High intragenomic, intergenomic, and phenotypic diversity in pulcherrimin-producing Metschnikowia yeasts indicates a special mode of genome evolution.

In molecular systematics, the delimitation of yeast species is based on the notion that the barcode differences are smaller within species than between them. The most widely used barcodes are segments of the chromosomal repeats coding for ribosomal RNAs that are homogenised in yeasts. The analysis of these segments of the type strains of ten species recently merged in Metschnikowia pulcherrima and 37 new isolates demonstrated that this is not the case in this species. The intragenomic diversity significantly exceeded the threshold gaps used to differentiate related yeast species. Large segments of the D1/D2 domains were not diverse within the genomes and could therefore be used to determine the taxonomic affiliation of the isolates. The genome structures of the isolates were compared by RAPD and the RFLP of the mitochondrial DNA. Both patterns were highly heterogeneous. The sequence analysis of the PUL4 gene (a member of the PUL gene cluster involved in pulcherrimin production) revealed very high intragenomic differences, suggesting that the genomes may be chimerised. Three phenotypic traits related to the antimicrobial antagonism characteristic of the species were also highly diverse and prone to reversible segregation resembling epigenetic processes (silencing and reactivation of regulators) rather than mutations and back-mutations. These features make M. pulcherrima unique among yeasts and indicate that it evolves in a non-standard way.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Candida massiliensis sp. nov. Isolated from a Clinical Sample.

The majority of Candida species are known as non-pathogenic yeasts and rarely involved in human diseases. However, recently case reports of human infections caused by non-albicans Candida species have increased, mostly in immunocompromised hosts. Our study aimed to describe and characterize as thoroughly as possible, a new species of the Metschnikowia clade, named here Candida massiliensis (PMML0037), isolated from a clinical sample of human sputum. We targeted four discriminant genetic regions: "Internal Transcribed Spacers" of rRNA, D1/D2 domains (28S large subunit rRNA) and part of the genes encoding Translation Elongation Factor 1-α and ß-tubulin2. The genetic data were compared to morphological characters, from scanning electron microscopy (TM 4000 Plus, SU5000), physiological, including the results of oxidation and assimilation tests of different carbon sources by the Biolog system, and chemical mapping by Energy-Dispersive X-ray Spectroscopy. Lastly, the in vitro antifungal susceptibility profile was performed using the E-test™ exponential gradient method. The multilocus analysis supported the genetic position of Candida massiliensis (PMML0037) as a new species of the Metschnikowia clade, and the phenotypic analysis highlighted its unique morphological and chemical profile when compared to the other Candida/Metschnikowia species included in the study.

Novel Metschnikowia yeasts from the gut of green lacewing in Japan.

Family Chrysopidae is known to harbor specific gut yeasts. However, no studies have been conducted outside of a limited number of these green lacewing species, and the diversity of yeasts in the family as a whole is not known. Therefore, we collected 58 Chrysopidae adults (9 species, 6 genera, 2 subfamilies) in Japan and isolated yeasts from all individuals. The results showed for the first time that not only subfamily Chrysopinae but also subfamily Apochrysinae have gut yeasts. We obtained 58 yeast isolates (one from each host individual), all of which were of the genus Metschnikowia. 28S rDNA- and ITS-based phylogenetic analysis showed that the isolates were divided into three clades, designated clade I, II, and III. Clade I contains two previously described Chrysopidae gut yeasts (M. picachoensis and M. pimensis) as well as a one of our new species named M. shishimaru. Clade II is a new clade, with at least two new species named M. kenjo and M. seizan. Clade III contains the previously described species M. noctiluminum, a Chrysopidae gut yeast, and one of our isolate (We have not described it as new species). However, the phylogenetic relationship between our isolate and M. noctiluminum was unclear. These results indicate that the Japanese Chrysopidae gut yeasts consist mainly of three undescribed species and that they are more unique than those found in previous surveys. The results of this study indicate that Chrysopidae gut yeasts are more diverse than previously thought and should be investigated in various geographical regions in the future.

Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the "milky disease" caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/µL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/µL) and internal transcribed spacer (ITS; 6.74 × 105 copies/µL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

First report of Metschnikowia bicuspidata infection in Chinese grass shrimp (Palaemonetes sinensis) in China.

The Chinese grass shrimp (Palaemonetes sinensis) was found with a white turbidity appearance in the Panjin area. After dissection, typical symptoms of milky disease with hemolymph emulsification and noncoagulation were observed; however, the pathogen was unknown. In this study, we aimed to isolate the pathogen of the diseased P. sinensis. We found that the pathogen could grow on the fungal medium Bengal red, and microscopic examination showed that it reproduced by budding. Molecular identification of the isolated and purified yeast strain LNMB2021 based on 26S rDNA sequence showed that the pathogenic pathogen was Metschnikowia bicuspidata (GenBank OK094821), with 98.74% homology with M. bicuspidata strain LNES0119 (GenBank OK073903) and 98.56% with M. bicuspidata strain Liao (GenBank MT856369). The results of an artificial infection test showed that M. bicuspidata caused the same clinical symptoms in P. sinensis, and the isolated pathogen was still the same, which proved that P. sinensis was a new host of M. bicuspidata. Histopathological analysis showed that there were obvious pathological changes in the hepatopancreas and muscle tissue of the diseased P. sinensis. Identification of the pathogen is essential for the prevention and control of the disease and the healthy culture of P. sinensis. Furthermore, considering the transmissibility and cross-host transmission of M. bicuspidata, its risk of infecting other aquatic animals deserves high attention.

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.

Genotypic variation in an ecologically important parasite is associated with host species, lake and spore size.

Genetic variation in parasites has important consequences for host­parasite interactions. Prior studies of the ecologically important parasite Metschnikowia bicuspidata have suggested low genetic variation in the species. Here, we collected M. bicuspidata from two host species (Daphnia dentifera and Ceriodaphnia dubia) and two regions (Michigan and Indiana, USA). Within a lake, outbreaks tended to occur in one host species but not the other. Using microsatellite markers, we identified six parasite genotypes grouped within three distinct clades, one of which was rare. Of the two main clades, one was generally associated with D. dentifera, with lakes in both regions containing a single genotype. The other M. bicuspidata clade was mainly associated with C. dubia, with a different genotype dominating in each region. Despite these associations, both D. dentifera- and C. dubia-associated genotypes were found infecting both hosts in lakes. However, in lab experiments, the D. dentifera-associated genotype infected both D. dentifera and C. dubia, but the C. dubia-associated genotype, which had spores that were approximately 30% smaller, did not infect D. dentifera. We hypothesize that variation in spore size might help explain patterns of cross-species transmission. Future studies exploring the causes and consequences of variation in spore size may help explain patterns of infection and the maintenance of genotypic diversity in this ecologically important system.

Metschnikowia pulcherrima represses aerobic respiration in Saccharomyces cerevisiae suggesting a direct response to co-cultivation.

The use of non-Saccharomyces species as starter cultures together with Saccharomyces cerevisiae is becoming a common practice in the oenological industry to produce wines that respond to new market demands. In this context, microbial interactions with these non-Saccharomyces species must be considered for a rational design of yeast starter combinations. Previously, transcriptional responses of S. cerevisiae to short-term co-cultivation with Torulaspora delbrueckii, Candida sake, or Hanseniaspora uvarum was compared. An activation of sugar consumption and glycolysis, membrane and cell wall biogenesis, and nitrogen utilization was observed, suggesting a metabolic boost of S. cerevisiae in response to competing yeasts. In the present study, the transcription profile of S. cerevisiae was analyzed after 3 h of cell contact with Metschnikowia pulcherrima. Results show an over-expression of the gluco-fermentative pathway much stronger than with the other species. Moreover, a great repression of the respiration pathway has been found in response to Metschnikowia. Our hypothesis is that there is a direct interaction stress response (DISR) between S. cerevisiae and the other yeast species that, under excess sugar conditions, induces transcription of the hexose transporters, triggering glucose flow to fermentation and inhibiting respiration, leading to an increase in both, metabolic flow and population dynamics.

Metschnikowia bicuspidate associated with a milky disease in Eriocheir sinensis and its effectitve treatment by Massoia lactone.

The pathogenic yeast strain LIAO causing the milky disease in the Chinese mitten crab belonged to one member of Metschnikowia bicuspidate which could grow well at different temperatures from 28 to 4 °C. It was also found that the pathogenic yeast strain LIAO could grow in the extracts of the muscle, gill, heart tissues, intestinal tracts of the healthy Chinese mitten crabs by using the reducing sugars, amino acids and other nutrients in them. Massoia lactone released from liamocins produced by Aureobasidium melanogenum had high anti-fungal activity against the pathogenic yeast strain LIAO and M. bicuspidate WCY isolated from the diseased marine crabs. The minimal inhibitory concentrations (MIC) and the minimal fungicidal concentration (MFC) in the liquid culture against the pathogenic yeast strain LIAO were 0.15 mg/mL and 0.34 mg/mL, respectively. Massoia lactone as a bio-surfactant could damage the cell membrane, even break the whole cells of the pathogenic yeast strain LIAO and cause cellular necrosis of the pathogenic yeast LIAO. Therefore, Massoia lactone could be used to effectively kill the pathogenic yeast strains and as an effectitve treatment for milky disease in the Chinese mitten crab.

The Genome-Wide Mutation Shows the Importance of Cell Wall Integrity in Growth of the Psychrophilic Yeast Metschnikowia australis W7-5 at Different Temperatures.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

Delineating yeast species with genome average nucleotide identity: a calibration of ANI with haplontic, heterothallic Metschnikowia species.

We determined pairwise average nucleotide identity (ANI) values for the genomes of 71 strains assigned to 36 Metschnikowia species, 28 of which were represented by multiple isolates selected to represent the range of genetic diversity of the species, and most of which were defined on the basis of reproductive isolation. Similar to what has been proposed for prokaryote species delineation, an ANI value of 95% emerged as a good guideline for the delineation of yeast species, although some overlap exists, whereby members of a reproductive community could have slightly lower values (e.g., 94.3% for M. kamakouana), and representatives of distinct sister species could give slightly higher values (e.g., 95.2% for the sister species M. drakensbergensis and M. proteae). Unlike what is observed in prokaryotes, a sizeable gap between intraspecific and interspecific ANI values was not encountered. Given the ease with which yeast draft genomes can now be obtained, ANI values are poised to become the new standard upon which yeast species may be delineated on genetic distance. As borderline cases exist, however, the delineation of yeast species will continue to require careful evaluation of all available data. We also explore the often-neglected distinction between phylogenetic relatedness and sequence identity through the analysis of a tree constructed from ANI' (100 - ANI) values.

The strange mitochondrial genomes of Metschnikowia yeasts.

While sequencing and characterizing the mitochondrial genomes of 71 strains from the yeast genus Metschnikowia [1] (close cousin to the model species Candida albicans), we uncovered one of the most extreme examples of mitochondrial genome architectural diversity observed to date. These Metschnikowia mitochondrial DNAs (mtDNAs) capture nearly the entire known gene-size and intron-content range for cox1 and cob across all eukaryotic life and show remarkable differences in structure and noncoding content. This genomic variation can be seen both among species and between strains of the same species, raising the question: why are Metschnikowia mitogenomes so malleable?

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars.

BACKGROUND: α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo- and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described. RESULTS: In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-D-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far. CONCLUSIONS: Mg/Mr-αGlu were heterologously overproduced and their biochemical and structural characteristics analysed. The recombinant α-glucosidases displayed excellent properties in terms of mild reaction conditions, in addition to pH and thermal stability. Besides, the enzymes produced a rare mixture of hetero-gluco-oligosaccharides by transglucosylation, mainly isomelezitose and trehalulose. These compounds are natural constituents of honey which purification from this natural source is quite unviable, what make these enzymes very interesting for the biotechnological industry. Finally, it should be remarked that these sugars have potential applications as food additives due to their suitable sweetness, viscosity and humectant capacity.

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.

Yeast communities of primary and secondary peat swamp forests in southern Thailand.

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

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).