Hi-C metagenomics facilitate comparative genome analysis of bacteria and yeast from spontaneous beer and cider.

Sequence-based analysis of fermented foods and beverages' microbiomes offers insights into their impact on taste and consumer health. High-throughput metagenomics provide detailed taxonomic and functional community profiling, but bacterial and yeast genome reconstruction and mobile genetic elements tracking are to be improved. We established a pipeline for exploring fermented foods microbiomes using metagenomics coupled with chromosome conformation capture (Hi-C metagenomics). The approach was applied to analyze a collection of spontaneously fermented beers and ciders (n = 12). The Hi-C reads were used to reconstruct the metagenome-assembled genomes (MAGs) of bacteria and yeasts facilitating subsequent comparative genomic analysis, assembly scaffolding and exploration of "plasmid-bacteria" links. For a subset of beverages, yeasts were isolated and characterized phenotypically. The reconstructed Hi-C MAGs primarily belonged to the Lactobacillaceae family in beers, along with Acetobacteraceae and Enterobacteriaceae in ciders, exhibiting improved quality compared to conventional metagenomic MAGs. Comparative genomic analysis of Lactobacillaceae Hi-C MAGs revealed clustering by niche and suggested genetic determinants of survival and probiotic potential. For Pediococcus damnosus, Hi-C-based networks of contigs enabled linking bacteria with plasmids. Analyzing phylogeny and accessory genes in the context of known reference genomes offered insights into the niche specialization of beer lactobacilli. The subspecies-level diversity of cider Tatumella spp. was disentangled using a Hi-C-based graph. We obtained highly complete yeast Hi-C MAGs primarily represented by Brettanomyces and Saccharomyces, with Hi-C-facilitated chromosome-level genome assembly for the former. Utilizing Hi-C metagenomics to unravel the genomic content of individual species can provide a deeper understanding of the ecological interactions within the food microbiome, aid in bioprospecting beneficial microorganisms, improving quality control and improving innovative fermented products.

Culturable yeast diversity in urban topsoil influenced by various anthropogenic impacts.

In urban ecosystems, processes associated with anthropogenic influences almost always lead to changes in soil micromycete complexes. The taxonomic structure of soil micromycete complexes is an important informative parameter of soil bioindication in the ecological control of urban environments. Unicellular fungi, such as culturable yeasts, are a very suitable and promising object of microbiological research for monitoring urban topsoil. This review aims to give an overview of the yeast communities in urban topsoil in different areas of Moscow (heating main area, household waste storage and disposal area, highway area) and to discuss the changes in the taxonomic structure of culturable yeast complexes depending on the type and intensity of anthropogenic impact.

Yeasts associated with mines on tree leaves in the urban areas / Levaduras asociadas a minas en hojas de árboles en zonas urbanas

Mines on tree leaves and undamaged leaves were studied to investigate yeast complexes in urban areas (Aesculus hippocastanum, miner — Cameraria ohridella; Betula verrucosa, miner — Caloptilia betulicola; Populus nigra, miner — Lithocolletis populifoliella; Quercus robur, miner — Tischeria companella; Salix caprea, miner — Trachys minuta; Syringa vulgaris, miner — Caloptilia syringella; Tilia cordata, miner — Phyllonorycter issikii; Ulmus laevis, miner — Carpatolechia fugitivella). The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media (GPY agar). Identification of yeast species was based on the ITS rDNA nucleotide sequence. The average abundance of yeasts during the first stages of mine formation in the internal tissues of leaves was 103 cfu/g. After 23–25 days, during the last stage of larval metamorphosis before mine destruction, the abundance of yeasts in the mines increased by two orders of magnitude to 105 cfu/g. No significant differences were observed in the abundance of yeasts in mines formed by different insects on different trees. A total of twelve yeast species were observed. The fast-growing ascomycetous yeasts Hanseniaspora uvarum and H. occidentalis dominated the mines. On undamaged leaves, the basidiomycetous yeasts Papiliotrema flavescens and Rhodotorula mucilaginosa, typical in the phyllosphere, dominated. The opportunistic yeast Candida parapsilosis was detected in the yeast complexes of all mines examined and was not found on the surface of leaves. Comparison of the relative abundance of yeast species between the studied mines and undamaged leaves using principal component analysis showed that all studied yeast communities in the mines were significantly different from the epiphytic yeast complexes of the undamaged leaves. Thus, miners in urban environments provoke the formation of short-lived endophytic yeast complexes with high abundance of Hanseniaspora. For leaf miners, the yeasts serve primarily as a food source for insect larvae rich in vitamins and amino acids. The adult leaf miners, in turn, contribute to the reproduction of the yeasts and create favorable conditions for their development.(AU)

Taxogenomic placement of Rasporella oleae and Rasporella dianae gen. and spp. nov., two insect associated yeast species.

During the course of independent studies in Europe, North America, and Africa, seven yeast strains were isolated from insect frass, decaying wood, tree flux, and olive oil sediment. Phylogenetic analysis of two barcoding DNA regions (internal transcribed spacer and the D1/D2 domain of the LSU rRNA gene) revealed that they belong to two closely related undescribed species distinct from all genera in the family Debaryomycetaceae. For reliable taxonomic placement the genomes of four strains of the two novel species and six type strains of closely related species were sequenced. Orthologous genes from 54 genomes of representatives of the Pichiomycetes and 23 outgroup taxa were concatenated to construct a fully supported phylogenetic tree. Consistent with the assumptions, we found that the two new species belong to a novel genus. In addition, the delimitation of the novel species was supported by genetic distance calculations from average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) values. The physiological characterization of the novel species was generally consistent with their genomic content. All strains had two alleles encoding secretory lipase in either two or three copies depending on the species. However, lipolytic activity was detected only in strains with three copies of the secretory lipase gene. Nevertheless, lipolytic activity might be related to their association with the insect gut. Based on these results, formal descriptions of the new genus Rasporella gen. nov. and of two new species Rasporella dianae sp. nov. (holotype UCDFST 68-643T , MycoBank no.: 850238) and Rasporella oleae sp. nov. (holotype ZIM 2471T , MycoBank no.: 850126) are provided.

Wild and partially synanthropic bird yeast diversity, in vitro virulence, and antifungal susceptibility of Candida parapsilosis and Candida tropicalis strains isolated from feces.

Yeast complexes in the fecal samples of wild (Dendrocopos major, Picus viridis) and partially synanthropic (Bombycilla garrulus, Garrulus glandarius, Pica pica, and Pyrrhula pyrrhula) birds were studied in a forest ecosystem during winter. A total of 18 yeast species were identified: 16 ascomycetes and two basidiomycetes belonging to five subphyla of fungi: Saccharomycotina (15), Pezizomycotina (1), Agaricomycotina (1), and Pucciniomycotina (1). Most yeast species were found in the fecal samples of P. pyrrhula (Candida parapsilosis, C. zeylanoides, Debaryomyces hansenii, Hanseniaspora uvarum, Metschnikowia pulcherrima, Meyerozyma carpophila, M. guilliermondii, Rhodotorula mucilaginosa); the lowest number of yeast species was observed in the feces of B. garrulus (C. parapsilosis, C. zeylanoides, Met. pulcherrima, and Rh. mucilaginosa). The opportunistic species of the genus Candida were found only in feces of partially synanthropic birds: C. parapsilosis was observed in the feces of B. garrulus, G. glandarius, P. pica, and P. pyrrhula; its relative abundance was 69.3%, 49.1%, 10.5%, and 1.1%, respectively; C. tropicalis was observed in the feces of P. pica and G. glandarius; its relative abundance was 54.6% and 7.1%, respectively. Strains of C. parapsilosis and C. tropicalis isolated from the feces of partially synanthropic birds were evaluated for their susceptibility to conventional antifungal agents (fluconazole, voriconazole, amphotericin B) and hydrolytic activity. A total of 160 strains were studied. Resistance to fluconazole was detected in 86.8% of C. parapsilosis strains and in 87% of C. tropicalis strains; resistance to voriconazole was detected in 71.7% of C. parapsilosis and in 66.7% of C. tropicalis strains, and the lowest percentage of resistant strains was detected to amphotericin B, 2.8% and 3.7% in C. parapsilosis and C. tropicalis strains, respectively. Multiresistance was detected in one strain of C. parapsilosis isolated from P. pica feces and in one strain of C. tropicalis isolated from G. glandarius feces. Phospholipase and hemolysin activities in the strains of C. parapsilosis were low (mean Pz values of 0.93 and 0.91, respectively); protease activity was moderate (mean Pz value of 0.53). The ability to produce hydrolytic enzymes was higher in the isolated strains of C. tropicalis. The mean Pz values of phospholipase and hemolysin activities were moderate (mean Pz values of 0.63 and 0.60, respectively), whereas protease activity was high (mean Pz value of 0.32). Thus, wild and partially synanthropic birds play an important role in disseminating of various yeast species. These yeasts can enter the topsoil via feces and contribute to the formation of allochthonous and uneven soil yeast diversity in natural ecosystems. In addition, partially synanthropic birds can be vectors of virulent strains of opportunistic Candida species from urban environments to natural biotopes.

The role of fruits as reservoirs for resistant and virulent strains of opportunistic yeasts.

Strains of the Candida parapsilosis and Meyerozyma guilliermondii species complexes isolated from the internal tissues of 26 fruit species from 21 countries were evaluated for their susceptibility to conventional antifungal compounds (fluconazole, voriconazole, amphotericin B) and hydrolytic activity. A total of 144 strains were studied. Resistance to at least one of the antifungal compounds tested was found in 26.4% of the endophytic strains examined. Most of the strains were insensitive to fluconazole. Multiresistance was detected only in two strains of C. parapsilosis sensu stricto from tropical apples. Phospholipase production and proteolytic and hemolytic activities were significantly higher in endophytes from tropical fruits. Resistant and virulent strains of opportunistic yeasts can thus spread worldwide via purchased fruit, which can harm people with a weakened immune status and children whose immune systems are not yet fully developed.

Yeasts associated with mines on tree leaves in the urban areas.

Mines on tree leaves and undamaged leaves were studied to investigate yeast complexes in urban areas (Aesculus hippocastanum, miner - Cameraria ohridella; Betula verrucosa, miner - Caloptilia betulicola; Populus nigra, miner - Lithocolletis populifoliella; Quercus robur, miner - Tischeria companella; Salix caprea, miner - Trachys minuta; Syringa vulgaris, miner - Caloptilia syringella; Tilia cordata, miner - Phyllonorycter issikii; Ulmus laevis, miner - Carpatolechia fugitivella). The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media (GPY agar). Identification of yeast species was based on the ITS rDNA nucleotide sequence. The average abundance of yeasts during the first stages of mine formation in the internal tissues of leaves was 103 cfu/g. After 23-25 days, during the last stage of larval metamorphosis before mine destruction, the abundance of yeasts in the mines increased by two orders of magnitude to 105 cfu/g. No significant differences were observed in the abundance of yeasts in mines formed by different insects on different trees. A total of twelve yeast species were observed. The fast-growing ascomycetous yeasts Hanseniaspora uvarum and H. occidentalis dominated the mines. On undamaged leaves, the basidiomycetous yeasts Papiliotrema flavescens and Rhodotorula mucilaginosa, typical in the phyllosphere, dominated. The opportunistic yeast Candida parapsilosis was detected in the yeast complexes of all mines examined and was not found on the surface of leaves. Comparison of the relative abundance of yeast species between the studied mines and undamaged leaves using principal component analysis showed that all studied yeast communities in the mines were significantly different from the epiphytic yeast complexes of the undamaged leaves. Thus, miners in urban environments provoke the formation of short-lived endophytic yeast complexes with high abundance of Hanseniaspora. For leaf miners, the yeasts serve primarily as a food source for insect larvae rich in vitamins and amino acids. The adult leaf miners, in turn, contribute to the reproduction of the yeasts and create favorable conditions for their development.

Diversity of Endophytic Yeasts from Agricultural Fruits Positive for Phytohormone IAA Production.

This study reports the diversity of cultivable endophytic yeasts from agricultural fruits that respond positively to the plant-promoting property of indole-3-acetic acid (IAA) production. The IAA synthesis by the strains was quantified with an Agilent 1100 series liquid chromatography system. IAA was present in the culture liquid of 72% of all 97 strains examined after three days of cultivation. The most active endophytic yeast strains in this study belonged to the species Aureobasidium pullulans, Candida zeylanoides, Hanseniaspora uvarum, Metschnikowia pulcherrima, Meyerozyma caribbica, Rhodotorula mucilaginosa, and Yarrowia galli. The highest IAA production was observed in the endophytic strain of A. pullulans (9109.19 ± 146.02 µg/g). No significant differences were found between IAA production in strains from agricultural products of different countries. However, the level of IAA production was strictly strain-specific. Our results suggest that the internal tissues of fruits may be a promising source for the isolation of plant-beneficial yeasts that can be used to promote plant growth.

Changes in urban soil yeast communities after a reduction in household waste during the COVID-19 pandemic.

The soils of streets, urban parks and suburban areas were examined for yeasts in the summer of 2020 on the territory of the southern cities of Russia and the Republic of Crimea: Krasnodar, Maykop, Sochi and Simferopol. The results of this study are compared with the results of a previous study carried out in these cities in 2019. This study was conducted three months after the lockdown due to the COVID-19 pandemic, which led to a sustained decline in household waste deposition in these areas. The number of tourists visiting these southern cities decreased significantly, and the number of walkers and visitors to urban parks fell sharply. In 2020, after the decline of household waste loads, the yeast abundance was slightly but reliably higher than in 2019. A total of 30 yeast species were observed - 11 ascomycetes and 19 basidiomycetes. This was more than in 2019 and was caused by twice as many autochthonous basidiomycetous yeast species (natural core community), which were found in urban soils only after the reduction in household waste in the environment - Apiotrichum dulcitum, A. laibachii, Saitozyma podzolica Solicoccozyma terricola. And at the same time, the proportion of clinically significant (opportunistic) yeasts, Candida sake and Meyerozyma guilliermondii, was much lower in 2020 than in 2019. Thus, the observed changes in yeast communities in urban soils could be a short-time response of the microbial community to a reduction in household waste.

Dramatic effect of black soldier fly larvae on fungal community in a compost.

BACKGROUND: Black soldier fly larvae (BSFL) offer very good prospects for the production of cheap and high-quality dietary protein. This insect is able to consume low-quality substrates, including food waste. The properties and safety of the compost obtained are largely determined by its microbiome. However, while the bacterial component of the BSFL-compost microbiome has been much studied, little is known about its fungal component. In plant-based rearing substrates both the biomass and the metabolic activity of fungi often exceed the biomass and the activity of prokaryotes. So, the purpose of this study was to investigate the fungal community of the compost produced by BSF larvae reared on a food waste substrate. RESULTS: Community structure was determined by metabarcoding of the ITS (internal transcribed spacer) region. Species composition and abundance were determined using the plating technique and subsequent identification of the isolated pure cultures. It was found that the primary mycobiome of the food waste substrate that was used consisted of 19 families, represented mainly by phytopathogenic and endophytic genera. Larva incubation led to the complete elimination of all mycelial fungi from the resulting compost. The final mycobiome consisted of only two yeast species, Pichia kudriavzevii and Diutina rugosa, with a total abundance of 1.2 × 107 CFU g-1 . CONCLUSIONS: The rearing of BSFL led to the complete elimination of mycelial fungi from its feed substrate. The final compost lacked harmful fungi, including molds. This information may be crucial for BSF compost utilization. The phenomenon was also an interesting aspect of zoomicrobial interactions in nature and agriculture. © 2021 Society of Chemical Industry.

Nomenclatural issues concerning cultured yeasts and other fungi: why it is important to avoid unneeded name changes.

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate.

Zygotorulaspora dagestanica sp. nov., a novel ascomycetous yeast species associated with the Georgian honeysuckle (Lonicera iberica M. Bieb.).

During an investigation of the yeast communities associated with wild fruit shrubs in Dagestan (Caucasus, Russia), four fermenting ascospore-producing yeast strains were isolated from leaves of the Georgian honeysuckle (Lonicera iberica M. Bieb.) and from soil underneath this plant. Phylogenetic analyses based on concatenated sequences of the ITS region and D1/D2 domains of the large subunit rRNA gene and concatenated sequences of the ribosomal DNA cystron, RPB2 and TEF1 genes showed that the isolated strains represented a new species of the genus Zygotorulaspora. The new species was placed in the basal position to other species of the clade and close to Zygotorulaspora mrakii. Based on the results of phylogenetic analyses and the phenotypic characteristics of the four studied strains, a novel species is described, for which the name Zygotorulaspora dagestanica sp. nov. is proposed. The holotype is KBP Y-4591T, three metabolically inactive cryopreserved isotype cultures are DSM 100088, VKM Y-3060 and VKPM Y-4318. The MycoBank number is MB 838285.

Yeasts in Feces of Pigeons (Columba livia) in the City of Moscow.

The yeast number and species diversity in feces of pigeons (Columba livia) were studied in various locations of the city of Moscow: parks, playgrounds and school grounds. The total number and species composition of yeasts in feces depended on the isolation temperature. The average yeast number at a cultivation temperature of 25 °C was 3.2 × 105 CFU/g, at 37 °C-2.5 × 106 CFU/g. At 37 °C, the number of yeasts was higher due to a more abundant growth of pathogenic and opportunistic yeast species. In total, 13 species of yeasts were isolated from feces: eight ascomycetes and five basidiomycetes. The study revealed high values of relative abundance and frequency of occurrence of pathogenic species Candida albicans and opportunistic yeasts, i.e. Diutina catenulata, Millerozyma farinosa, Pichia kudriavzevii, and Trichosporon asahii. Pigeon feces were found to present a constant source of "infection" of the urban environment with yeast species that are hazardous to human health, especially for immunosuppressive individuals.

Yamadazyma cocois f.a., sp. nov., an ascomycetous yeast isolated from coconuts.

During studies on the endophytic yeast communities associated with fruits from Vietnam, three fermenting yeast strains were isolated from fruits of the coconut palm (Cocos nucifera). Phylogenetic analysis based on the sequences of the ITS regions and D1/D2 domains of the large subunit rRNA gene showed that these strains represented a single species of the Yamadazyma clade that was distinct from the other related species. The new species represented a basal branch of the clade formed by the Yamadazyma species i.e. Y. insecticola and Y. takamatsuzukensis. Based on the phylogenetic analysis and phenotypic characteristics, the studied strains were assigned to a novel species of the genus Yamadazyma, for which the name Yamadazyma cocois f.a., sp. nov. is proposed. The holotype is VCIM 4241, with the ex-type cultures VTCC 920004=VKM Y-3049=KBP Y-6091 code 17-68. The MycoBank number is MB 834435.

Micromycetes as colonizers of mineral building materials in historic monuments and museums.

Complex of microfungi colonizing mineral building materials, i.e. limestone and plaster, in interiors of cultural heritage was characterized. Wide-scale investigation was carried out with fourteen objects studied. We have revealed a specific culturable community. We have analyzed role of obtained microfungi in biodeterioraton process on the basis of our tests (pH and water activity preferences, ability to solubilize CaCO3) and literature data (substrate preferences and enzyme activities). The species most actively developing in mineral materials in indoor environments were Acremonium charticola, Acremonium furcatum, Lecanicillium sp., Parengyodontium album, Purpureocillium lilacinum and Sarocladium kiliense. Considering this fact and their ability to develop successfully at extremely wide range of pH values from slightly acidic to alkaline ones and their high enzymatic activities we conclude that the listed species are of high interest in seeking the cause of biodeterioration. These species can actively develop in materials penetrating for years deep into the substrates and causing their deterioration in conditions of considerably heightened moisture content. In this group, A. charticola and Lecanicillium sp. were able to solubilize CaCO3.

Yeasts producing zeatin.

The present paper describes the first screening study of the ability of natural yeast strains to synthesize in culture the plant-related cytokine hormone zeatin, which was carried out using HPLC-MS/MS. A collection of 76 wild strains of 36 yeast species (23 genera) isolated from a variety of natural substrates was tested for the production of zeatin using HPLC-MS/MS. Zeatin was detected in more than a half (55%) of studied strains and was more frequently observed among basidiomycetous than ascomycetous species. The amount of zeatin accumulated during the experiment varied among species and strains. Highest zeatin values were recorded for basidiomycete Sporobolomyces roseus and ascomycete Taphrina sp. that produced up to 8,850.0 ng and 5,166.4 ng of zeatin per g of dry biomass, respectively. On average, the ability to produce zeatin was more pronounced among species isolated from the arctic-alpine zone than among strains from tropical and temperate climates. Our study also demonstrated that epiphytic strains and pigmented yeast species, typically for phyllosphere, are able to more often produce a plant hormone zeatin than other yeasts.

Yeast communities in Sphagnum phyllosphere along the temperature-moisture ecocline in the boreal forest-swamp ecosystem and description of Candida sphagnicola sp. nov.

The effects of the temperature-moisture factors on the phylloplane yeast communities inhabiting Sphagnum mosses were studied along the transition from a boreal forest to a swamp biotope at the Central Forest State Biosphere Reserve (Tver region, Russia). We tested the hypothesis that microclimatic parameters affect yeast community composition and structure even on a rather small spatial scale. Using a conventional plating technique we isolated and identified by molecular methods a total of 15 species of yeasts. Total yeast counts and species richness values did not depend on environmental factors, although yeast community composition and structure did. On average, Sphagnum in the swamp biotope supported a more evenly structured yeast community. Relative abundance of ascomycetous yeasts was significantly higher on swamp moss. Rhodotorula mucilaginosa dominated in the spruce forest and Cryptococcus magnus was more abundant in the swamp. Our study confirmed the low occurrence of tremellaceous yeasts in the Sphagnum phyllosphere. Of the few isolated ascomycetous yeast and yeast-like species, some were differentiated from hitherto known species in physiological tests and phylogenetic analyses. We describe one of them as Candida sphagnicola and designate KBP Y-3887(T) (=CBS 11774(T) = VKPM Y-3566(T) = MUCL 53590(T)) as the type strain. The new species was registered in MycoBank under MB 563443.

Ogataea cecidiorum sp. nov., a methanol-assimilating yeast isolated from galls on willow leaves.

Ten strains of a new endophytic ascospore-forming, methanol-assimilating yeast were isolated from the galls induced by sawflies on the leaves of willows in the Losiny Ostrov National Park (Moscow region). Standard phenotypical tests and phylogenetic analyses of 18S rRNA gene, 5.8S-ITS gene region and 26S rRNA gene (D1/D2 domains) sequences showed that the species belongs to the genus Ogataea. We describe it as Ogataea cecidiorum and designate type culture KBP Y-3846 (= CBS 11522(T) = VKM Y-2982(T) = VKPM Y-3482(T) = MUCL 52544(T) = NCAIM Y.01965(T)) as the type strain. The new species was registered in MycoBank under MB 515233.