A novel ascosporogenous yeast species, Zygosaccharomyces siamensis, and the sugar tolerant yeasts associated with raw honey collected in Thailand

Abstract

Diversity of yeasts in association with bees and their food sources has been explored during the last decade. In Thailand, there has been no study of yeast identification in honey and bees. Hence, a total of 186 yeast strains were isolated from 37 honey samples of 12 different bee species. On the basis of morphological and physiological characteristics, 55 representative strains were chosen and identified by sequence analysis of the 26S rDNA D1/D2 domain and the ITS region. The data were compared with the published sequences and the results showed the occurrence of 19 ascomycetous and 1 basidiomycetous yeast species. Six strains of the new species were isolated. Phylogenetic analysis of the 26S rDNA D1/D2 sequence revealed that they were conspecific and most closely related to Zygosaccharomyces mellis. Based on the ITS sequence, the new species was clustered with the type " and clearly distinguished from the type !. Sequence analysis of combined ITS-26S rDNA D1/D2 showed similar results. The occurrence of these two types, with a divergence of more than 1% in their sequences, and low DNA relatedness among them suggested that members of the type β can be regarded as separate species. An analysis of the morphological and physiological characteristics was performed. Ascospore formation was observed on acetate agar and Gorodkowa agar. The new Zygosaccharomyces species differed physiologically from Z. mellis in 4 assimilation tests. This data supports the hypothesis that the new species, Zygosaccharomyces siamensis, is a novel ascosporogenous yeast. The type strain is JCM 16825 T (=CBS 12273 T ) and a description is given here.

Zygosaccharomyces favi sp. nov., an obligate osmophilic yeast species from bee bread and honey

“…According to the classical guideline provided by Kurtzman and Robnett (1998) this amount of divergence suggests that these five strains are not conspecific with Z. gambellarensis. To place in closer context, D1/D2 sequence divergences among strains of the species pairs selected from the group of Z. bailii and the recently described two closely related yeast species Z. parabailii and Z. pseudobailli (Suh et al 2013) may be as small as 0-1 substitution, and Z. siamensis differs by 3 substitutions from Z. mellis (Saksinchai et al 2012). To further investigate the relationship among the five osmophilic strains recovered during this study and the type strain of Z. gambellarensis, the sequences of the ITS regions and the translation elongation factor-1a (EF-1a) gene of these strains were determined.…”

“…The attempt to sequence directly the amplicons obtained with Taq DNA polymerase enzyme by the primer pair ITS1 and ITS4 was unsuccessful, due to dual and multiple peaks on the chromatograms either in case of strains of the new species or in case of the type strain of Z. gambellarensis. It was supposed that the reason of failure was the presence of different ITS copies as shown for some other Zygosaccharomyces species (Gordon and Wolfe 2008;Saksinchai et al 2012;Solieri et al 2013). Therefore, prior to sequencing, PCR-amplified ITS fragments were inserted into pGEM-T vector system (Promega) and 8-12 randomly selected ITS clones from each strain were sequenced.…”

“…In the last decade several new species were added to the genus; Z. machadoi (Rosa and Lachance 2005), Z. gambellarensis (Torriani et al 2011), Z. siamensis (Saksinchai et al 2012), Z. sapae (Solieri et al 2013), Z. parabailii and Z. pseudobailii (Suh et al 2013), and the current number of the recognised Zygosaccharomyces species has risen to 12. The majority of the newly described Zygosaccharomyces species, Z. machadoi, Z. gambellarensis, Z. siamensis and Z. sapae formed a well-supported clade with Z. rouxii and Z. mellis, either based on the sequences of LSU D1/D2 domains of the rRNA gene alone (Rosa and Lachance 2005;Torriani et al 2011;Saksinchai et al 2012;Solieri et al 2013) or on the combined internal transcribed spacer (ITS) and D1/D2 sequence dataset (Suh et al 2013). Zygosaccharomyces rouxii is among the most xerophilic organisms (Pitt and Hocking 2009 and the references therein), but it also grows in/on culture media with high (a w [ 0.99) water activity.…”

Zygosaccharomyces sapae sp. nov., isolated from Italian traditional balsamic vinegar

“…An increasing number of examples have been recently reported on intragenomic polymorphism in ITS sequences within the same strain of fungal and yeast species (Wang & Yao, 2005;Fell et al, 2007;Kageyama et al, 2007;Jacques et al, 2009;Connell et al, 2010;Saksinchai et al, 2012). Z. sapae sp.…”

“…Finally, Z. sapae sp. nov. could be distinguished from Z. siamensis based on the ability of the latter species to assimilate erythritol (Saksinchai et al, 2012). When cultured on YPD medium under shaking conditions (James & Stratford, 2011); 5, Z. mellis (James & Stratford, 2011); 6, Z. bailii (James & Stratford, 2011).…”