Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Changes in microbial communities and their predictive functionalities during fermentation of toddy, an alcoholic beverage of India.

Toddy is a traditional mild-alcoholic drink of India, which is produced from fresh palm saps by natural fermentation. We studied the successional changes in bacterial and fungal communities during the natural fermentation (0 h-96 h) of toddy. During fermentation, alcohol content of the fermenting saps increased significantly from 0.6 %±0.15 to 5.6 %±0.02, pH decreased from 6.33 %±0.02-3.93 ± 0.01, volatile and titratable acidity acidity (g/100 mL) increased from 0.17 ± 0.02 (0 h) to 0.48 ± 0.02 (96 h) and 1.30 ± 0.005 (0 h) to 2.47 ± 0.005 (96 h), respectively. Total sugar content and ˚BRIX also decreased during the fermentation. Firmicutes (78.25 %) was the most abundant phylum followed by Proteobacteria (21.57 %). Leuconostoc was the most abundant genus in the early stages of fermentation. However, Lactobacillus and Gluconoacetobacter were found abundant with increase in pH during the later phases of fermentation (72 h-96 h). Ascomycota (99.02 %) was the most abundant fungal phylum. Hanseniaspora was the abundant yeast in the initial stages of fermentation, whereas the population of Saccharomyces increased significantly after 24 h of fermentation. Torulaspora, Lachancea and Starmerella showed their heterogeneous distribution throughout the fermentation. Computational analysis of metagenomes based on KEGG and MetaCyc databases showed different predictive functional profiles such as folate biosynthesis, glutathione metabolism, terpenoids biosynthesis and biosynthesis of amino acids with significant differences between the fresh palm saps and fermenting saps during toddy fermentation.

Circumscription of the genus Lepra, a recently resurrected genus to accommodate the "Variolaria"-group of Pertusaria sensu lato (Pertusariales, Ascomycota).

Pertusarialean lichens include more than 300 species belonging to several independent phylogenetic lineages. Only some of these phylogenetic clades have been comprehensively sampled for molecular data, and formally described as genera. Here we present a taxonomic treatment of a group of pertusarialean lichens formerly known as "Pertusaria amara-group", "Monomurata-group", or "Variolaria-group", which includes widespread and well-known taxa such as P. amara, P. albescens, or P. ophthalmiza. We generated a 6-locus data set with 79 OTUs representing 75 species. The distinction of the Variolaria clade is supported and consequently, the resurrection of the genus Lepra is followed. Thirty-five new combinations into Lepra are proposed and the new species Lepra austropacifica is described from mangroves in the South Pacific. Lepra is circumscribed to include species with disciform ascomata, a weakly to non-amyloid hymenial gel, strongly amyloid asci without clear apical amyloid structures, containing 1 or 2, single-layered, thin-walled ascospores. Chlorinated xanthones are not present, but thamnolic and picrolichenic acids occur frequently, as well as orcinol depsides. Seventy-one species are accepted in the genus. Although the distinction of the genus from Pertusaria is strongly supported, the relationships of Lepra remain unresolved and the genus is tentatively placed in Pertusariales incertae sedis.

Fodinomyces uranophilus gen. nov. sp. nov. and Coniochaeta fodinicola sp. nov., two uranium mine-inhabiting Ascomycota fungi from northern Australia.

Seven acidophilic/acidotolerant fungal strains were characterized from samples of process waters (raffinate) at one of Australia's largest uranium mines, the Ranger Mine in Northern Territory. They were isolated from raffinate, which typically were very acidic (pH 1.7-1.8) and contained high concentrations of total dissolved/colloidal salts (> 100 g/L). Five of the isolates correspond to two new acidotolerant Ascomycota fungi. The first is a member of a new genus, here described as Fodinomyces (Teratosphaeriaceae, Capnodiales, Dothideomycetes) and does not show clear close affiliation with any other described fungus in the scientific literature. The second belongs to the genus Coniochaeta (Coniochaetaceae, Coniochaetales, Sordariomycetes) and is closely related to Coniochaeta hansenii.

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies.

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.

Phylogeny and taxonomy of Aspicilia and Megasporaceae.

Phylogenetic analyses based on nuLSU and mtSSU indicate that Megasporaceae is monophyletic. Aspicilia species were distributed among three main well supported groups and one group with low support that included the type species; a division of the family into five genera is proposed. The old names Circinaria and Sagedia are reintroduced for groups not including A. cinerea, the type of Aspicilia. The monotypic Megaspora is closely related to Circinaria, while Lobothallia is the sister group of the other Megasporaceae genera. Aspicilia recedens and A. farinosa are transferred to Lobothallia. Species of the 'Sphaerothallia group' are nested in Circinaria. Aspilidea is not a member of Megasporaceae but seems to be more closely related to Ochrolechiaceae. Aspilidea myrinii is neotypified, and lectotypes are designated for Aspicilia gibbosa, A. leprosescens and Lecanora gibbosula.

Taxonomic revision of the Lecania cyrtella group based on molecular and morphological evidence.

This investigation elucidates relationships within the Lecania cyrtella group (Ramalinaceae, lichenized Ascomycota) by employing morphological, anatomical and molecular methods. The morphological studies included eleven species of Lecania, L. cyrtella, L. cyrtellina, L. dubitans, L. erysibe, L. hutchinsiae, L. leprosa, L. madida, L. prasinoides, L. sambucina, L. sordida and L. sylvestris, and a key to the species plus species descriptions are provided. Lecania madida, a new species from the Pacific Northwest of North America, L. leprosa, a new species from eastern Europe, and L. sordida, a new species from Europe, are described here. The known range of L. prasinoides is greatly extended to include the Baltic countries, Nordic countries and western Canada. Lectotypes are designated for L. cyrtella and L. sambucina. Molecular relationships within the group were examined with haplotype network estimations and phylogenetic reconstructions. Part of the IGS region as well as the complete ITS region were sequenced and analyzed. Both the haplotype network and the phylogenetic analyses indicate that the included species, as conceived in the morphological examinations, all are monophyletic.

Promiscuous DNA in the nuclear genomes of hemiascomycetous yeasts.

Abstract Transfer of fragments of mtDNA to the nuclear genome is a general phenomenon that gives rise to NUMTs (NUclear sequences of MiTochondrial origin). We present here the first comparative analysis of the NUMT content of entirely sequenced species belonging to a monophyletic group, the hemiascomycetous yeasts (Candida glabrata, Kluyveromyces lactis, Kluyveromyces thermotolerans, Debaryomyces hansenii and Yarrowia lipolytica, along with the updated NUMT content of Saccharomyces cerevisiae). This study revealed a huge diversity in NUMT number and organization across the six species. Debaryomyces hansenii harbors the highest number of NUMTs (145), half of which are distributed in numerous large mosaics of up to eight NUMTs arising from multiple noncontiguous mtDNA fragments inserted at the same chromosomal locus. Most NUMTs, in all species, are found within intergenic regions including seven NUMTs in pseudogenes. However, five NUMTs overlap a gene, suggesting a positive impact of NUMTs on protein evolution. Contrary to the other species, K. lactis and K. thermotolerans harbor only a few diverged NUMTs, suggesting that mitochondrial transfer to the nuclear genome has decreased or ceased in these phylogenetic branches. The dynamics of NUMT acquisition and loss are illustrated here by their species-specific distribution.

Why do some yeast species require niacin for growth? Different modes of NAD synthesis.

NAD holds a key position in metabolism and cellular regulatory events as a major redox carrier and a signalling molecule. NAD biosynthesis pathways have been reconstructed and compared in seven yeast species with completely sequenced genomes, including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Debaryomyces hansenii, Candida albicans, Yarrowia lipolytica and Schizosaccharomyces pombe. Both amino acid and nucleotide sequence similarity analysis in silico indicated that de novo NAD biosynthesis might not exist in K. lactis, C. glabrata and Schiz. pombe, while other species have the kynurenine pathway. It also showed that the NAD salvage pathway via nicotinic acid and nicotinic acid mononucleotide is conserved in all of these yeasts. Deletion of KlNPT1 (the gene for nicotinate phosphoribosyl-transferase) is lethal, which demonstrates that this salvage pathway, utilizing exogenous nicotinic acid, is the unique route to synthesize NAD in K. lactis. The results suggested that the basis of the variation of niacin requirements in yeasts lies in their different combinations of NAD biosynthesis pathways. The de novo pathway is absent but the salvage pathway is conserved in niacin-negative yeasts, while both pathways coexist in niacin-positive yeasts.

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese.

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis.

BACKGROUND: To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available. RESULTS: A dataset of 345,829 genes was extracted from 42 publicly available fungal genomes. Supertree methods were employed to derive phylogenies from 4,805 single gene families. We found that the average consensus supertree method may suffer from long-branch attraction artifacts, while matrix representation with parsimony (MRP) appears to be immune from these. A genome phylogeny was also reconstructed from a concatenated alignment of 153 universally distributed orthologs. Our MRP supertree and concatenated phylogeny are highly congruent. Within the Ascomycota, the sub-phyla Pezizomycotina and Saccharomycotina were resolved. Both phylogenies infer that the Leotiomycetes are the closest sister group to the Sordariomycetes. There is some ambiguity regarding the placement of Stagonospora nodurum, the sole member of the class Dothideomycetes present in the dataset. Within the Saccharomycotina, a monophyletic clade containing organisms that translate CTG as serine instead of leucine is evident. There is also strong support for two groups within the CTG clade, one containing the fully sexual species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus. The second major clade within the Saccharomycotina contains species whose genomes have undergone a whole genome duplication (WGD), and their close relatives. We could not confidently resolve whether Candida glabrata or Saccharomyces castellii lies at the base of the WGD clade. CONCLUSION: We have constructed robust phylogenies for fungi based on whole genome analysis. Overall, our phylogenies provide strong support for the classification of phyla, sub-phyla, classes and orders. We have resolved the relationship of the classes Leotiomyctes and Sordariomycetes, and have identified two classes within the CTG clade of the Saccharomycotina that may correlate with sexual status.

A surprisingly large RNase P RNA in Candida glabrata.

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

Effects of aliphatic acids, furfural, and phenolic compounds on Debaryomyces hansenii CCMI 941.

Debaryomyces hansenii is a polyol overproducing yeast that can have a potential use for upgrading lignocellulosic hydrolysates. Therefore, the establishment of its tolerance to metabolic inhibitors found in hydrolysates is of major interest. We studied the effects of selected aliphatic acids, phenolic compounds, and furfural. Acetic acid favored biomass production for concentrations <6.0 g/L. Formic acid was more toxic than acetic acid and induced xylitol accumulation (maximum yield of 0.21 g/g of xylose). All tested phenolics strongly decreased the specific growth rate. Increased toxicity was found for hydroquinone, syringaldehyde, and 4-methylcatechol and was correlated to the compound's hydrophobicity. Increasing the amount of furfural led to longer lag phases and had a detrimental effect on specific growth rate and biomass productivity.

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera.

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.

Reexamination of yeast strains classified as Torulaspora delbrueckii (Lindner).

Twenty-eight yeast strains presumed to represent Torulaspora delbrueckii were analyzed by randomly amplified polymorphic DNA-PCR analysis. Four strains (HUT 7161, IFO 1138, IFO 1145, and IFO 1956) that were considerably different from the type strain were further investigated. Morphological and physiological characteristics revealed that strains HUT 7161 and IFO 1145 belong to the genus Debaryomyces rather than the genus Torulaspora, and the former strain may represent Debaryomyces hansenii. Strains IFO 1138 and IFO 1956 were classified as either Saccharomyces castellii or Saccharomyces dairensis by identification keys involving physiological tests. On the basis of analysis of the sequences of two rRNA internal spacer regions, strains IFO 1138 and IFO 1956 were closely related to S. castellii and strains HUT 7161 and IFO 1145 were outside members of the genera Torulaspora, Zygosaccharomyces, and Saccharomyces.

Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences.

Partial 26S ribosomal DNA sequences of species assigned to the genera Hanseniaspora, Kloeckera, Dekkera, Brettanomyces, and Eeniella were determined. A phylogenetic analysis of the sequences showed that the genus Eeniella is derived within the genus Brettanomyces and that the genus Hanseniaspora (anamorph Kloeckera) is not closely related to the genus Dekkera (anamorph Brettanomyces). As a consequence, the name Eeniella is reduced to synonymy with the name Brettanomyces. In addition, our data do not support reassignment of certain Hanseniaspora species to the recently revived genus Kloeckeraspora.

Conspecificity of Hanseniaspora nodinigri and Hanseniaspora vineae: comparison by DNA reassociation.

Hanseniaspora nodinigri Lachance 1981, found in black knots (caused by Dibotryon morbosum) of Prunus virginiana, was described as a new species, some time after publication of the extensive study by Meyer et al. (1978) on the systematics of Hanseniaspora Zikes and its imperfect counterpart Kloeckera Janke. Lachance delimited the species from other members of the genus because of absence of growth on glucono-delta-lactone. He also stated that this species, although evidently similar in many ways to H. vineae van der Walt et Tscheuschner and H. osmophila (Niehaus) Phaff et al., has 'diverged from them, possibly in its adaptation to growth in association with black knots'.

Hanseniaspora nodinigri, a new yeast species found in black knots (Dibotryon morbosum) of Prunus virginiana.

The new yeast species Hanseniaspora nodinigri is described to accommodate members of the genus Hanseniaspora that are unable to assimilate glucono-sigma-lactone and isolated from stromatal tissue of black knots (Dobotryon morbosum) of chokecherry, Prunus virginiana. The newly described taxon shows much resemblance, by other criteria, to H. vineae van der Walt et Tscheuschner and H. osmophila (Niehaus) Phaff, Miller et Shifrine.

Systematics of Hanseniaspora Zikes and Kloeckera Janke.

The physiological and morphological characteristics of eighty-two strains of Hanseniaspora and Kloeckera, represented twenty-nine described species, were examined. These results along with DNA base composition and DNA/DNA reassociation experiments revealed that the genus Hanseniaspora comprises six distinct species, viz. H. valbyensis, H. uvarum, H. guilliermondii, H. occidentalis, H. osmophila and H. vineae, with K. japonica, K. apiculata, K. apis, K. javanica, K. corticis and K. africana, respectively, as their imperfect states.