The yeast genus Tortispora gen. nov., description of Tortispora ganteri sp. nov., Tortispora mauiana f.a., sp. nov., Tortispora agaves f.a., sp. nov., Tortispora sangerardonensis f.a., sp. nov., Tortispora cuajiniquilana f.a., sp. nov., Tortispora starmeri f.a., sp. nov. and Tortispora phaffii f.a., sp. nov., reassignment of Candida caseinolytica to Tortispora caseinolytica f.a., comb. nov., emendation of Botryozyma, and assignment of Botryozyma, Tortispora gen. nov. and Trigonopsis to the family Trigonopsidaceae fam. nov.

We describe the yeast genus Tortispora gen. nov., an early-diverging lineage in the Saccharomycetales that displays the formation of helical ascospores. The genus is based on 16 strains resembling Candida caseinolytica that were isolated from necrotic plant tissue in warm regions of the New World. Based on sequences of the D1/D2 domains of the nuclear large subunit rRNA gene, as well as other data, the strains are assigned to eight distinct species. The species are nutritionally specialized and share the unusual ability to hydrolyse casein and to grow on 1-butanol as sole carbon source. One species of the proposed new genus produces a simple ascus with a helical ascospore, whereas other species of the clade have failed to form ascospores. All species in the clade, including C. caseinolytica, are assigned to Tortispora gen. nov. The new binomials are Tortispora ganteri sp. nov., type species of the genus (SUB 86-469.5(T) = CBS 12581(T) = NRRL Y-17035(T)), Tortispora caseinolytica f.a., comb. nov. (UCD-FST 83-438.3(T) = CBS 7781(T) = NRRL Y-17796(T)), Tortispora mauiana f.a., sp. nov. (UWOPS 87-2430.3(T) = CBS 12803(T) = NRRL Y-48832(T)), Tortispora agaves f.a., sp. nov. (UWOPS 94-257.6(T) = CBS 12794(T) = NRRL Y-63662(T)), Tortispora sangerardonensis f.a., sp. nov. (UWOPS 00-157.1(T) = CBS 12795(T) = NRRL Y-63663(T)), Tortispora cuajiniquilana f.a., sp. nov. (UWOPS 99-344.4(T) = CBS 12796(T) = NRRL Y-63664(T)), Tortispora starmeri f.a., sp. nov. (G 91-702.5(T) = CBS 12793(T) = NRRL Y-63665(T)) and Tortispora phaffii f.a., sp. nov. (UWOPS 91-445.1(T) = CBS 12804(T) = NRRL Y-48833(T)). In addition, species formerly assigned to the genus Ascobotryozyma are reassigned to the genus Botryozyma. The genera Trigonopsis, Botryozyma and Tortispora are assigned to the family Trigonopsidaceae fam. nov.

Yeasts associated with nectarines and their potential for biological control of brown rot.

Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines ('Croce del Sud') from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast-like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov.

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran.

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C6H8O3 and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.

Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes.

The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis.

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

Biocontrol Potential of Metchnikowia pulcherrima Strains Against Blue Mold of Apple.

ABSTRACT Eight strains of Metschnikowia pulcherrima isolated over a 4-year period from an unmanaged orchard and selected for their biocontrol activity against blue mold (caused by Penicillium expansum) of apples were characterized phenotypically, genetically, and for their biocontrol potential against blue mold on apples. All strains grew well and only differed slightly in their growth in nutrient yeast dextrose broth medium at 1 degrees C after 216 h, but large differences occurred at 0 degrees C, with strain T5-A2 outgrowing other strains by more than 25% transmittance after 360 h. This strain was also one of the most resistant to diphenylamine (DPA), a postharvest antioxidant treatment. All strains required biotin for growth in minimum salt (MS) medium, although strain ST2-A10 grew slightly in MS medium containing riboflavin or folic acid, as did ST3-E1 in MS medium without vitamins. None of the strains produced killer toxins against an indicator strain of Saccharomyces cerevisiae. Analysis of Biolog data from YT plates for all eight strains using the MLCLUST program resulted in separation of the strains into one major cluster containing four strains and four scattered strains from which strain ST1-D10 was the most distant from all other strains. This was particularly apparent in 3-D and principle component analysis. Genetic differentiation of the eight strains using maximum parsimony analysis of nucleotide sequences from domain D1/D2 of nuclear large subunit (26S) ribosomal DNA resulted in detection of two clades. Strain ST1-D10 grouped with the type strain of M. pulcherrima but the remaining seven strains grouped separately, which might possibly represent a new species. All strains significantly reduced blue mold on mature Golden Delicious apples during 1 month of storage at 1 degrees C followed by 7 days at room temperature, but strains T5-A2 and T4-A2 were distinctly more effective under these conditions. Strain T5-A2 also was the most effective in tests on harvest mature apples treated with the lowest concentration of the antagonist and stored for 3 months at 0.5 degrees C. Populations of all eight strains increased in apple wounds by approximately 2 log units after 1 month at 1 degrees C followed by 5 days at 24 degrees C. Our results indicate that M. pulcherrima is an excellent candidate for biological control of postharvest diseases of pome fruit. The variation in phenotypic, genetic, and biocontrol characteristics among strains of M. pulcherrima isolated from the same orchard should make it possible to select antagonists with characteristics that are most desirable for postharvest application.

Three new species of Candida from apple cider: C. anglica, C. cidri and C. pomicola.

Three new anamorphic ascomycetous yeasts are described: Candida anglica (type strain NRRL Y-27079, CBS 4262), Candida cidri (type strain NRRL Y-27078, CBS 4241), and Candida pomicola (type strain NRRL Y-27083, CBS 4242). These three species were isolated from cider produced in the United Kingdom, and their identification was determined from unique nucleotide sequences in the species-specific D1/D2 domain of large subunit (26S) ribosomal DNA. Phylogenetic analysis of D1/D2 sequences placed C. anglica near Candida fragi, C. cidri near Pichia capsulata, and C. pomicola in the Pichia holstii clade.

Four new Candida species from geographically diverse locations.

Four new species of Candida are described based on their unique nucleotide sequences in the D1/D2 domain of large subunit (26S) ribosomal DNA. Candida peoriaensis (type strain NRRL YB-1497, CBS 8800) and C. ponderosae (type strain NRRL YB-2307, CBS 8801) are members of the Pichia anomala clade and were isolated in the U.S. from, respectively, the stump of an elm tree (Ulmus sp.) and from insect frass of a Ponderosa pine (Pinusponderosa). Candida ghanaensis (type strain NRRL YB-1486, CBS 8798) is a phylogenetically divergent species from soil in Ghana and appears related to the Dipodascus/Geotrichum clade. Candida litsaeae (type strain NRRL YB-3246, CBS 8799) was isolated from the frass of an insect-infested Litsaea polyantha tree from India, and is a divergent species that is most closely related to Candida ontarioensis.

Two new anamorphic yeasts: Candida germanica and Candida neerlandica.

Descriptions are given for the two new anamorphic ascomycetous yeasts Candida germanica (type strain NRRL Y-27064, CBS 4105) and Candida neerlandica (type strain NRRL Y-27057, CBS 434). The species were isolated, respectively, from the atmosphere over Germany and from pressed yeast cake in The Netherlands. Phylogenetic analysis of 26S domain D1/D2 ribosomal DNA sequences places C. germanica near Pichia philogaea, whereas C. neerlandica is a member of the Lodderomyces elongisporus/Candida albicans clade.

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.

Six new anamorphic ascomycetous yeasts near Candida tanzawaensis.

Six new species of the yeast genus Candida are described from their unique nucleotide sequences in the D1/D2 domain of 26S rDNA. Five of these species form a clade with Candida tanzawaensis, and the sixth is basal to this group. The new species and their sources of isolation are the following: Candida ambrosiae (type strain NRRL YB-1316, CBS 8844), from insect frass, rotted wood and mushroom fruiting bodies; Candida canberraensis (type strain NRRL YB-2417, CBS 8846), from soil; Candida caryicola (type strain NRRL YB-1499, CBS 8847), from a pignut hickory tree; Candida prunicola (type strain NRRL YB-869, CBS 8848), from exuded gum of a black cherry tree; Candida pyralidae (type strain NRRL Y-27085, CBS 5035), from insect frass; Candida xylopsoci (type strain NRRL Y-27066, CBS 6037), from insect frass.

Zygosaccharomyces kombuchaensis, a new ascosporogenous yeast from 'Kombucha tea'.

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.

Pichia hawaiiensis sp. nov., occurring in decaying bark of Charpentiera trees in the Hawaiian archipelago.

A description is given for Pichia hawaiiensis sp. nov., a nitrate-utilizing member of the genus Pichia E. C. Hansen emend. Kurtzman. Seven strains of the new species were isolated during the years 1972, 1973 and 1978 from rotting bark of the Hawaiian tree genera Charpentiera, Pisonia and Cheirodendron. P. hawaiiensis is heterothallic but appears to occur in nature mainly in the diploid state. Asci are deliquescent and produce up to four hat-shaped spores per ascus. Phylogenetic analysis of the 600 nucleotide D1/D2 domain of the 26S rDNA showed that P. hawaiiensis is most closely related to Pichia populi and Williopsis californica (syn. Hansenula californica). The type strain of P. hawaiiensis, isolated on the island of Hawaii from the rotting bark of Charpentiera sp. containing insect larvae, is strain UCD-FST 72-181T (= ATCC MYA-137T = CBS 8760T = NRRL Y-27270T).

Candida tartarivorans sp. nov., an anamorphic ascomycetous yeast with the capacity to degrade L(+)- and meso-tartaric acid.

An undescribed anamorphic yeast species of ascomycetous affinity, for which the name Candida tartarivorans is proposed, was isolated from dried wine lees in Portugal using a selective medium with L(+)-tartaric acid as the sole source of carbon and energy. The single isolate (IGC 4854T) showed the following characteristics: sympodial holoblastic conidiogenesis, absence of asci with ascospores, a negative colour reaction with Diazonium Blue B, production of elaborate pseudomycelium and ability to grow with inositol as sole source of carbon. Analysis of the physiological data pointed to a close relationship with other inositol-assimilating taxa, namely the genera Arxula, Stephanoascus, Sympodiomyces, Zygoascus and selected Candida species. Comparative analysis of the D1/D2 variable domain of the 26S rRNA gene of all available sequences for ascomycetous yeasts showed that strain IGC 4854T did not match with any other species in the database. The closest relative was Candida auringiensis Santa Maria, but the two species differed in 24 nucleotide positions. A description of the new species is given.

Four new yeasts in the Pichia anomala clade.

Four new yeasts are described that were recognized as novel from nucleotide substitutions in domain D1/D2 of 26S rDNA, a region that is sufficiently divergent to allow resolution of most ascomycetous yeast species. The new species and their type strains are as follows: Pichia maclurae NRRL Y-5377T (= CBS 8671T); Pichia misumaiensis NRRL Y-17389T (= CBS 8062T); Candida mycetangii NRRL Y-6843T (= CBS 8675T); and Candida ulmi NRRL YB-2694T (= CBS 8670T). The two Pichia species form spherical ascospores and are heterothallic. Phylogenetic analysis of domain D1/D2 sequences placed the four new species in the Pichia anomala clade.

Three new ascomycetous yeasts from insect-associated arboreal habitats.

A new species of Pichia and two new species of Candida are described and were determined to be genetically isolated from all other currently recognized ascomycetous yeasts from their sequence divergence in the species-variable D1/D2 domain of large subunit (26S) ribosomal DNA. The three species were primarily isolated from the frass of wood-boring insects living in pine and spruce trees. The new species and their type strains are the following: Pichia ramenticola NRRL YB-1985 (CBS 8699), mating type alpha (NRRL YB-3835, CBS 8700, mating type a), Candida piceae NRRL YB-2107 (CBS 8701), and Candida wyomingensis NRRL YB-2152 (CBS 8703). Pichia ramenticola and C. piceae assimilate methanol as a carbon source; P. ramenticola is the first known heterothallic ascomycetous yeast to utilize this compound.

Saccharomyces bulderi sp. nov., a yeast that ferments gluconolactone.

An unknown yeast species was isolated from maize silage and was determined to be novel on the basis of morphological and physiological characteristics, nucleotide sequence of domain D1/D2 of LSU rDNA and from its electrophoretic karyotype. The name for the proposed new species is Saccharomyces bulderi Middelhoven, Kurtzman et Vaughan-Martini (type strain CBS 8638, NRRL Y-27203, DBVPG 7127). S. bulderi is closely related to S. barnettii and S. exiguus from which it can be distinguished by having a double vitamin requirement of biotin and thiamine and by no or slow aerobic growth on raffinose, a sugar that on the contrary is fermented rapidly. Gluconolactone is rapidly fermented with ethanol, glycerol and carbon dioxide being the main products.

Pichia lachancei sp. nov., associated with several Hawaiian plant species.

A description is given of Pichia lachancei sp. nov., a new species of yeast that occurs in association with several Hawaiian plant species of the genera Tetraplasandra, Cheirodendron and Clermontia. The new species is heterothallic and occurs in nature in the haploid as well as the diploid state. Upon conjugation of complementary mating types, zygotes are formed that reproduce by budding as diploid cells. When placed on sporulation medium, four hat-shaped spores are produced which are rapidly released from the ascus. Phylogenetic analysis showed that P. lachancei is most closely related to Pichia rhodanensis and Pichia jadinii. The diploid type strain of P. lachancei, isolated from rotting bark of Tetraplasandra hawaiiensis on the island of Hawaii, is strain UCD-FST 79-9T (= ATCC 201914T = CBS 8557T = NRRL Y-27008T).

Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences.

Approximately 500 species of ascomycetous yeasts, including members of Candida and other anamorphic genera, were analyzed for extent of divergence in the variable D1/D2 domain of large subunit (26S) ribosomal DNA. Divergence in this domain is generally sufficient to resolve individual species, resulting in the prediction that 55 currently recognized taxa are synonyms of earlier described species. Phylogenetic relationships among the ascomycetous yeasts were analyzed from D1/D2 sequence divergence. For comparison, the phylogeny of selected members of the Saccharomyces clade was determined from 18S rDNA sequences. Species relationships were highly concordant between the D1/D2 and 18S trees when branches were statistically well supported.