Introduction

Methylotrophic yeasts, the yeasts that can utilize methanol as a sole source of carbon and energy, represent a relative small proportion of yeasts and belong to a limited number of yeast genera, including Ogataea (Yamada et al. 1994; Mikata and Yamada 1995; Suh et al. 2006), Komagataella (Yamada et al. 1995; Dlauchy et al. 2003; Kurtzman 2005), Kuraishia (Yamada et al. 1994; Péter et al. 2005), and related Candida species in these three groups (Lachance et al. 2011). In the recent years members of Ogataea increased rapidly, not only because of the transfer of many Pichia species to this genus after emendation of the genus description for nitrate assimilation, ascospores shape, ascospores number and multi-gene phylogenetic analyses, but also due to the discovery of many novel species in the past few years (Glushakova et al. 2010; Kurtzman and Robnett 2010; Limtong et al. 2008; Nagatsuka et al. 2008; Péter et al. 2008, 2009; Suh and Zhou 2010). In the yeasts, a taxonomic study, 5th edition 31 species are accepted in the genus Ogataea and 22 Candida species are placed in the Ogataea clade (Kurtzman et al. 2011b; Lachance et al. 2011). Subsequently, several additional members of the Ogataea clade have been proposed such as O. phyllophila, Candida chumphonensis, C. mattranensis (Koowadjanakul et al. 2011), C. rishirensis (Nakase et al. 2010), O. parapolymorpha (Kurtzman 2011a) and O. saltuana (Péter et al. 2011).

The external surface of plant leaves, which is usually referred to as the phylloplane or phyllosphere, has been recognized as an important habitat for epiphytic microorganisms (Fonseca and Inacio 2006; Phaff and Starmer 1987). Phylloplane are found to be colonized by members of both basidiomycetous and ascomycetous yeasts (Fonseca and Inacio 2006; Glushakova et al. 2007; Landell et al. 2010; Nakase et al. 2001; Slavikova et al. 2009). Although most common phylloplane yeasts are basidiomycetous species (Fonseca and Inacio 2006; Nakase et al. 2001) the occurrence of methylotrophic ascomycetous species were reported on leaves in Hungary (Péter et al. 2007) and Thailand (Koowadjanakul et al. 2011). This may be due to the fact that methanol on phyllopane derives by pectin demethylation inside the leaves and emitted to the surface through stomata during transpiration (Nemecek-Marshall et al. 1995) and methylotrophic yeasts utilize it for their growth (Péter et al. 2007).

During the investigation of methylotrophic yeasts on phylloplane of wine and table grapes and other plant species cultivated in Kanchanaburi province, Thailand, three strains that represent two novel species were isolated. Detailed analyses demonstrated that they belong to the genus Ogataea. In this report two novel Ogataea species are described.

Materials and methods

Yeast isolation

Green and undamaged leaves of wine and table grapes (23 samples) and other plant species (12 samples) were collected from fields in Kanchanaburi Research Station, Agro-Ecological System Research and Development Institute (N14°07′15.1′′ E099°19′05.6′′) of Kasetsart University located in Wang Dong sub-district, Mueang district, Kanchanaburi province, Thailand on 2 July 2009. Strain KM03T was isolated from the surface of a leaf from a mango tree (Mangifera indica, Family: Anacardiaceae) and strains KM13T and KM15 were obtained from the surface of leaves of two wine grapes (Vitis vinifera, Family: Vitaceae).

Methylotrophic yeasts were isolated from leaf surface by a technique involving three consecutive methanol enrichments as described by Limtong et al. (2004) but using 0.5 % (v/v) methanol-yeast nitrogen base (YNB) broth (0.67 % Difco yeast nitrogen base and 0.5 % (v/v) methanol) instead of 1 % (v/v) methanol-YNB broth. Three grams of cut leaves were aseptically placed in a 250 ml Erlenmeyer flask containing 50 ml enrichment broth and incubated on a rotary shaker at room temperature (27 ± 3 °C) for 4–5 days. After three consecutive enrichment cultivation a loopful of the enriched culture was streaked on 0.5 % (v/v) methanol-YNB agar and incubated at room temperature until yeast colonies appeared. Yeast colonies of different morphologies were picked and purified by cross streaking on yeast extract malt extract (YM) agar (0.3 % yeast extract, 0.3 % malt extract, 0.5 % peptone, 1 % glucose and 2 % agar). Purified yeast strains were suspended in YM broth supplemented with 10 % glycerol and maintained at −80 °C.

Yeast strains obtained were tested for growth on 0.5 % (v/v) methanol-YNB agar at room temperature (27 ± 3 °C).

DNA sequencing and phylogenetic analyses

The sequences of the D1/D2 and the ITS regions were determined from PCR products amplified from genomic DNA. Methods for DNA extraction and amplification of the D1/D2 region were described previously (Limtong et al. 2007). The ITS region was amplified with primers, ITS1 and ITS4, following the method of White et al. (1990). The PCR products were checked by agarose gel electrophoresis and purified by using the QIA quick purification kit (Qiagen, Germany). The purified products were sequenced commercially by Macrogen Inc. (Seoul, Korea) using primers, NL1 and NL4 for the D1/D2 region and ITS1 and ITS4 for the ITS region. The sequences were compared pairwise using a BLAST search (Altschul et al. 1997) and were aligned with the sequences of related species retrieved from GenBank using the multiple alignment program CLUSTAL_X version 1.81 (Thompson et al. 1997). The phylogenetic trees were constructed with the neighbor-joining, maximum-parsimony and maximum-likelihood methods using MEGA version 5 (Tamura et al. 2011). Confidence levels of the clades were estimated from bootstrap analysis (1,000 replicates) (Felsenstein 1985).

Examination of taxonomic characteristics

The strains were characterized morphologically, biochemically, and physiologically according to the standard methods described by Yarrow (1998). Mycelium formation was investigated on corn meal agar in slide culture at 25 °C for up to 7 days. Ascospore formation was investigated for individual strains or strain pairs on 5 % malt extract agar, Gorodkowa agar, Fowell’s acetate agar and corn meal agar at 15 and 25 °C for up to 4 weeks. Carbon assimilation tests were conducted in liquid medium as described by Yarrow (1998). Assimilation of nitrogen compounds was examined on solid media with starved inocula following the method of Nakase and Suzuki (1986). Growth at various temperatures was determined by cultivation of the strains in YM broth. Ubiquinones were extracted from cells that were cultivated in 500 ml Erlenmeyer flasks containing 250 ml of yeast extract peptone dextrose (YPD) broth (1 % yeast extract, 2 % peptone and 2 % dextrose) on a rotary shaker at 28 °C for 24–48 h and purified according to the methods described by Yamada and Kondo (1973) and Kuraishi et al. (1985). Isoprenologues were identified by HPLC as described previously (Limtong et al. 2007).

Results and discussion

Yeasts isolation, identification and novel species delineation

A total of 13 yeast strains were isolated from 35 leaf samples and seven strains grew well on 0.5 % (v/v) methanol-YNB agar at room temperature. Identification on the basis of similarities of the D1/D2 sequences revealed three strains (KM03T, KM13T and KM15) that represent novel species. Two strains were identified to be O. philodendri and the other two strains were closest to Candida sp. NRRL Y-12764, an undescribed species, but with 1 nucleotide substitution.

The sequences of the D1/D2 regions of KM13T and KM15 were identical and differed markedly from that of KM03T. In terms of pairwise sequence similarity the closest relative to KM03T was O. wickerhamii (GenBank EU011612) with 2.3 % nucleotide substitutions (13 nucleotide substitutions out of 566 nt). The two strains, KM13T and KM15, were closest to Candida sp. NRRL YB-1238 (CBS 12667; GenBank EU011607) with 1.9 % nucleotide substitutions (11 nucleotide substitutions out of 566 nt), differed from Candida suzukii (GenBank EU011610) and Candida nitratophila (GenBank EU011606) with 2.1 % nucleotide substitutions (12 nucleotide substitutions out of 566 nt), and differed from C. nanaspora (GenBank EU011602) with 2.4 % (13 substitutions and 1 indel out of 566 nt).

The nucleotide sequences of the ITS regions of these strains were also analyzed, and the sequences of KM03T, KM13T, KM15, O. wickerhamii CBS 4307T, Candida suzukii CBS 9253T, Candida nitratophila CBS 2027T, C. nanaspora CBS 7200T and Candida sp. NRRL YB-1238 (CBS 12667) were deposited as AB734093, AB734094, AB734095, JX546582, JX965187, JX965188, JX546583 and JX546581, respectively. In the ITS regions the sequences of KM03T exhibited 17 % nucleotide differences (91 nucleotide substitutions and 33 indels out of 722 nt) with O. wickerhamii (GenBank JX546582). Strains KM13T and KM15 that showed identical sequences differed by 8.5 % (43 nucleotide substitutions and 9 indels out of 617 nt) from Candida sp. NRRL YB-1238 (GenBank JX546581), 9.8 % (42 nucleotide substitutions and 22 indels out of 662 nt) from Candida suzukii (GenBank JX965187), 7.3 % (37 nucleotide substitutions and 11 indels out of 655 nt) from Candida nitratophila (GenBank JX965188) and 9.2 % (46 nucleotide substitutions and 12 indels out of 631nt) from C. nanspora (GenBank JX546583).

The phylogenetic trees based on the sequence of the D1/D2 region of the LSU rRNA gene constructed by the three methods namely neighbor-joining, maximum-parsimony and maximum-likelihood methods showed only slight differences in the topology of the closely related species but that the main topology of the clusters was the same (data not shown for maximum-parsimony and maximum-likelihood trees). They demonstrated that the three strains were in two separate clusters in the Ogataea clade (Fig. 1). Strain KM03T clustered with O. wickerhamii, the closest species in term of pairwise sequence similarities, and placed in a cluster that contains O. minuta, the type species of the genus. Strains KM13T and KM15 are located in the same position and form a cluster with C. suzukii, C. nitratophila, C. nanaspora, O. ramenticola and Candida sp. NRRL YB-1238 (CBS 12667).

Fig. 1
figure 1

Phylogenetic tree based on the sequences of the D1/D2 region of the LSU rRNA gene, showing the clustering of Ogataea kanchanaburiensis sp. nov. (KM03T) and Ogataea wangdongensis sp. nov. (KM13T and KM15), with respect to their closely related species. The phylogenetic trees were constructed with the neighbor-joining method using MEGA version 5 (Tamura et al. 2011). Numbers indicate percentages of bootstrap sampling, derived from 1,000 samples. The numbers in parentheses are GenBank accession numbers. Ambrosiozyma cicatricosa was the outgroup species in the analysis. Bar = 0.01 Knuc

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On the basis of the evidence from the molecular and other taxonomic criteria obtained during this study, we concluded that the three strains represent two novel Ogataea species although formation of ascospores was not observed in order to follow International Code of Nomenclature for algae, fungi, and plants concerning with the nomenclatural rules for fungi that the most important is the adoption of “one fungus, one name” which was enacted at the 18th International Botanical Congress in Melbourne, Australia in July 2011 (Miller et al. 2011). The name O. kanchanaburiensis sp. nov. (MB800996) is proposed for strain KM03T and the name O. wangdongensis sp. nov. (MB800997) is assigned to strains KM13T and KM15.

The novel species O. kanchanaburiensis sp. nov. and O. wangdongensis sp. nov. can be distinguished from each other and from their closely related recognized species, O. wickerhamii, and C. suzukii, C. nitratophila and C. nanaspora, respectively, not only on the basis of the sequences of the D1/D2 and the ITS regions but also by several phenotypic characteristics as shown in Table 1.

Table 1 Phenotypic characteristics that differentiate Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov. form the closely related species
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Members of the genus Ogataea have been found associated with plant material including tree bark, tree exudate, flower, leaf, gall on leaf, rotten wood insect and frass (Glushakova et al. 2010; Koowadjanakul et al. 2011; Kurtzman 2011a; Morais et al. 2004). Among these a few strains of the recognized species were reported to be isolated from plant leaves i.e. O. allantospora, O. nitratoaversa, O. phyllophila and O. saltuana (Koowadjanakul et al. 2011; Kurtzman 2011a; Péter et al. 2011). This study increases the number of Ogataea species isolated from phylloplane.

Description of Ogataea kanchanaburiensis Limtong, Kaewwichian and M. Groenewald sp. nov.

Growth in YM broth: After 3 days at 25 °C, cells are spherical (2.2–5.0 × 2.5–5.0 μm), and occur singly or in pairs (Fig. 2a). Budding is multilateral. Growth on YM agar: After 3 days at 25 °C, the streak culture is butyrous, cream-coloured, with a smooth surface and has an entire margin. Pseudohyphae and true hyphae are not formed in slide culture on corn meal agar after 7 days at 25 °C. Ascospores were not produced on 5 % malt extract agar, Fowell’s acetate agar, Gorodkowa agar and corn meal agar at 15 and 25 °C up to 6 weeks.

Fig. 2
figure 2

Budding cells on YM agar after 3 days at 25 °C. a Ogataea kanchanaburiensis sp. nov. (KM03T) b Ogataea wangdongensis sp. nov. (KM13T). Scale bar = 20 μm

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Fermentation of all carbon sources is negative. d-glucose, d-galactose, l-sorbose, α–α-trehalose, d-mannitol, d-glucono-1,5-lactone, succinate (weak), methanol, ethylamine HCl, l-lysine HCl and cadaverine are assimilated, but N-acetyl-d-glucosamine, d-ribose, d-xylose, l-arabinose, d-arabinose, l-rhamnose, sucrose, maltose, methyl-α-d-glucoside, cellobiose, salicin, melibiose, lactose, raffinose, melezitose, inulin, soluble starch, glycerol, erythritol, galactitol, myo-inositol, 2-ketogluconate, 5-ketogluconate, d-gluconate, d-glucuronate, d-galacturonate, ribitol, d-glucitol, dl-lactate, citrate, ethanol, potassium nitrate and sodium nitrite are not assimilated. Growth in vitamin free medium is positive (slow). Growth on medium containing 50 % (w/v) glucose is positive but on medium containing 60 % (w/v) glucose or 10 % (w/v) sodium chloride/5 % (w/v) glucose are negative. Growth with 0.01 % cycloheximide and 0.1 % cycloheximide are negative. Growth at 37 °C (slow) is positive, but at 40 °C is negative. Starch like compounds are not produced. Diazonium blue B color and urease reactions are negative. The major ubiquinone is Q-7.

Holotype

KM03T is the holotype of O. kanchanaburiensis (MB800996). The strain was isolated from leaves of mango tree (Mangifera indica Linn.), Kanchanaburi Research Station (N14°07′15.1′′E099°19′05.6′′), Wang Dong sub district, Mueang District, Kanchanaburi province collected on 2 July 2009. A living culture from the type strain was deposited at the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand, as BCC 47626T; NITE Biological Resources Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Chiba, Japan, as NBRC 108603T and CBS-KNAW Fungal Biodiversity Center (CBS), Utrecht, The Netherlands as CBS 12673T.

Etymology

The species epithet kanchanaburiensis refers to Kanchanaburi province, Thailand, where the strain was isolated.

Description of Ogataea wangdongensis Limtong, Kaewwichian and M. Groenewald sp. nov.

Growth in YM broth: After 3 days at 25 °C, cells are spherical (2–4 × 2–4 μm), and occur singly or in pairs or in groups (Fig. 2b). Budding is multilateral. Growth on YM agar: After 3 days at 25 °C, the streak culture is butyrous, white to cream-coloured, with an umbonate surface and has an undulate margin. Pseudohyphae and true hyphae are not formed in slide culture on corn meal agar after 7 days at 25 °C. Ascospores were not produced by individual strains or strains paired on 5 % malt extract agar, Fowell’s acetate agar, Gorodkowa agar and corn meal agar at 15 and 25 °C up to 6 weeks.

Fermentation of d-glucose is positive (slow) but negative for d-galactose, sucrose, maltose, lactose, trehalose and raffinose. Assimilation of d-glucose, d-galactose, l-sorbose, N-acetyl-d-glucosamine, d-xylose, l-arabinose, l-rhamnose, α–α-trehalose, glycerol, erythritol, ribitol, d-glucitol, d-mannitol, d-glucono-1,5-lactone, dl-lactate, succinate (weak), methanol, ethanol (positive/slow), potassium nitrate, sodium nitrite, ethylamine HCl, l-lysine HCl, and cadaverine are positive, but d-ribose, d-arabinose, sucrose, maltose, methyl-α-d-glucoside, cellobiose, salicin, melibiose, lactose, raffinose, melezitose, inulin, soluble starch, galactitol, myo-inositol, 2-ketogluconate, 5-ketogluconate, d-gluconate, d-glucuronate, d-galacturonate and citrate are not assimilated. Growth in vitamin free medium is positive (slow). Growth on media containing 50 % (w/v) glucose, 60 % (w/v) glucose or 10 % (w/v) sodium chloride/5 % (w/v) glucose is positive. Growth with 0.01 % cycloheximide and 0.1 % cycloheximide is negative. Growth at 37 °C (slow) is positive, but at 40 °C is negative. Starch like compounds are not produced. Diazonium blue B color and urease reactions are negative. The major ubiquinone is Q-7.

Holotype

KM13T is the holotype of O. wangdongensis (MB800997). The strain was isolated from leaves of wine grape (Vitis vinifera), Kanchanaburi Research Station (N14°07′15.1′′ E099°19′05.6′′), Wang Dong sub district, Mueang District, Kanchanaburi province collected on 2 July 2009. The living culture from type was deposited at the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand, as BCC 42667T; NITE Biological Resources Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Chiba, Japan, as NBRC 107781T and CBS-KNAW Fungal Biodiversity Center (CBS), Utrecht, The Netherlands as CBS 12674T.

Etymology

The species epithet wangdongensis refers to Wang Dong sub district, Mueang District, Kanchanaburi province, Thailand, where the strain was isolated.