Massive isolation and identification of <Emphasis Type="Italic">Saccharomyces paradoxus</Emphasis> yeasts from plant phyllosphere

Year-round studies of epiphytic yeast communities revealed that the number of ascosporogenous yeasts of the genus Saccharomyces inhabiting living and decaying leaves of some plants increased considerably in certain short periods (at the beginning of summer and in winter). Massive isolation of saccharomycetes was performed from 11 plant species; earlier, these yeasts had been revealed mainly in sugar-rich substrates. The isolates were identified as Saccharomyces paradoxus based on their physiological properties and RELP analysis of 5.8S-ITS. Possible reasons for short-term increases in the number of saccharomycetes in plant phyllosphere are discussed.     

Yeast population of the Kindo Peninsula lichens

Yeast abundance and species diversity in the lichens collected at the Kindo Peninsula (Karelia) were studied. A total of 14 lichen species analyzed belonged to the genera Bryoria, Cladonia, Hypogymnia, Icmadophila, Nephroma, Peltigera, and Ramalina. Abundance of cultured yeasts in lichens was intermediate between soil and phyllosphere. The average yeast number on lichens was ~2.5 × 10³ CFU/g, while it exceeded 8 × 10³ CFU/g on plants and reached only 1 × 10³ CFU/g in soil. Yeast population of different parts of Cladonia lichens was found to vary significantly in abundance, species diversity, and community structure. The highest yeast abundance and diversity were revealed in the growth zone. Fifteen yeast species were isolated from lichens, including 6 basidiomycetous and 9 ascomycetous ones. Unlike soils and plants, yeast population of lichens consisted mainly of ascomycetous species, with predominance of Candida sphagnicola and anamorphous yeasts of the genus Dothiora. These results show that yeasts from different taxonomic and ecological groups are a necessary component of lichens; conditions favoring the preservation and development of specific yeast communities differing from the typical soil and phyllosphere yeast complexes are formed in the lichens of northern taiga forests.     

Endophytic yeasts in <Emphasis Type="Italic">Malus domestica</Emphasis> and <Emphasis Type="Italic">Pyrus communis</Emphasis> fruits under anthropogenic impact

Yeast abundance and species diversity on the surface and in inner tissues of Malus domestica and Pyrus communis fruits under high anthropogenic impact in the city of Moscow (Russia) were studied. Results demonstrated that abundance of epiphytic yeasts on the fruits increased gradually, reaching the maximum of 3.2 × 10⁴ CFU/g on mature fruits. During summer, abundance of endophytes did not change significantly (variation near 2.5 × 10³ CFU/g) until complete maturation, while in September their numbers increased to 10⁴ CFU/g. Basidiomycetous yeasts (Filobasidium wieringae, F. magnum, Rhodotorula glutinis, and Rhodosporidiobolus colostri) predominated on the fruit surface. Ascomycetous species were the most diverse group inside the fruits, which quantitatively increased through maturation. It was found that the share of opportunistic species Candida parapsilosis in internal tissues was significant during the entire period of fruit formation and development under anthropogenic impact in the city. Specific properties of epiphytic and endophytic yeast communities developing in natural ecological niches under synanthropic conditions and anthropogenic impact are discussed.     

Massive isolation of anamorphous ascomycete yeasts Candida oleophila from plant phyllosphere

Many years of research has confirmed a wide distribution of anamorphous ascomycete yeasts in the phyllosphere of diverse plants of the Moscow oblast. Based on the standard morphological and physiological criteria, on the results of restriction analysis of the 5.8S-ITS rDNA region, and on the sequencing of the D1D2 region of 26S rDNA, these yeasts were identified as Candida oleophila Montrocher. Previous isolation of this species has been rare, possibly due to its incorrect identification. This species, together with phytobiotic basidiomycete yeasts, was shown to be dominant in the yeast epiphytic communities on the surface parts of plants. The relative abundance of C. oleophila is highest on plant fruits and increases significantly by the end of the vegetation period. Wide occurrence of this yeast species on fruits and in the phyllosphere may be related to its ability to compete with rapidly growing phytopathogenic fungi.     

Characterization of yeast groupings in the phyllosphere of <Emphasis Type="Italic">Sphagnum</Emphasis> mosses

Significant differences were revealed in the taxonomic structure of the epiphytic yeast communities formed on Sphagnum mosses and on the leaves of vascular plants. On mosses, low abundance of red yeasts was found (the most typical epiphytes on vascular plant leaves), along with a relatively high content and diversity of nonpigmented dimorphic basidiomycetes related to the order Leucosporidiales. The species composition of epiphytic yeasts from mosses is different from that of both forest and meadow grasses and of the parts of vascular plants submerged in the turf. The specific composition of the Sphagnum mosses yeast community is probably determined by the biochemical characteristics of this environment, rather than by the hydrothermal regime in the turf.     

Consortia formed by yeasts and acetic acid bacteria <Emphasis Type="Italic">Asaia</Emphasis> spp. in soft drinks

Yeast strains and acetic acid bacteria were isolated from spoiled soft drinks with characteristic flocs as a visual defect. Polymerase chain reaction and amplification of a partial region of the LSU rRNA gene identified the bacteria as Asaia spp. Sequence analysis of the D1/D2 region of the 26S rDNA in turn identified the yeast isolates as Wickerhamomyces anomalus, Dekkera bruxellensis and Rhodotorula mucilaginosa. The hydrophobicity and adhesion properties of the yeasts were evaluated in various culture media, taking into account the availability of nutrients and the carbon sources. The highest hydrophobicity and best adhesion properties were exhibited by the R. mucilaginosa cells. Our results suggest that Asaia spp. bacterial cells were responsible for the formation of flocs, while the presence of yeast cells may help to strengthen the structure of co-aggregates.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Identification of yeasts during alcoholic fermentation of <Emphasis Type="Italic">tchapalo</Emphasis>, a traditional sorghum beer from Côte d’Ivoire

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d’Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d’Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.     

Morphological and molecular-genetic distinctions between adult mosquitoes <Emphasis Type="Italic">Culex torrentium</Emphasis> Martini and <Emphasis Type="Italic">C. pipiens</Emphasis> Linnaeus (Diptera,Culicidae) from Moscow Province

Morphological, biological, and molecular-genetic characteristics of Culex pipiens and C. torrentium, collected from two sites in Moscow and five sites in Moscow Province, were studied. Our results demonstrated the absence of any differences between females of these species in the R ₂/R ₂₊₃ ratio. Figures and photographs of the male genitalia are given. Differences between the species in the mDNA cytochrome oxydase I gene and the 1TS2 region of the rDNA were revealed. 90–100% of C. pipiens and 0% of C. torrentium were infested with the endosymbiotic bacterium Wolbachia pipientis.     

South Brazilian wines: culturable yeasts associated to bottled wines produced in Rio Grande do Sul and Santa Catarina

A comprehensive understanding of the presence and role of yeasts in bottled wines helps to know and control the organoleptic quality of the final product. The South Region of Brazil is an important wine producer, and the state of “Rio Grande do Sul” (RS) accounts for 90% of Brazilian wines. The state of “Santa Catarina” (SC) started the production in 1975, and is currently the fifth Brazilian producer. As there is little information about yeasts present in Brazilian wines, our main objective was to assess the composition of culturable yeasts associated to bottled wines produced in RS and SC, South of Brazil. We sampled 20 RS and 29 SC bottled wines produced between 2003 and 2011, and we isolated culturable yeasts in non-selective agar plates. We identified all isolates by sequencing of the D1/D2 domain of LSU rDNA or ITS1-5.8 S-ITS2 region, and comparison with type strain sequences deposited in GenBank database. Six yeast species were shared in the final product in both regions. We obtained two spoilage yeast profiles: RS with Zygosaccharomyces bailii and Pichia membranifaciens (Dekkera bruxellensis was found only in specific table wines); and SC with Dekkera bruxellensis and Pichia manshurica. Knowledge concerning the different spoilage profiles is important for winemaking practices in both regions.     

Yeasts in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> latex

Yeast abundance and species diversity in the latex of rubber tree Hevea brasiliensis (Willd. ex Juss.) Müll. Arg., on its green leaves, and in soil below the plant were studied. The yeasts present in the fresh latex in numbers of up to 5.5 log(CFU/g) were almost exclusively represented by the species Candida heveicola. This species was previously isolated from Hevea latex in China. In the course of natural modification of the latex (turned from liquid to solid form), yeast diversity increased, while yeast abundance decreased. The yeasts in thickened and solidified latex were represented by typical epiphytic and ubiquitous species: Kodamea ohmeri, Debaryomyces hansenii, Rhodotorula mucilaginosa, and synanthropic species Candida parapsilosis and Cutaneotrichosporon arboriformis. The role of yeasts in latex modification at the initial stages of succession and their probable role in development of antifungal activity in the latex are discussed.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.     

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow region

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5?50 μg/mL) and are probably plant symbionts.     

Yeasts of Nikitsky Botanical Garden plants

Abundance and taxonomic structure of yeast communities on the surface and in the tissues of various plants of the Nikitsky Botanical Garden was studied. A total of 22 yeast species were isolated, including rare and new species. Yeast numbers on the studied plant substrates were varied within a broad range (8 × 10²?2.5 × 10⁷ CFU/g), reaching the maximum in Verbascum thapsus flowers, on the surface and inside the fruits of Rubus sp. and Ficus carica. Epiphytic and endophytic yeast communities of Ficus carica fruits were studied in dynamics, from fruit formation until their complete maturation and senescence. Specific properties of the yeast communities of the Nikitsky Botanical Garden plants and the features of the yeast communities from fig fruits are discussed in the paper.     

Analysis of 5S rDNA changes in synthetic allopolyploids <Emphasis Type="Italic">Triticum</Emphasis> × <Emphasis Type="Italic">Aegilops</Emphasis>

Changes of 5S rDNA at the early stage of allopolyploidization were investigated in three synthetic allopolyploids: Aegilops sharonensis × Ae. umbellulata (2n = 28), Triticum urartu × Ae. tauschii (2n = 28), and T. dicoccoides × Ae. tauschii (2n = 42). Fluorescent in situ hybridization (FISH) revealed quantitative changes affecting separate loci of one of the parental genomes in S₃ plants of each hybrid combination. Southern hybridization with genomic DNA of the allopolyploid T. urartu × Ae. tauschii (TMU38 × TQ27) revealed a lower intensity of signals from Ae. tauschii fragments compared with those derived from T. urartu. This confirmed the signal reduction revealed for chromosome 1D of this hybrid by FISH. Neither Southern hybridization nor PCR testing of 5–15 plants of the S₂-S₃ generations revealed an appearance of new 5S rDNA fragments or a complete disappearance of parental fragments from the allopolyploids under study. No changes were found by aligning nine 5S rDNA sequences of the allopolyploid TMU38 × TQ27 with corresponding sequences of the parental species. The similarity between one of the synthetic allopolyploids examined and a natural allopolyploid with the same genome composition points to an early formation of the 5S rDNA organization unique for each allopolyploid.     

Molecular characterization of eukaryotic algal communities in the tropical phyllosphere based on real-time sequencing of the 18S rDNA gene

Backgroud

Foliicolous algae are a common occurrence in tropical forests. They are referable to a few simple morphotypes (unicellular, sarcinoid-like or filamentous), which makes their morphology of limited usefulness for taxonomic studies and species diversity assessments. The relationship between algal community and their host phyllosphere was not clear. In order to obtain a more accurate assessment, we used single molecule real-time sequencing of the 18S rDNA gene to characterize the eukaryotic algal community in an area of South-western China.

Result

We annotated 2922 OTUs belonging to five classes, Ulvophyceae, Trebouxiophyceae, Chlorophyceae, Dinophyceae and Eustigmatophyceae. Novel clades formed by large numbers sequences of green algae were detected in the order Trentepohliales (Ulvophyceae) and the Watanabea clade (Trebouxiophyceae), suggesting that these foliicolous communities may be substantially more diverse than so far appreciated and require further research. Species in Trentepohliales, Watanabea clade and Apatococcus clade were detected as the core members in the phyllosphere community studied. Communities from different host trees and sampling sites were not significantly different in terms of OTUs composition. However, the communities of Musa and Ravenala differed from other host plants significantly at the genus level, since they were dominated by Trebouxiophycean epiphytes.

Conclusion

The cryptic diversity of eukaryotic algae especially Chlorophytes in tropical phyllosphere is very high. The community structure at species-level has no significant relationship either with host phyllosphere or locations. The core algal community in tropical phyllopshere is consisted of members from Trentepohliales, Watanabea clade and Apatococcus clade. Our study provided a large amount of novel 18S rDNA sequences that will be useful to unravel the cryptic diversity of phyllosphere eukaryotic algae and for comparisons with similar future studies on this type of communities.

     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

<Emphasis Type="Italic">Pattersoniomyces tillandsiae</Emphasis> gen. et comb. nov.: linking sexual and asexual morphs of the only known smut fungus associated with Bromeliaceae

The enigmatic species Ustilago tillandsiae is the only known smut fungus associated with Bromeliaceae. Its generic position is evaluated by morphological, physiological, and molecular phylogenetic analyses using large subunit rDNA sequences. Phylogenetic analyses resolved U. tillandsiae as a member of the Ustilaginales in a sister relationship to the lineage containing Tranzscheliella species. However, U. tillandsiae differs from Tranzscheliella species by the development of sori in flowers, a different structure of sori and a different type of spore ornamentation. Consequently, a new genus Pattersoniomyces is described to accommodate U. tillandsiae. The new combination Pattersoniomyces tillandsiae is substantiated. In the sexual stage (teleomorph), this species infects bromeliads: Tillandsia flabellata, Tillandsia leiboldiana, and Tillandsia sp. in Central America between southern Mexico and Costa Rica. The yeast stage (anamorph) of P. tillandsiae was found associated with the phylloplane of Canistrum improcerum and in water tanks (phytotelmata) of Vriesea minarum, two bromeliads occurring in northeast and southeast Brazil, respectively. The link between the teleomorph and anamorphic strains is supported by identical sequences of the D1/D2 domains of the large subunit rDNA. Pattersoniomyces represents the tenth endemic smut genus to the Americas, but the only one that occurs in both North and South America, being a truly neotropical genus. The host plant families of Ustilaginales are extended to the Bromeliaceae. As far as we know, Pattersoniomyces represents the single event of a host jump from Cyperaceae or Poaceae to Bromeliaceae, apparently without further species radiation on multiple bromeliad species and genera growing in South America.     

Genetic relationship and biological status of the industrially important yeast <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.