Taxonomy of the Yeast Genus Metschnikowia: a Correction and a New Variety

Metschnikowia isolates from ponds on Chatham Island, New Zealand, previously erroneously reported as M. zobellii, are described herein as M. bicuspidata var. chathamia var. n.     

Electron Micrograph Study of the Asci and Ascospores of Metschnikowia Kamienski

Internal and surface structures of asci and ascospores were studied by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) to establish the character and number of ascospores within the ascus of Metschnikowia krissii. Enzyme digestion with snail gut enzymes and SEM examination suggested the presence of a single ascospore enclosed in a thick sheath of epiplasmic materials. Two closely associated ascospores without an epiplasmic sheath were clearly distinguishable from asci of M. bicuspidata var. chathamia when similarly treated. Ultramicrotomy and TEM established conclusively that M. krissii produced a single ascospore per ascus. Neither SEM nor TEM revealed any morphological detail of the ascospores of taxonomic significance.     

Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb. nov

The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis. Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species. Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M. continentalis, Metschnikowia lochheadii, Metschnikowia sp. UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses. Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants. In the other species, some recombinants acquired the ability to form asci directly from single cells. These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids. Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent. In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement. In some cases, what appeared to be a recombinant mitochondrial genome was observed. Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor. Further evidence that C. ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively. C. ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene. The intron was absent in M. borealis, M. hawaiiensis, and other species in the genus Metschnikowia.     

Infections with the ascomycete fungus Metschnikowia typographi sp.nov. in the bark beetles Ips typographus and Ips amitinus (Coleoptera, Scolytidae)

The ascomycete fungus Metschnikowia typographi sp.nov. is described. It infects the spruce bark beetles Ips typographus L. and Ips amitinus Eichl. Masses of vegetative cells and navicular asci (I. typographus 13-17 x 2 microns; I. amitinus 17-22 x 2 microns) were found in cells of the midgut epithelium and in the body cavity of infected beetles. Each ascus contains two needle-shaped ascospores flattened in the central part, 0.5-1.5 x 0.3 x 13-15 microns and pointed at both ends. The parasitic species of Metschnikowia, M. bicuspidata, M. artemiae, M. unicuspidata, M. wickerhami and M. typographi are discussed as a special group of the genus characterized by morphological characters.     

Metschnikowia arizonensis and Metschnikowia dekortorum,two new large-spored yeast species associated with floricolous beetles

Two new haplontic heterothallic species of Metschnikowia were discovered in flowers and associated beetles. Metschnikowia arizonensis was recovered from flowers of cholla cactus (Opuntia echinocarpa) and a specimen of Carpophilus sp. (Coleoptera: Nitidulidae) found in these flowers, in Arizona. Metschnikowia dekortorum was isolated in specimens of the nitidulid beetle Conotelus sp. captured in flowers of two species of Ipomoea in northwestern Guanacaste Province, Costa Rica. The sexual cycle of these yeasts is typical of the large-spored Metschnikowia species, but the asci and spores are intermediate in size between these and other members of the genus. The physiology is consistent with that of most Metschnikowia species except that both species fail to utilize lysine as sole nitrogen source. Also, M. arizonensis utilizes fewer carbon compounds than most species and exhibits considerable variability among strains at this level. Partial ribosomal DNA large-subunit (D1/D2) sequences suggest that M. arizonensis and M. dekortorum are moderately related sister species whose positions are intermediate between the large-spored species Metschnikowia and Metschnikowia hibisci. The type cultures are: M. arizonensis, strains UWO(PS)99-103.3.1=CBS 9064=NRRL Y-27427 (h(+), holotype) and UWO(PS)99-103.4=CBS 9065=NRRL Y-27428 (h(-), isotype); and M. dekortorum, strains UWO(PS)01-142b3=CBS 9063=NRRL Y-27429 (h(+), holotype) and UWO(PS)01-138a3=CBS 9062=NRRL Y-27430 (h(-), isotype).     

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.     

Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts

The asci of Metschnikowia species normally contain two ascospores (never more), raising the question of whether these spores are true meiotic products. We investigated this problem by crossing genetically-marked strains of the haploid, heterothallic taxa Metschnikowia hawaiiensis, Metschnikowia continentalis var. continentalis, and M. continentalis var. borealis. Asci were dissected and the segregation patterns for various phenotypes analyzed. In all cases (n = 47) both mating types (h+ and h-) were recovered in pairs of sister spores, casting further uncertainty as to whether normal meiosis takes place. However, the segregation patterns for cycloheximide resistance and several auxotrophic markers were random, suggesting that normal meiosis indeed occurs. To explain the lack of second-division segregation of mating types, we concluded that crossing-over does not occur between the mating-type locus and the centromere, and that meiosis I is tied to spore formation, which explains why the number of spores is limited to two. The latter assumption was also supported by fluorescence microscopy. The second meiotic division takes place inside the spores and is followed by the resorption of two nuclei, one in each spore.     

Metschnikowia lochheadii and Metschnikowia drosophilae, two new yeast species isolated from insects associated with flowers

Two new haplontic heterothallic species of Metschnikowia were isolated from floricolous insects and flowers. Metschnikowia lochheadii was recovered from insects found in various flowers on the Hawaiian Islands of Kauai and Maui, and from Conotelus sp. (Coleoptera: Nitidulidae) in northwestern Guanacaste Province, Costa Rica. The morphology, physiology, and sexual cycle are typical of the large-spored Metschnikowia species, and the partial ribosomal DNA large subunit (D1D2) sequences suggest that the new species is most closely related to Candida ipomoeae. Metschnikowia lochheadii is nearly indistinguishable from its ascogenous relatives and conjugates freely with Metschnikowia continentalis, forming sterile asci. It also exhibits asymmetric mating with Metschnikowia hawaiiensis. Metschnikowia drosophilae was found in morning glory (Ipomoea sp.) flowers and associated Drosophila bromeliae on Grand Cayman Island. Its nutritional profile is atypical of the genus, being the only species that does not utilize sucrose or maltose as carbon sources, and one of the few that does not utilize melezitose. D1D2 sequences show that Metschnikowia drosophilae is a sister species to Candida torresii, to which it bears considerable similarity in nutritional profile. The type cultures are: Metschnikowia lochheadii, strains UWO(PS)00-133.2 = CBS 8807 (h+, holotype) UWO(PS)99-661.1 = CBS 8808 (h-, isotype); and Metschnikowia drosophilae, strains UWO(PS)83-1135.3 = CBS 8809 (h+, holotype) and UWO(PS)83-1143.1 = CBS 8810 (h-, isotype).     

Metschnikowia bicuspidata dominates in Taiwanese cold-weather yeast infections of Macrobrachium rosenbergii

At water temperatures below 17 degrees C, yeast infections often occurred in 6 to 11 mo old giant freshwater prawn Macrobrachium rosenbergii (8 to 12 cm) in Taiwan from May 2001 to December 2003, with a cumulative mortality of 20 to 95%. Gross signs of disease included milky hemolymph, a yellow exoskeleton, opaque muscles, and a swollen hepatopancreas (HP). Histopathology included marked edema and extensive necrotic lesions associated with large numbers of yeast aggregates and inflammation within the muscles, HP, and other internal organs such as the heart, ovary, and intestine. Yeast cell numbers isolated from various tissues ranged from 4.5 x 10(8) to 9.0 x 10(9) colony forming units (CFU) per 100 mg. From diseased prawns from 12 affected farms, the presence of Metschnikowia bicuspidata (98.4% prevalence), Saccharomyces cerevisiae (0.8% prevalence), and Candida albicans (0.8% prevalence) was confirmed by biochemical tests and sequencing of PCR products from the D1/D2 domain of 26S rDNA. Experimental infection with these isolates caused gross signs and histopathological changes similar to those observed in naturally infected prawns, and lethal doses (LD50) were 3.8 x 10(3), 2.0 x 10(3), and 4.3 x 10(3) CFU prawn-1, respectively. Although the results of this study revealed that M. bicuspidata may be the major cause of yeast infections in the giant freshwater prawns in Taiwan, this is the first time that S. cerevisiae and C. albicans are also reported as pathogens.     

Catching speciation in the act: Metschnikowia bowlesiae sp. nov., a yeast species found in nitidulid beetles of Hawaii and Belize

We describe the species Metschnikowia bowlesiae sp. nov. based on the recovery of six isolates from Hawaii and Belize. The species belongs to the Metschnikowia arizonensis subclade of the large-spored Metschnikowia clade. The isolates are haploid and heterothallic. Both Hawaiian strains had the mating type h ⁺ and the Belizean strains were h ^?. Paraphyletic species structures observed in some ribosomal DNA sequence analyses suggest that M. bowlesiae sp. nov. might represent an intermediate stage in a succession of peripatric speciation events from Metschnikowia dekortorum to Metschnikowia similis and might even hybridize with these species. The type of M. bowlesiae sp. nov. is strain UWOPS 04-243x5 (CBS 12940^T, NRRL Y-63671) and the allotype is strain UWOPS 12-619.1 (CBS 12939^A, NRRL Y-63670).     

Metschnikowia santaceciliae,Candida hawaiiana,and Candida kipukae,three new yeast species associated with insects of tropical morning glory

A new haplontic heterothallic species of Metschnikowia and two related asexual yeast species were discovered in morning glory flowers and associated insects. Metschnikowia santaceciliae came from Conotelus (Coleoptera: Nitidulidae) and other insect species associated with flowers of Ipomoea indica (purple morph) in Costa Rica. Candida hawaiiana and Candida kipukae were found in I. indica (syn. I. acuminata) and its insects in Hawai'i, and the former was also isolated in a specimen of Conotelus collected on Merremia tuberosa (Convolvulaceae) in Costa Rica. The three species have nearly identical physiological profiles, typical of the genus Metschnikowia. The sequences of the D1/D2 domains of their large subunit ribosomal DNA confirm that the species belong to the Metschnikowia clade, even though they share a very low degree of inter-relatedness. M. santaceciliae is a sister species to Metschnikowia continentalis. C. kipukae is a basal member of the large-spored Metschnikowia subclade, and C. hawaiiana has a weak affinity to Metschnikowia agaves. Two of the three species appear to be endemic. The type cultures are: Metschnikowia santaceciliae, strains UWO(PS)01-517a1=CBS 9148=NRRL Y-27475 (h(+, holotype) and UWO(PS)01-520a1=CBS 9149=NRRL Y-27476 (h-, isotype); Candida hawaiiana, strain UWO(PS)91-698.3=CBS 9146=NRRL Y-27473; Candida kipukae, strain UWO(PS)00-669.2=CBS 9147=NRRL Y-27474.     

Heterogeneity of the yeasts Zygowilliopsis californica: Z. californica var. dimennae comb. nov., stat. nov. and Z. californica var. fukushimae comb. nov., stat. nov

A significant heterogeneity of the species Zygowilliopsis californica was revealed using RFLP-analysis of the PCR-amplified rDNA fragment spanning the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. Phylogenetic analysis of the nucleotide sequences of ITS1 and ITS2 rDNA differentiated three varieties: Z. californica var. californica, Z. californica var. dimennae, and Z. californica var. fukushimae. The most variable was the ITS 2 region, where 7-26 nucleotide substitutions were revealed. The varieties formed semisterile hybrids with meiotic segregation of control markers. The limits of the phylogenetic species concept are discussed.     

Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots

A new ascosporic yeast, Metschnikowia fructicola (type strain NRRL Y-27328, CBS 8853), is described and was isolated from grapes grown in central Israel. Preliminary tests indicate the new species has biocontrol activity against Botrytis rot of stored grapes. Phylogenetic analysis of domain D1/D2 26S rDNA sequences showed M. fructicola to be a sister species of M. pulcherrima.     

Synonymy of Metschnikowia pulcherrima and Torulopsis burgeffiana

Torulopsis burgeffiana is placed in the genus Metschnikowia because it has been shown to have a perfect stage corresponding with this genus. Due to the characteristic shape of the ascus and ascospores, T. burgeffiana is to be considered a synonym of M. pulcherrima. The physiological differences which exist between strains are not sufficiently important to maintain a separate species.     

Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles

Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the digestive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect hosts were associated with sugary substances of plants, a typical habitat for yeasts in this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient to consider them as new species. Two of the novel yeast species are described as Metschnikowia noctiluminum (NRRL Y-27753^T) and M. corniflorae spp. nov. (NRRL Y-27750^T) based in part on production of needle-shaped ascospores, which are found in most Metschnikowia species. Sexual reproduction was not observed in the third new yeast, Candida chrysomelidarum sp. nov. (NRRL Y-27749^T). A fourth isolate, NRRL Y-27752, was not significantly distinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group.     

Isolation of heterothallic yeast strains ofMetschnikowia Kamienski and their mating reactions withChlamydozyma Wickerham spp

Isolation ofMetschnikowia bicuspidata var.australis in the haplophase from Antarctic waters, andM. bicuspidata var.zobellii in haplo-and diplophases at Chatham Is. N.Z., led to the study of mating reactions within theChlamydozyma - Metschnikowia complex. The results indicate the phylogenetic relationships between species. The plant-associatedCh. pulcherrima. Ch. reukaufii, andM. zygota are distinct species; when cross-mated they will conjugate but not produce asci. These three species are closely related toM. bicuspidata; when they are mated withM. bicuspidata and its varieties (var.australis and var.zobellii) asci are produced without spores. The taxonomic status ofM. bicuspidata and its varieties is based on less frequent formation of ascospores between than within taxa.The new varietyM. bicuspidata var.australis and the new combinationsM. zygota andM. bicuspidata var.zobellii are proposed.Contribution No. 925 from the Institute of Marine Sciences, University of Miami, Florida.     

Differentiation between aquatic and terrestrial <Emphasis Type="Italic">Metschnikowia</Emphasis> species of based on their sensitivity to <Emphasis Type="Italic">Pichia membranifaciens</Emphasis> mycocins

Aquatic Metschnikowia species (M. australis, M. bicuspidata, M. krissii, and M. zobellii) are sensitive to 10 out of 12 mycocins secreted by Pichia membranifaciens strains. Terrestrial species M. pulcherrima and M. reukaufii are resistant to all these mycocins, while M. gruessii and M. lunata are sensitive to one of them. The yeast described as Torula rubifaciens is also sensitive to this mycocin.     

Metschnikowia hawaiiensis sp. nov., a heterothallic haploid yeast from Hawaiian morning glory and associated drosophilids

A new haploid, heterothallic yeast species was isolated repeatedly from morning glory (Ipomoea acuminata) flowers and from two associated drosophilid species, Scaptomyza calliginosa and Drosophila floricola, in a Hawaiian kipuka. Haploid strains of this organism multiply asexually by budding and, under nutrient deprivation, by the formation of long germ tubes that develop into branching true mycelia. Mating compatibility is controlled by two alleles of a single locus. Plasmogamy between compatible strains is followed by the development of very large elongate asci bearing vestiges of the zygotes and the formation in each ascus of two unusually large aciculate ascospores similar to those formed by members of the genus Metschnikowia. Membership in the genus Metschnikowia is supported by the physiological profile of the yeast, which is typical of the genus but not identical to the profile of any previously described species. The name Metschnikowia hawaiiensis is proposed to emphasize the geographic origin of the new species, not its habitat, which has not been determined precisely. The holotype strain of M. hawaiiensis is strain UWO(PS) 87-2167.2 (= ATCC 76059 = CBS 7432), and the isotype strain is strain UWO(PS) 87-2203.2 (= ATCC 76058 = CBS 7433).     

Biogeography and population structure of the Neotropical endemic yeast species <Emphasis Type="Italic">Metschnikowia lochheadii</Emphasis>

The genetic structure of populations of the out-crossing haplontic yeast species Metschnikowia lochheadii was investigated. The species is associated with floricolous beetles in Central America and Hawaii. The objective was to determine whether sexual reproduction is prevalent and to what extent the geographic distribution of genotypes can be viewed as historical. The genetic markers examined include the mating type (h (+) or h (-)) and nine polymorphic DNA loci. The data were used to assess population structuring based on F (ST) and linkage disequilibrium and the distribution of alleles using parsimony haplotype networks. In Central America, M. lochheadii is subdivided into sexually active demes between which gene flow is limited. Isolates from five Hawaiian islands had identical haplotypes, confirming that the species has undergone a founder effect concomitant with the recent import of a nitidulid beetle into the archipelago.