Zygosaccharomyces Species Research Articles

Further evidences of an emerging stingless bee-yeast symbiosis.

Symbiotic interactions between microorganisms and social insects have been described as crucial for the maintenance of these multitrophic systems, as observed for the stingless bee Scaptotrigona depilis and the yeast Zygosaccharomyces sp. SDBC30G1. The larvae of S. depilis ingest fungal filaments of Zygosaccharomyces sp. SDBC30G1 to obtain ergosterol, which is the precursor for the biosynthesis of ecdysteroids that modulate insect metamorphosis. In this work, we find a similar insect-microbe interaction in other species of stingless bees. We analyzed brood cell samples from 19 species of stingless bees collected in Brazil. The osmophilic yeast Zygosaccharomyces spp. was isolated from eight bee species, namely Scaptotrigona bipunctata, S. postica, S. tubiba, Tetragona clavipes, Melipona quadrifasciata, M. fasciculata, M. bicolor, and Partamona helleri. These yeasts form pseudohyphae and also accumulate ergosterol in lipid droplets, similar to the pattern observed for S. depilis. The phylogenetic analyses including various Zygosaccharomyces revealed that strains isolated from the brood cells formed a branch separated from the previously described Zygosaccharomyces species, suggesting that they are new species of this genus and reinforcing the symbiotic interaction with the host insects.

Isolation and Identification of some Yeasts from some Plants

The present study included isolating 71 local isolates of yeasts during three months, starting from 1/10/2021 to 1/1/2022 from different plant sources, as the fruits were obtained from local markets in Mosul, Kirkuk, Erbil and Dohuk and from home gardens. The isolates were diagnosed after culturing on solid nutrient medium MEA (Malt Extract Agar Medium) based on phenotypic tests including colony color, shape, diameter, nature of its edges, height, texture, brightness, surface shape, culture and microscopy at 40X powers to observe the shape of yeast cells and measure their sizes Using (E 10X) ocular and biochemical including Diazonium Blue B (DBB) Color test, growth in 25˚C and 37˚C, assessing ability to utilize nitrate as a sole nitrogen source, assessing preservative resistance of glacial acetic acid, growth at reduced water activities in high carbohydrate levels, growth at reduced water activities and high level of sodium chloride and ability mycelium formation test. The results showed that it belongs to 17 different species of yeasts Debaromyces, Rhodotorula, Pichia, Candida, Kluyveromyces, Geotrichum, Kloeckera, Saccharomyces, Cryptococcus, Zygosaccharomyces and Trichosporon.

The chromosomal evolutionary lineage of the genus Zygosaccharomyces.

Genome ploidy of Zygosaccharomyces rouxii is an intriguing topic in the field of industrial yeast research. However, the evolutionary relationship between the genome of Z. rouxii and other Zygosaccharomyces species is complex and not completely understood. In this study, we determined the genome sequences of Z. rouxii NCYC 3042, also referred to as "Z. pseudorouxii," and Z. mellis CBS 736T. We also conducted comparative analysis of the yeast genomes of a total of 21 strains, including 17 strains of nine Zygosaccharomyces species. This comparative genomics revealed that 17 Zygosaccharomyces strains are classified into four groups consisting of nine genome types: (i) Z. rouxii, Z. mellis, Z. sapae, Z. siamensis, and "Candida versatilis" t-1 belong to the Group Rouxii sharing four related genome types (Rouxii-1 to Rouxii-4), (ii) Z. bailii, Z. parabailii, and Z. pseudobailii belong to the Group Bailii sharing three related genome types (Bailii-1 to Bailii-3), (iii and iv) Z. bisporus and Z. kombuchaensis belong to the Groups Bisporus and Kombuchaensis, respectively, which each have haploid genomes. The Zygosaccharomyces genome seems to have acquired complexity and diversity through evolutionary events such as interspecies hybridization, reciprocal translocation, and diploidization of these nine genome types.

Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.

Biodiversity of Zygosaccharomyces species in food systems

AbstractsZygosaccharomycesspecies are among the most problematic food spoilage yeasts. The two most infamous species areZygosaccharomyces baliiandZygosaccharomyces rouxii, although they may also take a positive role during the production of some fermented foods. DNA sequence based yeast identification aided by freely available reference databases of barcoding DNA sequences has boosted the description rate of novel yeast species in the last two decades. The genusZygosaccharomyceshas been considerably expanded as well. Especially the number of the extremely osmotolerantZygosaccharomycesspecies, related toZ. rouxiiand regularly found in high-sugar foods, has enlarged. A brief account of recent developments in the taxonomy and biodiversity of this important food associated genus is given in this review.

Effects of Environmental factors on the Growth and Proliferation of Yeasts

Yeast is one of the organisms that grow and survive in diverse conditions. The influence of environmental factors on its growth and proliferation were evaluated. The yeast used for the study was isolated from honey using Sabouraud Dextrose Agar and pour plate methods. The effects of temperature, pH and ultraviolet radiation on the growth of Rhodotorula, Debaryomyces, Zygosaccharomyces and Candida species were determined using standard methods. The growths of all the yeast isolates were greatly affected at 80oC. Rhodotorulla species showed least growth at 50oC, but optimal growth was recorded at 30oC after 96 h of incubation. A similar trend was observed in Debaromyces species. Zygosaccharomyces species recorded high reduction in growth at 60oC after 96 h of incubation, though there were no significant differences in the growths of the isolates at 60oC and 80oC. Candida species recorded the least effect of temperature at 30oC and highest at 60oC and 80oC after 96 h of incubation. The results of the effect of pH on growth of the yeasts showed that Rhodotorula and Zygosaccharomyces had optimum growth at pH of 5 and 4 respectively. All the isolates showed increase in growth with increase in incubation time. Exposure of the isolates to UV-rays negatively affected the growths of the isolates. The growths of the yeasts decreased with increase in exposure time. This study showed that yeast can survive adverse temperature and pH and can as well survive UV-ray exposure.

Microbial Diversity and Physicochemical Characteristics of the Maotai-Flavored Liquor Fermentation Process.

The grains fermented in Maotai-flavored liquor can be classified into two groups: high-temperature fermented grains generated by stacking and grains fermented in pits. The Maotai-flavor making process enriches a special microbiota from the fermented grains, which makes related studies more difficult. The use of modern molecular techniques to detect and analyze diversity and changes in total bacterial (16S rRNA sequencing) and fungal (internal transcribed spacer (ITS) sequencing) counts have helped to overcome the shortcomings of traditional technological research. The total RNA extracted by Fe₃O₄-SiO₂ nanoparticles was retrieved into DNA by MagBeads Total RNA Extraction Kit. However, discrepancies exist in the microbial community structures at different fermentation periods. Dominant bacteria include Escherichia-Shigella, Lactobacillus, Clostridium, and Streptococcus species and dominant fungi include Alternaria, Ciliophora, Pyrenochaetopsis, Cyphellophora, Aspergillus, Issatchenkia, Pichia, Candida, and Zygosaccharomyces species. Analyses of the enzymatic activity of samples at different fermentation stages have revealed that some existing bacilli influence amylase activity. Here, to investigate flavor changes during each fermentation round, the liquor quality at all rounds was comprehensively assessed based on the corresponding physicochemical properties, which were summarized by sensory evaluation. These results suggested that the liquor quality in the third, fourth, and fifth fermentation rounds was superior.

Study on the characterisation and application of synthetic peptide Snakin-1 derived from potato tubers – Action against food spoilage yeast

The demand for more natural forms of food preservation by consumers has risen in recent years. Food spoilage by microorganisms, in particular food spoilage yeast, has been reported in different food matrices. Zygosaccharomyces species, for example, are well known for their spoilage in high sugar content foods. In the battle against food spoilage, natural antimicrobial peptides derived from plants could be a potential new source of food preservatives. The antimicrobial peptide investigated in this study was Snakin-1, derived from potato tubers. A synthetic form of Snakin-1 was synthesised based on the amino acid sequence of its natural peptide. The peptide was characterised for its inhibitory effect against a number of food spoilage yeast. The peptide's minimum inhibitory concentration was generated and its mechanism of action against Zygosaccharomyces bailii was examined. Membrane permeabilisation caused by the presence of the peptide was observed at the highest concentrations applied, and its ability to withstand high salt concentrations, thermal treatment and a range of pH was also investigated. The peptide's safety for human consumption was studied and no adverse effects were found. Finally, the peptide was applied into different beverages commonly spoiled by Zygosaccharomyces bailii in order to demonstrate the potential application of this synthetic plant antimicrobial peptide.

Variations in mating-type-like (MTL) loci direct PCR-based tracking of Zygosaccharomyces strains formed by mating.

Variations of chromosomal structures and nucleotide sequences around mating-type-like (MTL) loci among Zygosaccharomyces species have been reported. We have analyzed these differences in more detail and, on the basis of PCR- and next-generation sequencing data, we describe the MTL loci on chromosomes C and F for Z. rouxii type-strain NBRC1130, Z. rouxii NBRC0740 and Zygosaccharomyces sp. NBRC1876. We developed a mating strategy for Zygosaccharomyces sp. NBRC1876 and Z. rouxii NBRC0740, and found that the mated stains could be identified from parental strains on the basis of nucleotide sequence variations of the MTL loci. We further obtained evidence that Zygosaccharomyces sp. NBRC1876 is a natural interspecies hybrid between Z. rouxii and a related species.

Ethyl Carbamate Formation Regulated by Lactic Acid Bacteria and Nonconventional Yeasts in Solid-State Fermentation of Chinese Moutai-Flavor Liquor.

This study aimed to identify specific microorganisms related to the formation of precursors of EC (ethyl carbamate) in the solid-state fermentation of Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R2 = 0.772, P < 0.01). Differences in urea production and degradation were found at both species and functional gene levels by metatranscriptomic sequencing and culture-dependent analysis. Lactobacillus spp. could competitively degrade arginine through the arginine deiminase pathway with yeasts, and most Lactobacillus species were capable of degrading urea. Some dominant nonconventional yeasts, such as Pichia, Schizosaccharomyces, and Zygosaccharomyces species, were shown to produce low amounts of urea relative to Saccharomyces cerevisiae. Moreover, unusual urea degradation pathways (urea carboxylase, allophanate hydrolase, and ATP-independent urease) were identified. Our results indicate that EC precursor levels in the solid-state fermentation can be controlled using lactic acid bacteria and nonconventional yeasts.

Yeast Biodiversity in Vineyards and Wineries and Its Importance to the South African Wine Industry. A Review

The art of winemaking is as old as human civilization and the use of yeast in this complex ecological and biochemical process dates back to ancient times. Traditionally, yeasts associated with grape berries were simply allowed to ferment the sugars to ethanol, carbon dioxide and other minor, but important, metabolites. Spontaneous fermentations are still being used in boutique wineries that depend more on vintage variability. Various microbes found on the surface of grape skins and the indigenous microbiota associated with winery surfaces participate in these natural wine fermentations. Yeasts of the genera Kloeckera, Hanseniaspora and Candida predominate in the early stages, followed by several species of Metschnikowia and Pichia (including those species that were previously assigned to the genus Hansenula) in the middle stages when the ethanol rises to 3-4%. The latter stages of natural wine fermentations are invariably dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. However, other yeasts, such as species of Brettanomyces, Kluyveromyces, Schizosaccharomyces, Torulaspora and Zygosaccharomyces also may be present during the fermentation and can occur in the resultant wine. By contrast, the rule, rather than the exception, for modern wineries depending on reliable fermentation and the production of wines with predictable quality, is the use of specially selected starter cultures of Saccharomyces. However, the use of such cultures may not necessarily prevent the growth and metabolic activity of indigenous, winery associated strains of S. cerevisiae or other wild yeasts such as Kloeckera apiculata, Hanseniaspora uvarum, Candida stellata and Torulaspora delbrueckii. It is therefore clear that both spontaneous and inoculated wine fermentations are affected by the diversity of yeasts associated with the vineyard (natural habitat) and winery (man-made niche). In light of this, focused taxonomic surveys within an ecological framework are essential to preserve and exploit the hidden oenological potential of the untapped wealth of yeast biodiversity in our wine-producing regions. To achieve this, yeast taxonomists need to continue to isolate and characterize new yeast species and strains, while wine microbiologists develop improved identification techniques that differentiate more efficiently among individual strains. At the same time such biological surveys will complement strain development and the current international effort of molecular biologists to assign a biological function to the products of each of the 6000 genes identified by computer analysis of the nucleotide sequence of the 16 chromosomes of a laboratory strain of S. cerevisiae. Furthermore, only when we have a much better understanding of yeast biodeversity, biogeography, ecology and the interaction within yeast communities will we be able to optimally harness gene technology that will benefit both the wine producer and the consumer.

Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex.

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.

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

Five yeast strains representing a hitherto undescribed yeast species were isolated from bee bread and honey in Hungary. They are obligate osmophilic, i.e. they are unable to grow in/on high water activity culture media. Following isogamous conjugation, they form 1-4 spheroid or subspheroid ascospores in persistent asci. The analysis of the sequences of their large subunit rRNA gene D1/D2 domain placed the new species in the Zygosaccharomyces clade. In terms of pairwise sequence similarity, Zygosaccharomyces gambellarensis is the most closely related species. Comparisons of D1/D2, internal transcribed spacer and translation elongation factor-1α (EF-1α) gene sequences of the five strains with that of the type strain of Z. gambellarensis revealed that they represent a new yeast species. The name Zygosaccharomyces favi sp. nov. (type strain: NCAIM Y.01994(T)=CBS 13653(T)=NRRL Y-63719(T)=ZIM 2551(T)) is proposed for this new yeast species, which based on phenotype can be distinguished from related Zygosaccharomyces species by its obligate osmophilic nature. Some intragenomic sequence variability, mainly indels, was detected among the ITS copies of the strains of the new species.

Advances in the control of the spoilage caused by Zygosaccharomyces species on sweet wines and concentrated grape musts

Zygosaccharomyces is one of the most damaging foodborne spoilage yeast, and tools for controlling its growth are limited due to its reported resistance to preservatives and osmotic stress. Zygosaccharomyces contamination may result in spoilage of grape juice concentrates or in re-fermentation and CO2 production in sweet wines so, biological control of these yeasts could be important. In this study, thirty-four strains belonging to Zygosaccharomyces have been identified and tested against a panel of eighteen killer yeasts. Here, for the first time, PMKT is proven to be effective against different strains of Zygosaccharomyces. Cultures in media composed by sweet wine and concentrated grape juice showed that PMKT has the ability to inhibit Zygosaccharomyces, while Saccharomyces cerevisiae was fully resistant in such conditions indicating that PMKT could be used to avoid the Zygosaccharomyces spoilage. Additionally, PMKT activity was determined in the presence of potassium sorbate and potassium metabisulphite showing unexpected synergistic effects with the latter, and making possible the promising application of reducing preservative concentrations in beverages.

Rapid identification of Zygosaccharomyces with genus-specific primers

There has been a recent and rapid increase in the number of species of the genus Zygosaccharomyces which now comprises Z. bailii, Z. bisporus, Z. gambellarensis, Z. kombuchaensis, Z. lentus, Z. machadoi, Z. mellis, Z. parabaillii, Z. pseudobailii, Z. pseudorouxii, Z. rouxii, Z. sapae, and Z. siamensis. Z. pseudorouxii is an unofficial name given to isolates closely related to the newly-described species Z. sapae. The Zygosaccharomyces genus contains species that are important as food and beverage spoilage organisms and others are associated with fermentations and sweet foodstuffs, such as honey. Their economic significance means that the ability to identify them rapidly is of significant importance. Although Z. rouxii and Z. bailii have been genome-sequenced the extent of sequence data for the others, especially the newly-discovered species, is sometimes extremely limited which makes identification slow. However, parts of the ITS1/5.8S/ITS2 rDNA region contain sequences of sufficient similarity within the genus and of sufficient difference with outgroups, to be potential regions for the design of genus-wide specific primers. We report here the development of genus-specific primers that can detect all the major Zygosaccharomyces species including all those associated with foods; the rare and localised species Z. machadoi and Z. gambellarensis are not detected. The size of the single amplicon produced varies between species and in some cases is sufficiently different to assign provisional species identification. Sequence data from rDNA regions are available for virtually all described yeast species in all genera, thus, prior to having sufficient sequence data from structural genes, rDNA regions may provide more generally suitable candidates for both genus-specific and species-specific primer design.

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

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 β and clearly distinguished from the type α. 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 16825T (=CBS 12273T) and a description is given here.

Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species

Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732 T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732 T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732 T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732 T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.

A new putative Zygosaccharomyces yeast species isolated from traditional balsamic vinegar

The taxonomic status and species number of the genus Zygosaccharomyces have rapidly changed in the last years. In this study, two new osmotolerant Zygosaccharomyces strains isolated from traditional balsamic vinegar, viz. ABT301 and ABT601, were investigated to elucidate their taxonomic relationships with Zygosaccharomyces rouxii species. A multi-gene sequence approach was employed, including regions of the rDNA repeat [5.8S, two internal transcribed spacers (ITS) and the 26S D1/D2 domain], COX2 mitochondrial gene and two nuclear genes (SOD2 and HIS3). Cloning and sequence analysis of 5.8S-ITS rDNA revealed that these strains bear an unusual polymorphism for this region. Three highly divergent 5.8S-ITS sequences were detected, one identical to Z. rouxii, the other two showing some relatedness to Z. mellis. Sequence and gene number polymorphism was also observed for the protein-encoding nuclear genes SOD2 and HIS3, as two copies for each gene different from those found in Z. rouxii were detected. Analysis of the D1/D2 26S domain showed that ABT301 and ABT601 have only one type of D1/D2 sequence statistically different from that of Z. rouxii. The findings obtained in this work suggest that the genomic background of strains ABT301 and ABT601 is different from the other Zygosaccharomyces species. We speculated that they could belong to a new putative species related to Z. rouxii.

Zygosaccharomyces machadoi sp. n., a yeast species isolated from a nest of the stingless bee Tetragonisca angustula

A new yeast species, Zygosaccharomyces machadoi, was discovered in garbage pellets of the stingless bee Tetragosnica angustula in Brazil. Analysis of the sequence of the D1/D2 domains of the large-subunit rDNA showed that the new species is related to Zygosaccharomyces rouxii. Z. machadoi probably causes food spoilage in hives of stingless bees. It assimilates only a few carbon sources such that it is difficult to distinguish it from other Zygosaccharomyces species based on conventional physiological tests. The type strain of Z. machadoi is UFMG-J01-63.2T (CBS10264T).