Biosynthesis of silver nanoparticles with antimicrobial and anticancer properties using two novel yeasts.

AgNPs are nanomaterials with many potential biomedical applications. In this study, the two novel yeast strains HX-YS and LPP-12Y capable of producing biological silver nanoparticles were isolated. Sequencing of ribosomal DNA-ITS fragments, as well as partial D1/D2 regions of 26S rDNA indicated that the strains are related to species from the genus Metschnikowia. The BioAgNPs produced by HX-YS and LPP-12Y at pH 5.0-6.0 and 26 °C ranged in size from 50 to 500 nm. The antibacterial activities of yeast BioAgNPs against five pathogenic bacteria were determined. The highest antibacterial effect was observed on P. aeruginosa, with additional obvious effects on E. coli ATCC8099 and S. aureus ATCC10231. Additionally, the BioAgNPs showed antiproliferative effects on lung cancer cell lines H1975 and A579, with low toxicity in Beas 2B normal lung cells. Therefore, the AgNPs biosynthesized by HX-YS and LPP-12Y may have potential applications in the treatment of bacterial infections and cancer.

New Isolated Metschnikowia pulcherrima Strains from Apples for Postharvest Biocontrol of Penicillium expansum and Patulin Accumulation.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

Biodiversity of yeasts isolated during spontaneous fermentation of cool climate grape musts.

Biodiversity of native yeasts, especially in winemaking, has hidden potential. In order to use the value of non-Saccharomyces strains in wine production and to minimise the possibility of its deterioration, it is necessary to thoroughly study the yeast cultures present on grape fruits and in grape must, as well as their metabolic properties. The aim of the study was to characterise the yeast microbiota found during spontaneous fermentation of grape musts obtained from grape varieties 'Rondo', 'Regent' and 'Johanniter'. Grapes from two vineyards (Srebrna Góra and Zadora) located in southern Poland were used for the research. Succession of subsequent groups of yeasts was observed during the process. Metschnikowia pulcherrima yeasts were identified both at the beginning and the end of the process. Hanseniaspora uvarum, Wickerhamomyces onychis and Torulaspora delbrueckii strains were also identified during the fermentation. Torulaspora delbrueckii and Wickerhamomyces onychis strains were identified only in grape musts obtained from grapes of the Zadora vineyard. These strains may be characteristic of this vineyard and shape the identity of wines formed in it. Our research has provided specific knowledge on the biodiversity of yeast cultures on grapes and during their spontaneous fermentation. The research results presented indicate the possibility of using native strains for fermentation of grape musts, allowing to obtain a product with favourable chemical composition and sensory profile.

Catching speciation in the act-act 2: Metschnikowia lacustris sp. nov., a sister species to Metschnikowia dekortorum.

The isolation of a single yeast strain in the clade containing Metschnikowia dekortorum, in the Amazon biome of Brazil, incited us to re-examine the species boundaries within the clade. The strain (UFMG-CM-Y6306) was difficult to position relative to neighbouring species using standard barcode sequences (ITS-D1/D2 rRNA gene region). Mating took place freely with α strains of M. bowlesiae, M. dekortorum, and M. similis, but two-spored asci, indicative of a fertile meiotic progeny, were formed abundantly only with certain strains of M. dekortorum. Accordingly, we examined mating success among every phylotype in the clade and constructed a phylogeny based on a concatenation of 100 of the largest orthologous genes annotated in draft genomes. The analyses confirmed membership of the Amazonian isolate in M. dekortorum, but also indicated that the species should be subdivided into two. As a result, we retain three original members of M. dekortorum in the species, together with the new isolate, and reassign six isolates recovered from Mesoamerican lacustrine habitats to Metschnikowia lacustris sp. nov. The type is UWOPS 12-619.2T (isotype CBS 16250T). MycoBank: MB 833751.

The yeast community of Conotelus sp. (Coleoptera: Nitidulidae) in Brazilian passionfruit flowers (Passiflora edulis) and description of Metschnikowia amazonensis sp. nov., a large-spored clade yeast.

Species of the nitidulid beetle Conotelus found in flowers of Convolvulaceae and other plants across the New World and in Hawaii consistently harbour a yeast community dominated by one or more large-spored Metschnikowia species. We investigated the yeasts found in beetles and flowers of cultivated passionfruit in Rondônia state, in the Amazon biome of Brazil, where a Conotelus species damages the flowers and hinders fruit production. A sample of 46 beetles and 49 flowers yielded 86 and 83 yeast isolates, respectively. Whereas the flower community was dominated by Kodamaea ohmeri and Kurtzmaniella quercitrusa, the major yeasts recovered from beetles were Wickerhamiella occidentalis, which is commonly isolated from this community, and a novel species of large-spored Metschnikowia in the arizonensis subclade, which we describe here as Metschnikowia amazonensis sp. nov. Phylogenetic analyses based on barcode sequences (ITS-D1/D2) and a multigene alignment of 11,917 positions (genes ura2, msh6, and pmt2) agreed to place the new species as a sister to Metschnikowia arizonensis, a rare species known only from one locality in Arizona. The two form sterile asci when mated, which is typical of related members of the clade. The α pheromone of the new species is unique but typical of the subclade. The type of M. amazonensis sp. nov. is UFMG-CM-Y6309T (ex-type CBS 16156T , mating type a), and the designated allotype (mating type α) is UFMG-CM-Y6307A (CBS 16155A ). MycoBank MB 833560.

Metschnikowia miensis f.a., sp. nov., isolated from flowers in Mie prefecture, Japan.

Four yeast strains (RIFY 10001T, RIFY 10002, RIFY 10003, and RIFY 10004) were isolated from flowers growing in fields of mustard and broad beans in Japan. Ascospore formation was not observed. Sequence analysis of the D1/D2 domain of the large subunit ribosomal RNA (LSU rRNA) gene of the four strains indicated that they belong to the genus Metschnikowia and are closely related to Metschnikowia hawaiiana strain CBS 9146T and Metschnikowia orientalis strain CBS 10331T. The D1/D2 domain of the LSU rRNA gene and internal transcribed spacer regions of strain RIFY 10001T were 85.7% identical to those of M. hawaiiana strain CBS 9146T. All four strains were distinguished from the M. hawaiiana strain CBS 9146T by their inability to ferment glucose. Hence, these four strains are novel species and were named as Metschnikowia miensis (holotype: RIFY 10001T; isotypes: NBRC 112445T = CBS 14749T).

Metschnikowia baotianmanensis f.a., sp. nov., a new yeast species isolated from the gut of the rhinoceros beetle Allomyrina dichotoma.

Four strains, NYNU 15610, NYNU 15612, NYNU 15613 and NYNU 15615, of a novel ascomycetous yeast were isolated from the gut of Allomyrina dichotoma (Coleoptera: Scarabeidae) collected from two different localities in Henan Province, Central PR China. The four strains shared identical sequences in both of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer regions. Sequence analyses revealed that this novel species represents a member of the genus Metschnikowia. It differed from its closest known species Metschnikowia zobellii, Metschnikowiaaustralis and Metschnikowia bicuspidata, by 8.4-9.2 % sequence divergence (33-40 nt substitutions and 7-12 gaps over 509 bases) in the D1/D2 sequences. The formation of ascospores was not observed on various sporulation media. In contrast to M. zobellii, M. australis and M. bicuspidata, the novel yeast species was unable to assimilate succinate, ethanol, ethylamine, cadaverine and 10 % NaCl plus 5 % glucose, but was able to grow in vitamin-free medium. The name Metschnikowia baotianmanensis f.a., sp. nov. is proposed to accommodate these strains, with NYNU 15613 as the holotype.

Exploring the diversity of a collection of native non-Saccharomyces yeasts to develop co-starter cultures for winemaking.

The inoculation of Saccharomyces cerevisiae starter cultures in grape musts is a common practice in wineries worldwide; however, native non-Saccharomyces yeast species are increasingly investigated as co-starters to augment the complexity and regionality of wine. In this study, an extensive collection of non-Saccharomyces yeasts from high-sugar matrices was created and screened with the aim to discover new strains with potentially positive oenological traits. After mining >400 yeasts from 167 samples collected across multiple Italian regions, the isolates were identified based on RAPD-PCR analysis and ITS sequencing. About one quarter of them, belonging to the genera Starmerella, Lachancea and Metschnikowia, were picked up for an in-depth molecular and physiological characterization, since these yeasts were well strewed and have a good oenological reputation. Following the genotyping, stress tolerance assays, enzymatic activity trials and single inoculum fermentations, a huge diversity was acknowledged within and between the species. Strains of S. bacillaris showed a high tolerance to ethanol and increased glycerol production, L. thermotolerans reduced volatile acidity while increasing total acidity with lactic acid, and Metschnikowia spp. exhibited remarkable aroma-related enzymatic activities, which are all prized features in winemaking. Since most of the characteristics analyzed were species and strain dependent, the obtained results are valuable for the selection of a new generation of co-starters for attempting mixed fermentation strategies aimed to improve the overall quality of regional wine.

Biocontrol capability of local Metschnikowia sp. isolates.

This study set out to isolate and identify epiphytic yeasts producing pulcherrimin, and to evaluate their potential as biological control agents (BCAs). We isolated Metschnikowia sp. strains from flowers and fruits collected in Poland. The plant material had been collected between April to September 2017 from two small orchards where traditional organic management is employed. We identified the essential phenotypic features of the yeast, including assimilation and enzymatic profiles, stress resistance, adhesion properties, and antimicrobial activity against various fungi involved in crop and/or food spoilage. Yeast screening was performed using YPD agar supplemented with chloramphenicol and Fe(III) ions. Taxonomic classification was determined by sequence analysis of the D1/D2 domains of the large subunit rRNA gene. The isolates were identified as Metschnikowia andauensis and Metschnikowia sinensis. The yeast isolates were further characterized based on their enzymatic and assimilation profiles, as well as their growth under various stress conditions. In addition, the hydrophobicity and adhesive abilities of the Metschnikowia isolates were determined using a MATH test and luminometry. Their antagonistic action against molds representing typical crop spoiling microflora was also evaluated. The assimilation profiles of the wild isolates were similar to those displayed by collection strains of M. pulcherrima. However, some of the isolates displayed more beneficial phenotypic properties, especially good growth under stress conditions. Several of the epiphytes grew well over a wider range of temperatures (8-30 °C) and pH levels (3-9), and additionally showed elevated tolerance to ethanol (8%), glucose (30%), and peroxides (50 mM). The hydrophobicity and adhesion of the yeast cells were strain- and surface-dependent. The tested yeasts showed potential for use as BCAs, with some exhibiting strong antagonism against molds belonging to the genera Alternaria, Botrytis, Fusarium, Rhizopus, and Verticillium, as well as against yeasts isolated as food spoilage microbiota.

Metschnikowia maroccana f.a., sp. nov., a new yeast species associated with floral nectar from Morocco.

Wild flowers, and in particular, nectar of flowers, have been shown to be a rich reservoir of yeast biodiversity. In a taxonomic study of yeasts recovered from floral nectar in Morocco, nine strains were found to represent a novel species. Morphological and physiological characteristics and sequence analyses of the D1/D2 region of the large subunit rRNA gene as well as the internal transcribed spacer region showed that the novel species belonged to the genus Metschnikowia. The name Metschnikowia maroccana f.a., sp. nov. (EBDCdVMor24-1T=CBS 15053T=NRRL Y-63972T) is proposed to accommodate this new species. Metschnikowia maroccana was isolated from floral nectar of Teucrium pseudochamaepitys, Teucrium polium and Gladiolus italicus. The ascosporic state of the novel species was not found. Metschnikowia maroccana was phylogenetically distinct from any currently recognized species and forms a well-supported subclade (bootstrap value 81 %) containing species associated with flowers and flower-visiting insects, including Metschnikowia gruessii, Metschnikowia lachancei and Metschnikowia vanudenii. The close genealogical relationship of M. maroccana with the M. gruessii clade is also consistent with the striking similarity of their 'aeroplane' cells morphologies and the lack of utilization of the α-glucoside trehalose. The ecology of these novel species and its probable endemicity are discussed.

Genomic diversity of a nectar yeast clusters into metabolically, but not geographically, distinct lineages.

Both dispersal limitation and environmental sorting can affect genetic variation in populations, but their contribution remains unclear, particularly in microbes. We sought to determine the contribution of geographic distance (as a proxy for dispersal limitation) and phenotypic traits (as a proxy for environmental sorting), including morphology, metabolic ability and interspecific competitiveness, to the genotypic diversity in a nectar yeast species, Metschnikowia reukaufii. To measure genotypic diversity, we sequenced the genomes of 102 strains of M. reukaufii isolated from the floral nectar of hummingbird-pollinated shrub, Mimulus aurantiacus, along a 200-km coastline in California. Intraspecific genetic variation showed no detectable relationship with geographic distance, but could be grouped into three distinct lineages that correlated with metabolic ability and interspecific competitiveness. Despite ample evidence for strong competitive interactions within and among nectar yeasts, a full spectrum of the genotypic and phenotypic diversity observed across the 200-km coastline was represented even at a scale as small as 200 m. Further, more competitive strains were not necessarily more abundant. These results suggest that dispersal limitation and environmental sorting might not fully explain intraspecific diversity in this microbe and highlight the need to also consider other ecological factors such as trade-offs, source-sink dynamics and niche modification.

Solving the puzzle of yeast survival in ephemeral nectar systems: exponential growth is not enough.

Flower nectar is a sugar-rich ephemeral habitat for microorganisms. Nectar-borne yeasts are part of the microbial community and can affect pollination by changing nectar chemistry, attractiveness to pollinators or flower temperature if yeast population densities are high. Pollinators act as dispersal agents in this system; however, pollination events lead potentially to shrinking nectar yeast populations. We here examine how sufficiently high cell densities of nectar yeast can develop in a flower. In laboratory experiments, we determined the remaining fraction of nectar yeast cells after nectar removal, and used honeybees to determine the number of transmitted yeast cells from one flower to the next. The results of these experiments directly fed into a simulation model providing an insight into movement and colonization ecology of nectar yeasts. We found that cell densities only reached an ecologically relevant size for an intermediate pollination probability. Too few pollination events reduce yeast inoculation rate and too many reduce yeast population size strongly. In addition, nectar yeasts need a trait combination of at least an intermediate growth rate and an intermediate remaining fraction to compensate for highly frequent decimations. Our results can be used to predict nectar yeast dispersal, growth and consequently their ecological effects.

Wine yeasts identification by MALDI-TOF MS: Optimization of the preanalytical steps and development of an extensible open-source platform for processing and analysis of an in-house MS database.

Saccharomyces cerevisiae is the most important yeast species for the production of wine and other beverages. In addition, nowadays, researchers and winemakers are aware of the influence of non-Saccharomyces in wine aroma complexity. Due to the high microbial diversity associated to several agro-food processes, such as winemaking, developing fast and accurate methods for microbial identification is demanded. In this context, MALDI-TOF MS mass fingerprint provides reliable tool for fast biotyping and classification of microorganisms. However, there is no versatile and standardized method for fungi currently available. In this study, an optimized sample preparation protocol was devised for the biotyping of yeasts of oenological origin. Taking into account that commercially available reference databases comprise almost exclusively clinical microorganisms, most of them bacteria, in the present study a database of yeasts isolated from vineyards and wineries was created, and its accuracy was tested using industrial and laboratory yeast strains. In addition, the implementation of a program for MALDI-TOF MS spectra analysis has been developed as an extensible open-source platform for MALDI data processing and analysis with statistical techniques that has arisen from our previous experience working with MALDI data. The software integrates two R packages for raw MALDI data preprocessing: Continuous Wavelet Transform (CWT)-based algorithm and MassSpecWavelet. One of the advantages of the CWT is that it can be directly applied to a raw spectrum, without prior baseline correction. Mass fingerprints of 109 S. cerevisiae strains and 107 non-Saccharomyces isolates were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed for strain, species, and genus-level differentiation. As a reference method, for S. cerevisiae differentiation at strain level, the analysis of the polymorphism in the inter-delta region was chosen. The data revealed that MALDI-TOF MS can be used for the rapid and accurate identification of S. cerevisiae and non-Saccharomyces isolates at genus and species level. However, S. cerevisiae differentiation at strain level was not successfully achieved, and the differentiation among Metschnikowia species was also difficult.

Isolation and Characterization of an Atypical Metschnikowia sp. Strain from the Skin Scraping of a Dermatitis Patient.

A yeast-like organism was isolated from the skin scraping sample of a stasis dermatitis patient in the Mycology Unit Department of Medical Microbiology, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The isolate produced no pigment and was not identifiable using chromogenic agar and API 20C AUX. The fungus was identified as Metschnikowia sp. strain UM 1034, which is close to that of Metschnikowia drosophilae based on ITS- and D1/D2 domain-based phylogenetic analysis. However, the physiology of the strain was not associated to M. drosophilae. This pathogen exhibited low sensitivity to all tested azoles, echinocandins, 5-flucytosine and amphotericin B. This study provided insight into Metschnikowia sp. strain UM 1034 phenotype profiles using a Biolog phenotypic microarray (PM). The isolate utilized 373 nutrients of 760 nutrient sources and could adapt to a broad range of osmotic and pH environments. To our knowledge, this is the first report of the isolation of Metschnikowia non-pulcherrima sp. from skin scraping, revealing this rare yeast species as a potential human pathogen that may be misidentified as Candida sp. using conventional methods. Metschnikowia sp. strain UM 1034 can survive in flexible and diverse environments with a generalist lifestyle.

Nectar yeasts of the Metschnikowia clade are highly susceptible to azole antifungals widely used in medicine and agriculture.

The widespread use of azole antifungals in medicine and agriculture and the resulting long-persistent residues could potentially affect beneficial fungi. However, there is very little information on the tolerance of non-target environmental fungi to azoles. In this study, we assessed the susceptibility of diverse plant- and insect-associated yeasts from the Metschnikowia clade, including several ecologically important species, to widely used medical and agricultural azoles (epoxiconazole, imazalil, ketoconazole and voriconazole). A total of 120 strains from six species were tested. Minimum inhibitory concentrations (MICs) were determined by the EUCAST broth microdilution procedure after some necessary modifications were made. The majority of species tested were highly susceptible to epoxiconazole, ketoconazole and voriconazole (>95% of strains showed MICs ≤ 0.125 mg l(-1)). Most strains were also very susceptible to imazalil, although MIC values were generally higher than for the other azoles. Furthermore, certain Metschnikowia reukaufii strains displayed a 'trailing' phenotype (i.e. showed reduced but persistent growth at antifungal concentrations above the MIC), but this characteristic was dependent on test conditions. It was concluded that exposure to azoles may pose a risk for ecologically relevant yeasts from the Metschnikowia clade, and thus could potentially impinge on the tripartite interaction linking these fungi with plants and their insect pollinators.

Species coexistence in simple microbial communities: unravelling the phenotypic landscape of co-occurring Metschnikowia species in floral nectar.

Identifying the ecological processes that underlie the distribution and abundance of species in microbial communities is a central issue in microbial ecology and evolution. Classical trade-off based niche theories of resource competition predict that co-occurrence in microbial communities is more likely when the residing species show trait divergence and complementary resource use. We tested the prediction that niche differentiation explained the co-occurrence of two yeast species (Metschnikowia reukaufii and M. gruessii) in floral nectar. Assessment of the phenotypic landscape showed that both species displayed a significantly different physiological profile. Comparison of utilization profiles in single versus mixed cultures indicated that these two species did not compete for most carbon and nitrogen sources. In mixed cultures, M. reukaufii grew better in sucrose solutions and in the presence of the antimicrobial compound digitonin than when grown as pure culture. M. gruessii, on the other hand, grew better in mixed cultures in glucose and fructose solutions. Overall, these results provide clear evidence that M. reukaufii and M. gruessii frequently co-occur in nectar and that they differ in their phenotypic response to variation in environmental conditions, suggesting that niche differentiation and resource partitioning are important mechanisms contributing to species co-occurrence in nectar yeast communities.

Non-Saccharomyces yeasts protect against epithelial cell barrier disruption induced by Salmonella enterica subsp. enterica serovar Typhimurium.

UNLABELLED: The human gastrointestinal epithelium makes up the largest barrier separating the body from the external environment. Whereas invasive pathogens cause epithelial barrier disruption, probiotic micro-organisms modulate tight junction regulation and improve epithelial barrier function. In addition, probiotic strains may be able to reduce epithelial barrier disruption caused by pathogenic species. The aim of this study was to explore non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Benchmarking against established probiotic strains, we evaluated the ability of four nonpathogenic yeast species to modulate transepithelial electrical resistance (TER) across a monolayer of differentiated human colonocytes (Caco-2 cells). Further, we assessed yeast modulation of a Salmonella Typhimurium-induced epithelial cell barrier function insult. Our findings demonstrate distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function. While the established probiotic yeast Saccharomyces boulardii increased TER across a Caco-2 monolayer by 30%, Kluyveromyces marxianus exhibited significantly stronger properties of TER enhancement (50% TER increase). In addition, our data demonstrate significant yeast-mediated modulation of Salmonella-induced epithelial cell barrier disruption and identify K. marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates distinct patterns of non-Saccharomyces yeast modulation of epithelial cell barrier function in vitro. Further, our data demonstrate significant yeast-mediated modulation of Salmonella Typhimurium-induced epithelial cell barrier disruption and identify Kluyveromyces marxianus and Metschnikowia gruessii as two non-Saccharomyces yeasts capable of protecting human epithelial cells from pathogen invasion. This study is the first to demonstrate significant non-Saccharomyces yeast-mediated epithelial cell barrier protection from Salmonella invasion, thus encouraging future efforts aimed at confirming the observed effects in vivo and driving further strain development towards novel yeast probiotics.

Yeast diversity on grapes in two German wine growing regions.

The yeast diversity on wine grapes in Germany, one of the most northern wine growing regions of the world, was investigated by means of a culture dependent approach. All yeast isolates were identified by sequence analysis of the D1/D2 domain of the 26S rDNA and the ITS region. Besides Hanseniaspora uvarum and Metschnikowia pulcherrima, which are well known to be abundant on grapes, Metschnikowia viticola, Rhodosporidium babjevae, and Curvibasidium pallidicorallinum, as well as two potentially new species related to Sporidiobolus pararoseus and Filobasidium floriforme, turned out to be typical members of the grape yeast community. We found M. viticola in about half of the grape samples in high abundance. Our data strongly suggest that M. viticola is one of the most important fermenting yeast species on grapes in the temperate climate of Germany. The frequent occurrence of Cu. pallidicorallinum and strains related to F. floriforme is a new finding. The current investigation provides information on the distribution of recently described yeast species, some of which are known from a very few strains up to now. Interestingly yeasts known for their role in the wine making process, such as Saccharomyces cerevisiae, Saccharomyces bayanus ssp. uvarum, Torulaspora delbrueckii, and Zygosaccharomyces bailii, were not found in the grape samples.

The expanding large-spored Metschnikowia clade: Metschnikowia matae sp. nov., a yeast species with two varieties from the Brazilian Atlantic Forest.

Fifty-two yeast isolates from flowers and associated nitidulid beetles of the Brazilian Atlantic Forest (Mata Atlântica) region were found to represent a new species in the large-spored Metschnikowia clade. The species is heterothallic, haploid, and allogamous, and produces asci with two aciculate ascospores that can reach 80 µm in length, as is typical in the clade. Analysis of sequences of the ribosomal RNA gene cluster indicates that the new species is closely related to Metschnikowia lochheadii, which ranges across Central America to northern Brazil, occurs as an adventive species in Hawaii, but is rarely found in central Brazil. The species is not readily distinguishable from relatives based on morphology or growth responses, but is well delineated from M. lochheadii on reproductive isolation. Based on an intron splice site PCR screen, we selected 26 isolates for further study. The sequence of the region that includes the complete internal transcribed spacer/5.8S rRNA gene segment as well as the D1/D2 domains of the large subunit rRNA gene contained three polymorphic segments and 14 haplotypes were identified. Of these, a single divergent isolate from the southernmost of four sampled localities exhibited diminished mating success when crossed with others. We describe two varieties, Metschnikowia matae var. matae sp. nov. var. nov. (type UFMG-CM-Y395(T), CBS 13986(T), NRRL Y-63736(T); allotype UFMG-CM-Y391(A), CBS 13987(A), NRRL Y-63735(A)) and Metschnikowia matae var. maris sp. nov. var. nov. (type UFMG-CM-Y397(T), CBS 13985(T), NRRL Y-63737(T)). We also report on the discovery of the h (+) mating type of Candida ipomoeae and transfer of the species to Metschnikowia ipomoeae comb. nov. (allotype UWOPS 12-660.1(A), CBS 13988(A), NRRL Y-63738(A)).

The impact of nectar chemical features on phenotypic variation in two related nectar yeasts.

Floral nectars become easily colonized by microbes, most often species of the ascomycetous yeast genus Metschnikowia. Although it is known that nectar composition can vary tremendously among plant species, most probably corresponding to the nutritional requirements of their main pollinators, far less is known about how variation in nectar chemistry affects intraspecific variation in nectarivorous yeasts. Because variation in nectar traits probably affects growth and abundance of nectar yeasts, nectar yeasts can be expected to display large phenotypic variation in order to cope with varying nectar conditions. To test this hypothesis, we related variation in the phenotypic landscape of a vast collection of nectar-living yeast isolates from two Metschnikowia species (M. reukaufii and M. gruessii) to nectar chemical traits using non-linear redundancy analyses. Nectar yeasts were collected from 19 plant species from different plant families to include as much variation in nectar chemical traits as possible. As expected, nectar yeasts displayed large variation in phenotypic traits, particularly in traits related to growth performance in carbon sources and inhibitors, which was significantly related to the host plant from which they were isolated. Total sugar concentration and relative fructose content significantly explained the observed variation in the phenotypic profile of the investigated yeast species, indicating that sugar concentration and composition are the key traits that affect phenotypic variation in nectarivorous yeasts.