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Based on IGS-PCR RFLP polymorphism, we previously detected two Z. rouxii strains (CECT 11923 and CECT 10425) that clustered with hybrid strains (NCYC 1682, NCYC 3060 and NCYC 3061). Given the recently recognized important industrial role of hybrids, their detection is very useful. Based on the IGS1 rDNA region alignment of hybrid strains and the Z. rouxii CECT 11923 and CECT 10425, in this work, we developed a pair of Zygosaccharomyces hybrid-specific primers, HibZF/HibZR. Positive amplicons were only obtained in the Zygosaccharomyces spp. hybrids included in this study and the CECT 11923 and CECT 10425 strains analyzed here. In the present study, we applied molecular tools to highlight the nature of these strains; they are quite different from each other as well as from Z. rouxii type strain. Based on the presence of two heterologous copies of nuclear-encoded genes (SOD2 and HIS3), the sequences of divergent 5.8S-ITS rDNA, D1/D2 26S rDNA copies and, the amplification with species-specific primer for Z. rouxii and Z. pseudorouxii, we hypothesize that the CECT 11923 strain might be a hybrid strain. Whereas, CECT 10425, the sequence analysis of 5.8S-ITS rDNA and D1/D2 26S rDNA copies presented 99–100% sequence identity with Zygosaccharomyces sp. NBRC 10669 (LN849119.1) and Z. sapae ABT 301T. Nevertheless, we discard that it could be a Z. sapae strain based on the results obtained in this study. Namely, the amplification with hybrid-specific primer designed in this study, the number of divergent copies of HIS3 (2), the fact that it only possesses one SOD2 gene and the amplification with species-specific primer for Z. pseudorouxii, therefore it could be a new species or a hybrid strain.

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only four were used in this work. All microsatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction, which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair. Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool.

Unlike previously reported methods that need a combination of several typing techniques, we have 22 developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage 23 species. Strains belonging to other species have also been included for comparison. We have demonstrated 24 that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in 25 this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z.bailii. 26 We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns 27 with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS 28 rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of 29 Zygosaccharomyces in a separate cluster.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.

Four yeast strains were isolated from Spanish traditional fermented sausages (chorizo) spoiled by gas production. Using the classical identification procedures, they were identified as Debaryomyces hansenii. However, they fermented galactose and did not produce positive results in Debaryomyces differential medium (DDM), a growth medium highly specific for this species. Phylogenetic analysis showed identical sequences for the D1/D2 domain of the 26S rRNA gene and almost identical sequences for the 5.8S-ITS region with those of the recently described yeast species Candida cretensis. This result was confirmed by sequencing the gene encoding actin of the type and the new strains. Candida cretensis is a new species included in the so-called Candida kruisii clade that was described from a single strain, isolated from a decaying mushroom in Crete, Greece. The discovery of new strains of C. cretensis in fermented food expands its physiological and ecological diversity. With the description of these new strains isolated from food, three groups of strains can be distinguished within C. cretensis according to the restriction patterns of the intergenic spacer rRNA gene region and on the basis of some physiological properties that are of ecological relevance.

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.