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).

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)).

Demonstration of redox potential of Metschnikowia koreensis for stereoinversion of secondary alcohols/1,2-diols.

The present work reports the Metschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. Reaction is stereoselective and proceeds with sequential oxidation of (R)-secondary alcohols to the corresponding ketones and the reduction of the ketones to (S)-secondary alcohols. Method is applicable to a repertoire of racemic aryl secondary alcohols and 1,2-diols establishing a wide range of substrate specificity of M. koreensis. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98% ee), minimizing the requirement of multistep reaction and expensive cofactor.

New species of the genus Metschnikowia isolated from flowers in Indonesia, Metschnikowia cibodasensis sp. nov.

A novel species, Metschnikowia cibodasensis, is proposed to accommodate eight strains (ID03- 0093(T), ID03-0094, ID03-0095, ID03-0096, ID03-0097, ID03-0098, ID03-0099, and ID03-0109) isolated from flowers of Saurauia pendula, Berberis nepalensis, and Brunfelsia americana in Cibodas Botanical Garden, West Java, Indonesia. The type strain of M. cibodasensis is ID03- 0093(T) (= NBRC 101693(T) =UICC Y-335(T) = BTCC-Y25(T)). The common features of M. cibodasensis are a spherical to ellipsoidopedunculate shaped ascus, which contains one or two needleshaped ascospores, and lyse at maturity. Asci generally develop directly from vegetative cells but sometimes from chlamydospores. The neighbor-joining tree based on the D1/D2 domain of nuclear large subunit (nLSU) ribosomal DNA sequences strongly supports that M. cibodasensis (eight strains) and its closest teleomorphic species, M. reukaufii, are different species by a 100% bootstrap value. The type strain of M. cibodasensis, ID03-0093(T), differed from M. reukaufii NBRC 1679(T) by six nt (five substitutions and one deletion) in their D1/D2 region of nLSU rDNA, and by 18 nt (five deletions, four insertions, and nine substitutions) in their internal transcribed spacer regions of rDNA, respectively. Four strains representative of M. cibodasensis (ID03-0093(T), ID03-0095, ID03-0096, and ID03-0099) showed a mol% G+C content of 44.05 ± 0.25%, whereas that of M. reukaufii NBRC 1679(T) was 41.3%. The low value of DNADNA homology (5-16%) in four strains of M. cibodasensis and M. reukaufii NBRC 1679(T) strongly supported that these strains represent a distinct species.

Phylogenetic analysis of the angiosperm-floricolous insect-yeast association: have yeast and angiosperm lineages co-diversified?

Metschnikowia (Saccharomycetales, Metschnikowiaceae/Metschnikowia clade) is an ascomycetous yeast genus whose species are associated mostly with angiosperms and their insect pollinators over all continents. The wide distribution of the genus, its association with angiosperm flowers, and the fact that it includes some of the best-studied yeasts in terms of biogeography and ecology make Metschnikowia an excellent group to investigate a possible co-radiation with angiosperm lineages. We performed phylogenetic analyses implementing Bayesian inference and likelihood methods, using a concatenated matrix (≈2.6 Kbp) of nuclear DNA (ACT1, 1st and 2nd codon positions of EF2, Mcm7, and RPB2) sequences. We included 77 species representing approximately 90% of the species in the family. Bayesian and parsimony methods were used to perform ancestral character reconstructions within Metschnikowia in three key morphological characters. Patterns of evolution of yeast habitats and divergence times were explored in the Metschnikowia clade lineages with the purpose of inferring the time of origin of angiosperm-associated habitats within Metschnikowiaceae. This paper presents the first phylogenetic hypothesis to include nearly all known species in the family. The polyphyletic nature of Clavispora was confirmed and Metschnikowia species (and their anamorphs) were shown to form two groups: one that includes mostly floricolous, insect-associated species distributed in mostly tropical areas (the large-spored Metschnikowia clade and relatives) and another that comprises more heterogeneous species in terms of habitat and geographical distribution. Reconstruction of character evolution suggests that sexual characters (ascospore length, number of ascospores, and ascus formation) evolved multiple times within Metschnikowia. Complex and dynamic habitat transitions seem to have punctuated the course of evolution of the Metschnikowiaceae with repeated and independent origins of angiosperm-associated habitats. The origin of the family is placed in the Late Cretaceous (71.7 Ma) with most extant species arising from the Early Eocene. Therefore, the Metschnikowiaceae likely radiated long after the Mid-Cretaceous radiations of angiosperms and their diversification seems to be driven by repeated radiation on a pre-existing diverse resource.

Metschnikowia henanensis sp. nov., a new anamorphic yeast species isolated from rotten wood in China.

Four yeast strains were isolated from rotting wood samples collected from two sites in the Baotianman Nature Reserve and the Laojieling Nature Reserve in China. DNA sequence comparison and other taxonomic characteristics identified the strains as a single novel species of the genus Metschnikowia. The name Metschnikowia henanensis sp. nov. is proposed to accommodate these highly divergent organisms with the type strain BY-97(T) (= CICC 1982(T) = CBS 12677(T)). The novel species produced chlamydospores, but it did not exhibit ascospore formation in sporulation media for 4 weeks. Molecular phylogeny from the D1/D2 domains of the large subunit (LSU) rRNA gene sequences placed this new species in a basal position to the Metschnikowia viticola/Candida kofuensis/Metschnikowia noctiluminum subclade, and an undescribed Candida species namely strains IMB-EMP4 and IMB-EMP5 was a close sister to M. henanensis.

Global changes in gene expression of grapefruit peel tissue in response to the yeast biocontrol agent Metschnikowia fructicola.

To gain a better understanding of the molecular changes taking place in citrus fruit tissue following the application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. Using a cut-off of P < 0.05 and a 1.5-fold change difference as biologically significant, the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. Microarray results of selected genes were validated by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The data indicated that yeast application induced the expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. Moreover, suppression was correlated with significantly higher levels of hydrogen peroxide, superoxide anion and hydroxyl radical production in yeast-treated surface wounds. Interestingly, large amounts of hydrogen peroxide were detected inside yeast cells recovered from wounded fruit tissue, indicating the ability of the yeast to activate reactive oxygen species when it is in contact with plant tissue. This study provides the first global picture of gene expression changes in grapefruit in response to the yeast antagonist M. fructicola.