995) Schutz and Gafner (1994) Torok et al. (1993) Baleiras Couto et al. (1996) Ibeas et al. (1996) Pearson and Mckee (1992) Pearson and Mckee (1992)

Baleiras Couto, (1996); Quesada and Cenis (1995)

Lopandic et al. (1996)

Quesada and Cenis (1995)

Baleiras Couto et al. (1994)

Quesada and Cenis (1995)

Quesada and Cenis (1995)

Quesada and Cenis (1995)

Quesada and Cenis (1995)

Versaud and Hallet (1995)

Versaud and Hallet (1995)

Versaud and Hallet (1995)

Querol et al. (1992a); Querol et al. (1994) Belloch et al. (1997) Guillamon et al. (1997) Ibeas et al. (1996)

Constant! et al (1997); Guillamon et al. (1998) Guillamon et al. (1998) Guillamon et al. (1998) Esteve-Zarzoso et al. (1999)

focussed primarily on the D1/D2 domain of the 26S rDNA (Kurtzman and Robnett 1998) and on the 18S subunit (James et al. 1997). The techniques have gained substantially from the introduction of the polymerase chain reaction, direct sequencing, and the availability of numerous nucleotide databases, which contain sequence information on a diverse range of organisms.

3.1.2 Restriction Fragment Length Polymorphism of RDNA

Recently, Esteve-Zarzoso et al. (1999) proposed a rapid and easy method for routine yeast identification, based on RFLPs of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and 2). They presented an initial database for the identification of more than 132 yeast species belonging to 25 genera, most isolated from food. This is the first available molecular method that provides information to identify a large number of yeast species in an easy and quick way. This database has been improved to identify 300 yeast species (http.//motor.edinfo.es/iata). Using this method, it is possible to identify yeasts from isolated colonies or directly from food samples (see Figure 1). In addition, the anamorph and teleomorph forms yielded the same patterns and all the strains of the same species also exhibit the same pattern. Guillamon et al. (1998) used this method to identify 33 wine yeast species and Fernandez-Espinar et al. (2000) identified all the species of the genus Saccharomyces, including the flor yeast responsible for biological ageing in the process of making "fino" sherry wine. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae, due to the presence of a 24-bp deletion located in the ITS1 region. Esteve-Zarzoso and Peris-Toran (2001) detected this specific "flor" pattern in all the strains isolated from the velum of sherry wines from wineries located in Jerez, Spain.

Using the same methodology, but amplifying a different region (18S rRNA and ITS1) Dlauchy et al. (1999) also constructed a database of restriction fragment patterns to identify 128 species associated mainly with food, and fermented drinks.

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