Lipomyces orientalis sp. nov., a yeast species isolated from soil in Vietnam

Yeasts of the genus Lipomyces are true soil inhabitants and have a worldwide distribution. The genus Lipomyces and associated genera (Dipodascopsis, Zygozyma, Myxozyma) represent a unique branch in the evolution of the ascomycetes (van der Walt, 1992). Currently 11 species of Lipomyces are recognized, Lipomyces doorenjongii, L. japonicus, L. kockii, L. kononenkoae, L. lipofer, L. mesembrius, L. spencermartinsiae, L. starkeyi, L. tetrasporus, L. yamadae and L. yarrowii (van der Walt et al., 1997; Smith, 1998; van der Walt et al., 1999). Despite the worldwide distribution of the genus, some geographical and geochemical preferences have been recorded for some species. For example, L. lipofer is restricted to temperate zones (Canada, Russia) whereas L. kononenkoae and L. spencermartinsiae can be commonly found in ferric soils of the tropics (Mali, Nigeria, Trinidad) (Babjeva & Gorin, 1987). In a survey of the diversity of soil yeasts in Vietnam, strains representing a novel species of the genus Lipomyces were isolated.

A total of 52 soil samples from different locations in Vietnam were collected and analysed (see Supplementary Fig. S1 for locations of sampling sites). For isolation of lipomycetaceous yeasts, soil particles were sprinkled over agar plates containing nitrogen-depleted medium (l⁻¹: 20 g glucose, 20 g agar, 0.85 g KH₂PO₄, 0.15 g K₂HPO₄, 0.5 g MgSO₄.7H₂O, 0.1 g NaCl, 0.1 g CaCl₂.6H₂O, 0.5 mg H₃BO₃, 0.04 mg CuSO₄.H₂O, 0.1 mg KI, 0.2 mg FeCl₃.6H₂O, 0.4 mg MnSO₄.H₂O, 0.2 mg Na₂MoO₄.2H₂O, 0.4 mg ZnSO₄.7H₂O). The plates were incubated at 25 °C for 2–3 weeks until characteristic slimy growth of Lipomyces was detected. Yeast colonies were purified on the nitrogen-depleted medium and then on malt/glucose agar (1 °Bx malt broth, 1 % glucose, 2 % agar). A protocol describing the preparation of the malt broth is available as supplementary material in IJSEM Online.

Lipomycetaceous yeasts were found in 24 of the 52 samples. From these, 73 strains were isolated. They all sporulated strongly on diluted malt/glucose agar after 3–4 weeks of incubation. The strains were divided into groups based on the mode of ascospore formation. Twenty-four strains were found to be of the ‘tetrasporus' type (i.e. they formed asci containing one to four ascospores). The remaining strains produced asci containing mainly more than eight ascospores. Among the ‘tetrasporus' group, 19 strains produced striated ascospores and the other five produced roughened ascospores (Fig. 1a⇓). Closer examination by scanning electron microscopy (SEM) revealed that ascospores of the latter five strains had warty surfaces (Fig. 1b⇓ and Supplementary Fig. S2) and, thus, they differed morphologically from all known Lipomyces species.

Physiological tests were carried out on the latter five strains using standard methods for yeast characterization (Yarrow, 1998). Sporulation of strains was obtained on 10-fold diluted malt/glucose agar (0.1 °Bx malt broth, 0.1 % glucose, 2 % agar). The morphology of vegetative cells and ascospore formation was studied using a light microscope (Eclipse E-600; Nikon). Photographs were taken with a digital video camera (TK-C1380E; JVC). For SEM, strongly sporulated biomass was collected and fixed with 10 % glutaraldehyde for 2 h. The biomass was then treated with increasing concentrations of acetone (25, 50, 75 and 100 %) and dried. The samples were coated with gold and viewed with scanning electron microscope (JSM-5410LV; JEOL) at 15 000 kV.

The five novel strains differed from the standard description of L. tetrasporus in three assimilation tests (assimilation of propane-1,2-diol, butane-2,3-diol and nitrite). These five lipomycetaceous strains were isolated from three soil samples collected at Thanh Hoa (strain MD66-1), Quang Ninh (strains MD88-4 and MD 89-4) and Nha Trang (strains Lip 51 and Lip 95^T) (Table 1⇓). The distances between these locations are roughly 260, 940 and 1000 km.

Extraction and purification of the coenzyme Q system were performed according to Yamada & Kondo (1973). Reversed-phase HPTLC was used for identification of coenzyme Q. Reversed-phase HPTLC plates (silica gel 60 for nano TLC; Merck) were prepared by dipping in n-hexane containing 5 % liquid paraffin. The plates were air-dried. Coenzyme Q samples and standards were spotted on the plates, which were then developed with methanol/isopropanol (1 : 1) saturated with liquid paraffin. Visualization was done by dipping the plates in 0.2 % KMnO₄ for 5 min. This procedure ensures good separation of CoQ-6, CoQ-7, CoQ-8, CoQ-9 and CoQ-10. To separate CoQ-10 and CoQ-10(H₂), a developing system containing acetone/acetonitrile (1 : 1) saturated with liquid paraffin was used. The major ubiquinone of the novel strains was Q-9.

For DNA extraction, strain Lip 95^T was grown on YMA (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 1 % glucose, 2 % agar) for 3 days at 25 °C. One loopful of cells was transferred to a microfuge tube containing 1 ml 2× SSC and heated at 99 °C for 10 min. Cells were collected and washed once with 1 ml sterile deionized water by centrifugation. To the cell pellet, about 75 μl glass beads (0.2–0.5 mm in diameter), 75 μl phenol/chloroform and 100 μl water were added. The tube was shaken at 1400 r.p.m. for 10 min and then centrifuged at 10 000 g for 10 min. The upper layer was transferred to a new microfuge tube and used directly as template for PCR. Due to the well-known inhibition effect of slimy substances of Lipomyces on the PCR (Nishimura et al., 2002), care is needed regarding the age of culture and the cultivation medium.

The ITS-D1/D2 region was amplified using primers ITS1 and NL4 on a GeneAmp PCR System 9700 (PE Applied Biosystems). The PCR conditions were as follows: an initial denaturation step of 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 52 °C for 40 s and 72 °C for 60 s, with a final extension step at 72 °C for 7 min. The PCR product was purified by using a QIAEX II agarose gel extraction kit (Qiagen). The DNA was sequenced by the dideoxy chain-termination method using primers ITS1, ITS4, NL1 and NL4 described by Kurtzman & Robnett (1998) and Esteve-Zarzoso et al. (1999). A model 377 automated DNA sequencer (Applied Biosystems) was used. For phylogenetic analyses, the resulting sequences were compared with known sequences in GenBank using the web-based nucleotide-nucleotide blast search engine hosted by the National Center for Biotechnology Information () (Altschul et al., 1997). The most similar sequences were edited using the program BioEdit (Hall, 1999) and aligned using clustal x version 1.81 (Thompson et al., 1997). The fixed-gap and floating-gap penalty values used were respectively 10 and 0.2. Phylogenetic trees were constructed from distance data transformed according to Kimura (1980), using the neighbour-joining method (Saitou & Nei, 1987) in clustal x. Bootstrap analyses were performed from 1000 random resamplings (Felsenstein, 1985). The phylogenetic tree was displayed using TreeExplorer version 1.21 (which can be downloaded from ). Sequences from Stephanoascus smithiae strains NRRL Y-17849 (GenBank accession no. U76531) and CBS 5657 (AJ606463) were used as the outgroups for the D1/D2 and ITS trees, respectively.

ITS and 26S rDNA D1/D2 sequences were obtained for strain Lip 95^T. In the phylogenetic tree constructed based on D1/D2 sequences, Lip 95^T clustered with L. kononenkoae, L. spencermartinsiae, L. starkeyi and L. tetrasporus but formed an out-branch (see Supplementary Fig. S3 in IJSEM Online). In the D1/D2 region, Lip 95^T was closest to L. tetrasporus but differed from the latter at six positions (five substitutions and one insertion). In the ITS tree, Lip 95^T clustered with L. tetrasporus (Fig. 2⇓). They differed from each other at 16 positions in the ITS region. Based on the distinctive phenotypic characteristics and phylogenetic position amongst established taxa, a novel species of Lipomyces, Lipomyces orientalis sp. nov., is proposed to accommodate the new isolates.