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Torulaspora quercuum sp. nov. and Candida pseudohumilis sp. nov., novel yeasts from human and forest habitats

Qi-Ming Wang, Jianping Xu, Huamin Wang, Juan Li, Feng-Yan Bai
DOI: http://dx.doi.org/10.1111/j.1567-1364.2009.00567.x 1322-1326 First published online: 1 December 2009

Abstract

Strains XZ-46A, XZ-105, XZ-129 and XZ-281T isolated from the oral cavities of healthy Tibetan volunteers were revealed to represent two novel ascomycetous yeast species by molecular taxonomic characterizations. Strain XZ-281T was most closely related to Candida humilis, but differed from the type strain of the species by eight (1.2%) substitutions in the 26S rRNA gene D1/D2 domain and by >100 (>20%) mismatches in the internal transcribed spacer (ITS) region. Strains XZ-46A, XZ-105 and XZ-129 had identical or similar D1/D2 and ITS sequences with each other and with strain 17YFT isolated from a leaf of an oak tree (Quercus sp.). The closest relative of this group was Torulaspora microellipsoides. They differed from the type strain of the species by five (0.9%) substitutions in the D1/D2 domain and >70 (>15%) mismatches in the ITS region. A sexual state was observed in strain 17YFT, but not in the other four oral strains. An anamorphic name Candida pseudohumilis sp. nov. is proposed for strain XZ-281T (=AS 2.3956T=CBS 11404T) and a teleomorphic name Torulaspora quercuum sp. nov. is proposed for strain 17YFT (=AS 2.3768T=CBS 11403T) and the other three oral strains.

Keywords
  • Torulaspora quercuum sp. nov.
  • Candida pseudohumilis sp. nov.
  • oral yeasts

Introduction

Yeasts are common members of the microbial community of the skin, oral cavity and intestinal and urogenital tracts of human beings. Some of the yeasts, predominantly Candida species, are opportunistic pathogens to humans (Odds, 1988; Kleinegger, 1996; Kam & Xu, 2002). Many of the yeasts can cause infections ranging from mild, superficial irritations such as oral and vaginal candidiasis to fatal, systemic diseases in immunocompromised or critically ill patients (Odds, 1988; Eggimann, 2003). Therefore, investigations on the transient or persistent members of yeast flora in humans and on the genetic relationships of the commensal species or strains with those associated with various infections will considerably aid our understanding of the ecology, evolution and epidemiology of human fungal infections.

During our survey of the oral yeast flora in healthy persons from different geographical and ethnical origins in China, two hitherto undescribed species were identified from the yeast strains isolated from the oral cavities of 356 healthy local volunteers in Tibet. Interestingly, one group of strains was conspecific to a teleomorphic yeast strain isolated from a plant leaf collected in another region located in northeast China, about 3300 km away from Tibet. The two new species are described as Torulaspora quercuum sp. nov. and Candida pseudohumilis sp. nov., respectively.

Materials and methods

Yeast strains

The yeast strains studied are listed in Table 1. The four oral strains were isolated using the method described in Xu & Mitchell (2003). Strain 17YFT was isolated from a wilting leaf on a tree of Quercus sp. collected in Changbai Mountain, Jilin Province, northeast China, during the survey of Saccharomyces species distribution in natural environments. The enrichment method as described in Wang & Bai (2008) was used for yeast isolation.

View this table:
Table 1

Yeast strains studied

SpeciesStrainOther designationSource
Torulaspora quercuumXZ-46AAS 2.4013Oral cavity of a healthy Tibetan female in Shigatse, Tibet
XZ-105AS 2.3957Oral cavity of a healthy Tibetan female in Shannan, Tibet
XZ-129AS 2.4014Oral cavity of a healthy Tibetan female in Shannan, Tibet
17YFTAS 2.3768T CBS 11403TLeaf of Quercus sp., Changbai Mountain, Jilin, northeast China
Candida pseudohumilisXZ-281TAS 2.3956T CBS 11404TOral cavity of a healthy Tibetan male in Nyingchi, Tibet

Phenotypic and molecular characterization

Morphological, physiological and biochemical characteristics were examined according to standard methods used for yeast taxonomy (Yarrow, 1998). Assimilation tests of carbon and nitrogen compounds were investigated using liquid and solid media, respectively. Nuclear DNA was extracted using the method of Makimura (1994). The internal transcribed spacers (ITS, including the 5.8S rRNA gene) and the 26 rRNA gene D1/D2 domain sequences were determined by the method as described previously (Bai, 2002). The GenBank accession numbers for the ITS and D1/D2 sequences of strains 17YFT, XZ-46A and XZ-281T are FJ888524, FJ888525 and FJ888526, respectively.

Molecular phylogenetic analysis

Sequences were aligned using the clustal x program (Thompson, 1997). Phylogenetic trees were constructed using the neighbor-joining method (Saitou & Nei, 1987) based on evolutionary distances calculated from the Kimura-2-Parameter model (Kimura, 1980). Sites where gaps existed in any of the aligned sequences were excluded. Bootstrap analyses (Felsenstein, 1985) were performed from 1000 random resamplings.

Results and discussion

Sequence analyses

Strains XZ-46A, XZ-105, XZ-129 and 17YFT had identical D1/D2 sequences. The former three oral strains also had identical ITS sequences, but they differed from strain 17YFT by two substitutions in the ITS region. The sequence data thus suggest that the four strains are conspecific. In the tree constructed from the concatenated sequences of the ITS and D1/D2 regions, they were clustered in the Torulaspora clade (Fig. 1a). The closest relative of the new strains was Torulaspora microellipsoides. The new strains differed from the type strain of T. microellipsoides by five (0.9%) substitutions in the D1/D2 domain and by >70 (>15%) mismatches in the ITS region.

Figure 1

Phylogenetic tree drawn from a neighbor-joining analysis of the combined sequences of ITS (including 5.8S rRNA gene) region and 26S rRNA gene D1/D2 domain, depicting the relationships of Torulaspora quercuum sp. nov. (a) and Candida pseudohumilis sp. nov. (b) with closely related taxa. Bootstrap percentages over 50% from 1000 bootstrap replicates are shown. Reference sequences were from the type strains of the species retrieved from GenBank under the accession numbers indicated. Bold font denotes new species.

Strain XZ-281T had unique ITS and D1/D2 sequences. This strain was clustered in the Kazachstania clade, together with Candida humilis and Candida milleri (Kurtzman, 2003) (Fig. 1). The latter two taxa described may be the same or sister species (Kurtzman & Robnett, 2003). Strain XZ-281T differed from the type strains of C. humilis and C. milleri by eight to nine (1.2%) substitutions in the D1/D2 domain and by >100 (>20%) mismatches in the ITS region.

Taxonomy and ecology

Previous studies have shown that yeast strains with >1% substitution in the D1/D2 domain or ITS region usually represent separate species (Kurtzman & Robnett, 1998, 2003; Sugita, 1999; Fell, 2000; Scorzetti, 2002). The remarkable ITS sequence divergences of the new strains studied from their closely related described species indicated that they represent two novel yeast species.

Because the strains studied were phylogenetically located in two clades containing mainly teleomorphic ascomycetous yeast species, special efforts were made to induce their sexual states. Sporulation media including McClary acetate agar, 5% malt extract agar, corn meal agar and diluted (1 : 4 and 1 : 19) V8 juice agar were used and inoculated with single or mixed strains as described in Yarrow (1998). The cultures were incubated at 25 °C for 2 months and observed after 7 days, and then at weekly intervals. Ascosporulating cells with a tapered protuberance typical for the genus Torulaspora and persistent asci containing two to four spherical ascospores were observed in the culture of strain 17YFT on diluted V8 agar after 7 days. The sexual state was not observed for the four oral strains on any of the sporulation media used. Mixtures of the conspecific strains XZ-46A, XZ-105 and XZ-129 also failed to develop a sexual state.

A teleomorphic name T. quercuum sp. nov. is proposed for strain 17YFT. Because the three oral strains XZ-46A, XZ-105 and XZ-129 from Tibetan volunteers are conspecific with strain 17YFT, as revealed by rRNA gene sequence analysis, they are included in this new species. An anamorphic name C. pseudohumilis sp. nov. is proposed for strain XZ-281T. The two new species have phenotypic properties similar to their closest phylogenetic relatives. However, T. quercuum sp. nov. differs from T. microellipsoides in its inability to assimilate melibiose. Candida pseudohumilis sp. nov. differs from C. humilis slightly in its maximum growth temperature; the former cannot grow above 28 °C while the latter can grow at 30 °C.

The phenotypic characteristics and phylogenetic positions of the two new species suggest that they were likely transient members of the microbial community in the oral cavities of their hosts and may not be pathogenic to humans. None of the analyzed strains in either species can grow at 37 °C. The maximum growth temperatures of C. pseudohumilis sp. nov. and T. quercuum sp. nov. are 28 and 34 °C, respectively. In addition, their phylogenetically close relatives have not been reported to be pathogens of humans. The strains of the new species were most probably transmitted to the mouth of the Tibetans through food or drinks. Because of their specific climate and geography, Tibetans are known to consume large quantities of fermented vegetables, meat and dairy products, many of which may contain fermenting yeasts. The strains reported here for both new species have strong sugar fermentation abilities, similar to their close relatives.

Latin diagnosis of Torulaspora quercuum sp. nov. F.-Y. Bai, Q.-M. Wang & J. Xu

In media liquido YM post dies 3 ad 25 °C, cellulae ovoidae et ellipsoideae, 3.7–7.5 × 4.5–7.5 μm, singulae, binae et adhaerentes. Post 1 mensem sedimentum formatur. In agaro farinae Zea maydis confecto pseudomycelium non observatae. Asci inconjugatio fiunt. Ascosporae globosae, 2 vel 4 in quoque asco.

Glucosum, galactosum, sucrosum et raffinosum (lente) fermentantur at non maltosum nec lactosum. Glucosum, galactosum, sucrosum, maltosum (variabile), trehalosum, raffinosum, inulinum (lente), d-xylosum (lente), ethanolum, d-mannitolum, glucitolum et dl-lacticum (lente et exigue) assimilantur at non l-sorbosum, cellobiosum, lactosum, melibiosum, melezitosum, amylum solubile, l-arabinosum, d-arabinosum, d-ribosum, l-rhamnosum, d-glucosaminum, methanolum, glycerolum, erythritolum, ribitolum, galactitolum, α-methyl-d-glucosidum, salicinum, acidum succinicum, acidum citricum, inositolum nec hexdecane. Ammonium sulfatum, l-lysinum et cadaverinum (lente et exigue) assimilantur at non natrum nitrosum, kalium nitricum nec ethylaminum. Ad crescentiam vitaminae externae necessariae sunt. Maxima temperatura crescentiae: 34 °C. Materia amyloidea iodophila non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur. Typus: isolatus ex folio Quercus sp., 17YFT, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.3768T).

Description of Torulaspora quercuum sp. nov. F.-Y. Bai, Q.-M. Wang & J. Xu

In YM broth, after 3 days at 25 °C, the cells are ovoid or ellipsoid, 3.7–7.5 × 4.5–7.5 μm, and occur singly, in pairs or in groups (Fig. 2a). Budding is multilateral. After 1 month at 25 °C, sediment is present. Pseudohyphae are not observed in cultures grown on corn-meal agar. Sporulation is observed on V8 juice agar (1 : 19) after 7 days at 25 °C; persistent asci each containing two or four subglobose ascospores are formed directly from yeast cells or from ascosporulating cells with a tapered protuberance (Fig. 2b).

Figure 2

Torulaspora quercuum sp. nov. 17YFT yeast cells grown in YM broth for 3 days at 25°C (a), asci and ascospores formed on diluted (1 : 19) V8 juice agar after 7 days at 25°C (b) and Candida pseudohumilis sp. nov. XZ-281T yeast cells grown in YM broth for 3 days at 25°C (c). Scale bars=10 μm.

Glucose, galactose, sucrose and raffinose (delayed) are fermented; maltose and lactose are not fermented. Glucose, galactose, sucrose, maltose (variable), trehalose, raffinose, inulin (delayed), d-xylose (delayed), ethanol, d-mannitol, d-glucitol and dl-lactic acid (delayed and weak) are assimilated; l-sorbose, cellobiose, lactose, melibiose, melezitose, soluble starch, l-arabinose, d-arabinose, d-ribose, l-rhamnose, d-glucosamine, methanol, glycerol, erythritol, ribitol, galactitol, α-methyl-d-glucoside, salicin, succinic acid, citric acid, inositol and hexadecane are not assimilated. Ammonium sulfate, l-lysine and cadaverine hydrochloride (delayed and weak) are assimilated; potassium nitrate, sodium nitrite and ethylamine hydrochloride are not assimilated. Growth in vitamin-free medium is negative. The maximum growth temperature is 34 °C. Starch-like compounds are not produced. Diazonium blue B reaction is negative. Urease activity is negative. The type strain, 17YFT, was isolated from a leaf of Quercus sp. collected in Changbai Mountain, Jilin Province, northeast China, in August 2007. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, as AS 2.3768T (=CBS 11403T).

Etymology: The specific epithet quercuum refers to the genus of the plant (Quercus) from which the type strain was isolated.

Latin diagnosis of Candida pseudohumilis sp. nov. F.-Y. Bai, Q.-M. Wang & J. Xu

In media liquido YM post dies 3 ad 25 °C, cellulae ellipsoideae, oviodae, 2.5–5.0 × 5.0–6.2 μm, singulae et binae. Post 1 mensem sedimentum formatur. In agaro farinae Zea maydis confecto pseudomycelium non observatae.

Glucosum, galactosum, sucrosum et raffinosum fermentantur at non maltosum nec lactose. Glucosum, galactosum, sucrosum, trehalosum et raffinosum assimilantur at non l-sorbosum, maltosum, cellobiosum, lactosum, melibiosum, melezitosum, inulinum, amylum solubile, d-xylosum, l-arabinosum, d-arabinosum, d-ribosum, l-rhamnosum, d-glucosaminum, methanolum, ethanolum, glycerolum, erythritolum, ribitolum, galactitolum, d-mannitolum, glucitolum, α-methyl-d-glucosidum, salicinum, dl-lacticum, acidum succinicum, acidum citricum, inositolum nec hexdecane. Ammonium sulfatum assimilatur at non natrum nitrosum, kalium nitricum, l-lysinum, ethylaminum nec cadaverinum. Ad crescentiam vitaminae externae necessariae sunt. Maxima temperatura crescentiae: 28 °C. Materia amyloidea iodophila non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur. Typus: isolatus ex ostium, XZ-281T, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.3956T).

Description of Candida pseudohumilis sp. nov. F.-Y. Bai, Q.-M. Wang & J. Xu

In YM broth, after 3 days at 25 °C, the cells are ellipsoid or ovoid, 2.5–5.0 × 5.0–6.2 μm, and occur singly or in pairs (Fig. 2c). Budding is multilateral. After 1 month at 25 °C, sediment is present. Pseudohyphae are not observed in cultures grown on corn-meal agar.

Glucose, galactose, sucrose and raffinose are fermented; maltose and lactose are not fermented. Glucose, galactose, sucrose, trehalose and raffinose are assimilated; l-sorbose, maltose, cellobiose, lactose, melibiose, melezitose, inulin, soluble starch, d-xylose, l-arabinose, d-arabinose, d-ribose, l-rhamnose, d-glucosamine, methanol, ethanol, glycerol, erythritol, ribitol, galactitol, d-mannitol, d-glucitol, α-methyl-d-glucoside, salicin, dl-lactic acid, succinic acid, citric acid, inositol and hexadecane are not assimilated. Ammonium sulfate is assimilated; potassium nitrate, sodium nitrite, l-lysine, ethylamine hydrochloride and cadaverine hydrochloride are not assimilated. Growth in vitamin-free medium is negative. The maximum growth temperature is 28 °C. Starch-like compounds are not produced. Diazonium blue B reaction is negative. Urease activity is negative. The type strain, XZ-281T, was isolated from the mouth of a Tibetan male in Nyingchi, Tibet, in April 2007. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, as AS 2.3956T (=CBS 11404T).

Etymology: The specific epithet pseudohumilis refers to the similarity of the new species to C. humilis.

Acknowledgements

We thank Dr Wang Mu, Tibet Agricultural and Animal Husbandry College, Nyingchi, Tibet, for her help in oral sample collection. This study was supported by grants no. 30628002 and no. 30770048 from the National Natural Science Foundation of China (NSFC).

Footnotes

  • Editor: Cletus Kurtzman

References

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