Pichia cecembensis sp. nov. isolated from a papaya fruit (Carica papaya L., Caricaceae ) Bhaskar Bhadra1, R. Sreenivas Rao1, N. Naveen Kumar1, Preeti Chaturvedi1, Partha K. Sarkar2 & S. Shivaji1 1 Centre for Cellular and Molecular Biology, Hyderabad, India; and 2Shantha Biotechnics Limited, Medchal, Hyderabad, India Correspondence: S. Shivaji, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. Tel.: 100 91 40 27192504; fax: 100 91 40 27160591; e-mail: shivas@ccmb.res.in GenBank/EMBL accession numbers for the 26S rRNA gene D1/D2 domain and internal transcribed spacer (ITS)1–5.8S rRNA gene–ITS2 sequences of Pichia cecembensis sp. nov. YS16T are AM159112 and AM233511 respectively. Received 3 November 2006; revised 20 December 2006; accepted 28 December 2006. Abstract The ascogenous yeast YS16T was isolated from a decaying papaya fruit. Phenotypic traits such as multilateral budding, spheroidal or elongate shape, pseudohyphae formation, asci with one or more ascospores, ability to ferment D-glucose, inability to assimilate nitrate and the presence of Q7 ubiquinone suggest its affiliation to the genus Pichia. The nearest phylogenetic neighbor, based on D1/D2 domain sequence of the 26S rRNA gene and ITS region sequence, was identified as Issatchenkia orientalis (NRRL Y-5396T, a synonym of Pichia kudriavzevii) with similarities of 98.2% and 97% respectively. In addition to the difference in the D1/D2 and ITS region sequence, YS16T differs from I. orientalis with respect to a number of phenotypic traits. However, in the phylogenetic analysis, YS16T showed close relatedness to the P. membranifaciens clade. Thus, it is proposed to assign the status of a new species to YS16T, for which the name P. cecembensis sp. nov. is proposed. The type strain of P. cecembensis sp. nov. is YS16T ( = NRRL Y-27985T = JCM 13873T = CBS 10445T). DOI:10.1111/j.1567-1364.2007.00215.x Editor: Cletus Kurtzman Keywords Pichia ; Pichia cecembensis ; yeast; papaya; Issatchenkia . Introduction Fruits contain high levels of sugars, ample quantities of other nutrients, and sufficient water to support microbial growth. Therefore, microorganisms contaminate fruits in the fields and cause substantial spoilage. Several novel yeast strains have been isolated from both healthy and rotten fruits (Peter et al., 2005; Tournas & Katsoudas, 2005). During the course of a study to identify yeasts from decaying fruits of papaya (Carica papaya L.), several yeast strains, YSF9, YSF9A, YSF9B, YSF10, YSF10A, YS16T, YS16A, YS16B, YS104 and YS104A, were isolated. Detailed polyphasic taxonomic analysis indicated that the strains are representatives of the genera Kluyveromyces and Pichia. On the basis of biochemical characteristics and the sequence of the D1/D2 domain and the internal transcribed spacer (ITS)1–5.8S rRNA gene–ITS2 region, strain YS16T is proposed as a new species of the genus Pichia, and the name proposed is Pichia cecembensis sp. nov. FEMS Yeast Res xx (2007) 000–000 Materials and methods Isolation and identification Papaya fruits were collected from the local market in the city of Hyderabad, Andhra Pradesh, India, in the summer months (April and May) of the year 2005. A small piece from the rotten part of the fruit (c. 1 g) was sliced using a sterile scalpel blade, and suspended in 1 mL of sterile distilled water by vigorous vortexing. The resulting suspension (100 mL) was plated on Rose Bengal agar containing chloramphenicol (HiMEDIA, India). The medium contains mycological peptone (5 g L 1), dextrose (10 g L 1), monopotassium phosphate (1 g L 1), magnesium sulfate (0.5 g L 1), Rose Bengal (0.05 g L 1), chloramphenicol (0.1 g L 1) and agar (15.50 g L 1). Similarly, on the control plates, 100 mL of suspension prepared from a small piece of unspoiled papaya fruit was also plated. The plates were incubated at 28 1C for 48 h, and two or three representatives of each colony morphotype were purified by repeated 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 2 streaking on yeast malt (YM) agar plates [containing peptic digest of animal tissue (5 g L 1), yeast extract (3 g L 1), malt extract (3 g L 1), dextrose (10 g L 1) and agar (15 g L 1)]. For routine subculturing and maintenance, the strains were grown on YM agar/broth at 28 1C. Morphologic, physiologic and biochemical properties were determined for one representative of each group, as described by Yarrow (1998). All assimilation tests were performed twice, and the results were scored after 1 week and 4 weeks for delayed positive reaction. An AXIOPLAN microscope (Zeiss, Germany) was used to visualize the morphology of the vegetative cells and ascospores. Strains were grown on Potato Dextrose Rose Bengal agar (M938, HiMEDIA, India) (containing potato infusion, 200 g L 1; dextrose, 20 g L 1; Rose Bengal, 0.008 g L 1, agar, 15 g L 1) and corn-meal agar (M146, HiMEDIA, India), and visualized after 2–4 weeks for ascospore formation using a phase contrast microscope under a  100 oil immersion objective. Ascospores were also visualized using the fluorescent dye 4 0 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D8417, Sigma-Aldrich), which binds to DNA. Spores and the nucleus would be fluorescent following staining with DAPI, but other artefacts, such as oil droplets, would not be fluorescent. Assimilation of nitrogen compounds was investigated on liquid media with starved inocula (Nakase & Suzuki, 1986). Quinones were extracted from 500 mg of freeze-dried cells according to Collins et al. (1977), resolved by thin-layer chromatography, and identified using authentic standards as described earlier (Shivaji et al., 2004). For all the analyses, Issatchenkia orientalis NRRL Y-5396T (a synonym of P. kudriavzevii) was used as a control. The mol% G1C content of the DNA was determined using a Lambda 2 Spectrophotometer with the TEMPLAB 2.0 software package (Parkin-Elmer) (Shivaji et al., 2006). Genomic DNA was prepared as described below. Grouping of isolates Grouping of isolates was done on the basis of the random amplified polymorphic DNA (RAPD) analysis of the genomic DNA of two to three representatives of each colony morphotype. For this purpose, cells were harvested from stationary phase, suspended in lysis buffer [100 mM TrisHCl (pH 8.0) containing 2% Triton X-100, 1% sodium dodecyl sulfate, 1 mM EDTA), and lysed by vortexing with 0.3 g of glass beads (0.45–0.52 mm in diameter, Sigma), as proposed by Tavanti et al. (2005). The cell lysate was then used to prepare DNA according to the method of Makimura et al. (1994). The genomic DNA from each isolate was then subjected to RAPD analysis with two primers, namely (GTG)5 (Thanh et al., 2003) and T3B (Correia et al., 2004). The volume of each PCR was 50 mL, and it contained 25 pg of genomic DNA, 30 pmol of the primer, and the ‘PCR c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved B. Bhadra et al. amplifications core kit’ (containing 1  PCR buffer, 1.5 mM MgCl2, 1.2 mM dNTP mix and 2 U of Taq DNA Polymerase) (GENEI, Bangalore, India). PCR conditions with primer (GTG)5 were as follows: an initial denaturation of 94 1C for 2 min, followed by 30 cycles of 94 1C for 30 s, 50 1C for 30 s and 72 1C for 1.0 min, and a final extension step of 7 min at 72 1C. After amplification, 10 mL of the PCR product was loaded on a 1% agarose gel, electrophoresed in 1  TAE buffer (Sambrook et al., 1989), and documented. The PCR conditions employed for the T3B primer (5 0 -AGG TCG CGG GTT CGA ATC C-3 0 ) were as described by Correia et al. (2004): an initial denaturation of 94 1C for 2 min, followed by 30 cycles of 94 1C for 30 s, 47 1C for 30 s and 72 1C for 1.3 min, and a final extension step of 5 min at 72 1C. The isolates were then grouped on the basis of the similarity of their amplified products. A representative from each group was then subjected to detailed phylogenetic analysis. Phylogenetic analyses The DNA region containing the ITS including 5.8S rRNA gene (ITS1–5.8S rRNA gene–ITS2 region) and the D1/D2 domain of the 26S rRNA gene was amplified using the primer pairs ITS1 (5 0 -TCC GTA GGT GAA CCT GCG G-3 0 ) and NL4 (5 0 -GGT CCG TGT TTC AAG ACG G-3 0 ). PCR was performed for 30 cycles, with denaturation at 94 1C for 1 min, annealing at 47 1C for 45 s, and extension at 72 1C for 1.5 min. The PCR products (c. 1 kb) were purified with the QIA quick purification kit (QIAGEN) and were sequenced on an ABI 3700 DNA analyzer (Applied Biosystems), using the primers used for PCR and five other primers: NL1 (5 0 GCA TAT CAA TAA GCG GAG GAA AAG-3 0 ), NL2A (5 0 CTT GTT CGC TAT CGG TCT C-3 0 ), NL3A (5 0 -GAG ACC GAT AGC GAA CAA G-3 0 ), ITS3 (5 0 -GCA TCG ATG AAG AAC GCA GC-3 0 ), and ITS4 (5 0 -TCC TCC GCT TAT TGA TAT GC-3 0 ) (Lin et al., 1995; Kurtzman & Robnett, 1997). Sequences were manually corrected and aligned using CLUSTAL_X (Thompson et al., 1997). A neighbor-joining phylogenetic tree (Saitou & Nei, 1987) was constructed using MEGA 3.1 (Kumar et al., 2004), on the basis of evolutionary distance data that was determined with Kimura’s twoparameter model (Kimura, 1980). Bootstrap analysis (Felsenstein, 1985) was performed for 1000 replications. Reference sequences were retrieved from GenBank under the accession numbers in the tree. Results and discussion Physiology, phylogenetic position and identification In total, 52 yeast colonies appeared on Rose Bengal chloramphenicol plates when suspensions from the three FEMS Yeast Res xx (2007) 000–000 3 Pichia cecembensis sp. nov. decaying fruits were plated. However, when the suspension prepared from healthy fruit was plated, no yeast colonies appeared. On the basis of color and shape, the colonies could be grouped into four morphotypes. Two to three representatives of each morphotype were then purified, and 10 isolates (YSF9, YSF9A, YSF9B, YSF10, YSF10A, YS16T, YS16A, YS16B, YS104 and YS104A) were subjected to RAPD analysis using (GTG)5 as a primer. On the basis of similarity in the banding pattern of the PCR products, the 10 isolates could be grouped into four groups, namely group I (YS16T, YS16A and YS16B), group II (YS104 and YS104A), group III (YSF10 and YSF10A), and group IV (YSF9, YSF9A and YSF9B). Similar grouping results were obtained with both the (GTG)5 and T3B primers. One representative from each group, i.e. YSF9, YSF10, YS16T and YS104, were then subjected to detailed phylogenetic analysis. Phylogenetic analysis of yeasts has now become easier, with the availability of a database of 26S rRNA gene D1/D2 sequences (Kurtzman & Robnett, 1997, 1998; Fell et al., 2000). BLAST analysis of the nucleotide sequence of the D1/ D2 domain of the 26S rRNA gene of YSF9 (AM275340), YSF10 (AM275339) and YS104 (AM275341) showed 99.6–99.9% similarity with Kluyveromyces marxianus, P. kluyveri var. kluyveri and P. galeiformis, respectively. In the neighbor-joining phylogenetic tree, constructed using the D1/D2 sequences, YSF9, YSF10 and YS104 grouped with K. marxianus NRRL Y-8281T (U94924), P. kluyveri var. kluyveri NRRL Y-11519T (U75727) and P. galeiformis NRRL Y-75738T (U75738), respectively, with bootstrap support of 78–85% (data not shown). The strains also showed biochemical characteristics that were similar to those of their nearest phylogenetic relatives. Therefore, strains YSF9, YSF10 and YS104 were assumed to be strains of K. marxianus, P. kluyveri and P. galeiformis, respectively. The nucleotide sequence of the D1/D2 domain of the 26S rRNA gene and ITS1–5.8S–ITS2 region nucleotide sequences of YS16T, YS16A and YS16B were identical, suggesting that they are strains of the same species. Therefore, subsequent phylogenetic analyses were done using only YS16T. BLAST analysis of the nucleotide sequence of the D1/ D2 domain of YS16T (AM159112), representative of group I, indicated that the nearest phylogenetic neighbor is I. orientalis NRRL Y-5396T (U76347) (a synonym of P. kudriavzevii, Boidin et al., 1965), with which it showed a similarity of 98.2% (eight substitutions and two gaps in 562 nucleotides). The similarity with the other reported species of Issatchenkia, namely I. hanoiensis (AY163900, 93%), I. occidentalis (U76348, 94%), I. scutulata (AF325358, 93%) and I. terricola (U76345, 90%), ranged from 90% to 94%, and with P. membranifaciens (NRRL Y-2026T, 90.1%) and P. deserticola (NRRL Y-12918T, 90%), it was c. 90%. The phylogenetic tree constructed using the neighbor-joining method based on the D1/D2 sequence clearly shows that YS16T is related to FEMS Yeast Res xx (2007) 000–000 I. orientalis (U76347) and forms a distinct clade with a bootstrap support of 99% (Fig. 1). It is also observed that this clade of YS16T and I. orientalis (U76347) is closely related to the P. membranifaciens clade consisting of P. membranifaciens NRRL Y-2026T, P. deserticola NRRL Y-12918T, and Candida ethanolica NRRL Y-12615T, with a bootstrap support of 100% (Fig. 1). The phylogenetic tree also indicates that YS16T and the other species of Issatchenkia are not confined to a single clade but exhibit nonuniform distribution into subclades along with species belonging to the genera Pichia and Candida. This is in accordance with the earlier phylogenetic analysis of Issatchenkia species (Kurtzman & Robnett, 1998; Thanh et al., 2003). The ITS region sequence has also been used for phylogenetic analysis of yeasts (James et al., 1996; Bai et al., 2002; Scorzetti et al., 2002; Tavanti et al., 2005). The ITS1–5.8S–ITS2 region nucleotide sequence of YS16T (AM233511) showed 97% similarity with I. orientalis ATCC 24210T (AY939808) (Leinberger et al., 2005). Multiple sequence alignment (using CLUSTAL_W 1.83) based on the nucleotide sequence of the ITS1–5.8S rRNA gene–ITS2 region of strain YS16T indicated that YS16T differs from I. orientalis ATCC 24210T and shows 11 substitutions, three gaps and one insertion in 509 nucleotides. Furthermore, the alignment of the ITS1–5.8S rRNA gene–ITS2 regions of YS16T and I. orientalis indicated more changes in the ITS2 region than in the ITS1 region. YS16T also differed from P. membranifaciens CBS 107T (DQ104710) by 22.1%, from P. deserticola CBS 7119T (AY790539) by 21.2%, and from Can. ethanolica CBS 8041T (AY790538) by 19.5%, when the nucleotide sequences of the ITS1–5.8S rRNA gene–ITS2 regions were compared. Previous studies have shown that strains with 4 1% substitution in the D1/D2 domain usually represent separate species (Kurtzman & Robnett, 1997, 1998; Lu et al., 2004; Suh & Blackwell, 2004). Therefore, strain YS16T, which shows a 1.8% D1/D2 sequence difference and a 3% ITS1–5.8S–ITS2 region sequence difference from I. orientalis NRRL Y-5396T (U76347) (a synonym of P. kudriavzevii), is assumed to be a novel species of the genus Pichia. In addition to the observed nucleotide sequence differences, YS16T differs from I. orientalis NRRL Y-5396T with respect to some phenotypic traits. YS16T, unlike I. orientalis NRRL Y-5396T, assimilates D-arabinose, ribitol and galactitol (delayed) but not DL-lactate as the sole carbon source. YS16T also assimilates ethylamine as nitrogen source, but the assimilation is delayed (3–4 weeks) in comparison to I. orientalis NRRL Y-5396T, which exhibits growth in 4–5 days. Furthermore, YS16T does grows in the presence of 60% glucose, unlike I. orientalis NRRL Y-5396T, which does not grow. Thus, on the basis of the phenotypic differences and phylogenetic analysis, it is concluded that YS16T, which forms part of the P. membranifaciens clade, should be 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 4 B. Bhadra et al. Pichia cecembensis sp. nov. YS16A (DQ871595) 100 99 Pichia cecembensissp. nov. YS16B (DQ871596) Pichia cecembensis sp. nov. YS16T (AM159112) Issatchenkia orientalis NRRL Y-5396T (U76347) 54 Pichia membranifaciens NRRL Y-2026T (U75725) Candida ethanolica NRRL Y-12615T (U71073) 100 32 100 Pichia deserticola NRRL Y- 12918T (U75734) Candida pseudolambica NRRL Y-17318T (U71063) 55 Issatchenkia hanoiensis HB1.3.13T (AY163900) 30 Issatchenkia occidentalis NRRL Y-7552T (U76348) 19 Issatchenkia scutulata var. scutulata NRRL Y-7663 (AF325358) 67 59 Issatchenkia scutulata var.exigua NRRL Y-10920T (U76349) Pichia norvegensis CBS 1922T (AJ508574) Issatchenkia terricola NRRL YB-4310T (U76345) 32 Pichia fermentans NRRL Y-1619T (U75726) Saccharomyces cerevisiae NRRL Y-12632T (AY048154) 2% Fig. 1. Phylogenetic tree drawn using the neighbor-joining method with Kimura two-parameter model based on the of the D1/D2 domain of the 26S rRNA gene, depicting the relationships of Pichia cecembensis sp. nov. YS16T with the selected species of the genera Issatchenkia, Candida, Pichia and Saccharomyces. Bootstrap values calculated from 1000 replications are indicated in the branch nodes. The bar represents two substitutions per 100 nucleotides. Reference sequences were retrieved from GenBank under the accession numbers in parentheses. assigned to a novel species of the genus Pichia, and the name P. cecembensis sp. nov. is proposed. The type strain of P. cecembensis sp. nov. is YS16T (NRRL Y-27985T = JCM 13873T = CBS 10445T). Latin diagnosis of P. cecembensis Bhadra, Sreenivas Rao, et Shivaji, sp. nov. In medio liquido YM post dies 3 ad 28 1C, cellulae vegetativae globosae aut oblongae (5.0–8.5  2.5–4.5 mm), cellulae singulae, binae et aggregatae. Per gemmationem multipolarem reproducentes. Post 1 mensem ad 28 1C sedimentum formatur. In agaro YM post 1 mensem ad 28 1C, butyrosa, albida vel cremea, glabra, pauro hebia, margo glabro vel undulato. Ascosporae fiunt post dies 15 in corn-meal agaro. In agaro farinae Zea mays post dies 15 ad 28 1C pseudomycellium formantur. Ascosporae globosae, 1–2 in ascus. D-Glucosum fermentatur; D-galactosum, maltosum, sucrosum, lactosum, D-cellobiosum, raffinosum et D-xylosum non fermentantur. Assimilantur D-glucosum, D-arabinosum, glycerolum, ribitolum, galactitolum (lente), natrium succinatum et natrium citratum. D-Galactosum, L-sorboc 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved sum, D-xylosum, L-arabinosum, L-rhamnosum, sucrosum, maltosum, trehalosum, cellobiosum, salicinum, lactosum, raffinosum, melezitosum, erythritolum, glucitolum, Dmannitolum, D-glucuronatum, DL-lactatum, propane1,2-diolum et butane-2,3-diolum non assimilantur. Assimilantur ethylaminum (lente), L-lysinum et cadaverinum; non-assimilantur nitrosum, nitricum, creatininum, glucosaminum et imidazolum. Non crescit in medio 0.01% et 0.1% cycloheximido addito. Ureum non hydralysatur. Diazonium caeruleum B negativum. Crescere potest in temperatura 42 1C, crescit in 60% glucosum. Ubiquinonum majus: Q-7. G1C acidi deoxyribonucleati 42.5 (  0.5) mol%. Typus: YS16 ( = NRRL Y-27985T = JCM13873T = CBS 10445T), designat stirpem typicum. Isolata ex Carica papaya L. fructu (Caricaceae), Hyderabad, AP, India, depositata in Collectione Culturarum (ARS), Peoria, IL. Description of P. cecembensis Bhadra, Sreenivas Rao & Shivaji sp. nov. Pichia cecembensis sp. nov. [ce.cem.ben’sis. N.L. fem. adj. cecembensis, pertaining to CCMB, arbitrary adjective FEMS Yeast Res xx (2007) 000–000 5 Pichia cecembensis sp. nov. formed from the acronym of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India]. After 3 days in yeast-malt broth at 28 1C, cells are elongate (5.0–8.5  2.5–4.5 mm) and occur singly, in pairs or in groups (Fig. 2a). Budding is multilateral. After 1 month at 28 1C, sediment is present. On YM agar medium after 1 month at 28 1C, the streak culture is butyrous, white or cream and semi-glossy, with an entire to slightly undulating margin. Pseudohyphae (simple) formation observed on corn-meal agar plates after 15 days of incubation at 28 1C (Fig. 2b and c). Unconjugated, persistent asci containing one or two, rough or smooth, round ascospore(s) are seen (a) after 15 days of incubation at 28 1C in corn-meal agar and Potato Dextrose Rose Bengal agar (Fig. 2c–e). Ascospores were also visible as brightly fluorescent round structures using DAPI. The major ubiquinone of YS16T is Q7. Ferments D-glucose but not D-galactose, maltose, sucrose, lactose, D-cellobiose, raffinose and D-xylose. Assimilates D-glucose, D-arabinose, glycerol, ribitol, galactitol (delayed), succinate and citrate as the sole carbon source, but not D-galactose, L-sorbose, D-xylose, L-arabinose, L-rhamnose, sucrose, maltose, trehalose, cellobiose, salicin, lactose, raffinose, melezitose, erythritol, glucitol, D-mannitol, D-glucuronate, DL-lactate, propane-1,2-diol and butane-2,3diol. Uses ethylamine (delayed), L-lysine and cadaverine as the sole nitrogen source, but not nitrate, nitrite, creatinine, D-glucosamine and imidazole. Grows in vitamin-free medium and 60% glucose-containing medium. Urea is not hydrolyzed. Diazonium blue B reaction is negative and is sensitive to 0.01% and 0.1% cycloheximide. Growth at 42 1C is positive. Mol% G1C content of DNA is 42.5 (  0.5). The type strain YS16T (= NRRL Y-27985T = JCM13873T = CBS10445T) was isolated from papaya fruit (Carica papaya L.) collected from Hyderabad, India. Acknowledgements (c) (b) We thank the Council of Scientific and Industrial Research, Government of India, New Delhi, for funding. We also acknowledge Professor C. Kurtzman, ARS Culture Collection, for providing us with the type strains for analysis and for his valuable comments, which led to substantial improvement of the manuscript. References (d) (e) Fig. 2. Differential interference contrast micrograph of Pichia cecembensis sp. nov. YS16T grown at 28 1C for 48 h on yeast malt agar (a); pseudohyphae on corn-meal agar (b, c); ascospores on corn-meal agar (c) and Potato Dextrose Rose Bengal agar (d, e). The single arrow indicates an ascospore, and the double arrow indicates pseudohyphae. The bars represent 7 mm. FEMS Yeast Res xx (2007) 000–000 Bai FY, Zhao JH, Takashima M, Jia JH, Boekhout T & Nakase T (2002) Reclassification of the Sporobolomyces roseus and Sporidiobolus pararoseus complexes, with the description of Sporobolomyces phaffii sp. nov. Int J Syst Evol Microbiol 52: 2309–2314. Boidin J, Pignal MC & Besson M (1965) Le genre Pichia sensu lato (quatrieme contribution). Bull de la Societe Mycol de France 81: 566–606. Collins MD, Pirouz T, Goodfellow M & Minnikin DE (1977) Distribution of menaquinones in actinomycetes and corynebacteria. J Gen Microbiol 100: 221–230. Correia A, Sampaio P, Almeida J & Pais C (2004) Study of molecular epidemiology of candidiasis in Portugal by PCR fingerprinting of Candida clinical isolates. J Clin Microbiol 42: 5899–5903. Fell JW, Boekhout T, Fonseca A, Scorzetti G & Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50: 1351–1371. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 6 Felsenstein J (1985) Confidence limits on phylogenies: an approach using bootstrap. Evolution 39: 783–791. James SA, Collins MD & Roberts IN (1996) Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora. Int J Syst Bacteriol 46: 189–194. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16: 111–120. Kumar S, Tamura K & Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5: 150–163. Kurtzman CP & Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5 0 end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35: 1216–1223. Kurtzman CP & Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73: 331–371. Leinberger DM, Schumacher U, Autenrieth IB & Bachmann TT (2005) Development of a DNA microarray for detection and identification of fungal pathogens involved in invasive mycoses. J Clin Microbiol 43: 4943–4953. Lin D, Wu LC, Rinaldi MG & Lehmann PF (1995) Three distinct genotypes within Candida parapsilosis from clinical sources. J Clin Microbiol 33: 1815–1821. Lu HZ, Jia JH, Wang OM & Bai FY (2004) Candida aspargi sp. nov., Candida diospyri sp. nov. and Candida qinlingensis sp. nov., novel anamorphic, ascomycetous yeast species. Int J Syst Evol Microbiol 54: 1409–1414. Makimura K, Murayama SY & Yamaguchi H (1994) Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 40: 358–364. Nakase T & Suzuki M (1986) Bullera megalospora, a new species of yeast forming large ballistospores isolated from dead leaves of Oryza sativa, Miscanthus sinensis, and Sasa sp. in Japan. J Gen Appl Microbiol 32: 225–240. Peter G, Tornai-Lehoczki J, Suzuki M & Dlauchy D (2005) Metschnikowia viticola sp. nov., a new yeast species from grape. Antonie van Leeuwenhoek 87: 155–160. c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved B. Bhadra et al. Saitou N & Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425. Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Mannual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Scorzetti G, Fell JW, Fonseca A & Statzell-Tallman A (2002) Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res 2: 495–517. Shivaji S, Reddy GSN, Raghavan PUM, Sarita NB & Delille D (2004) Psychrobacter salsus sp. nov. and Psychrobacter adeliensis sp. nov. isolated from fast ice from Adelie Land, Antarctica. Syst Appl Microbiol 27: 628–635. Shivaji S, Chaturvedi P, Suresh K, Reddy GS, Dutt CB, Wainwright M, Narlikar JV & Bhargava PM (2006) Bacillus aerius sp. nov., Bacillus aerophilus sp. nov., Bacillus stratosphericus sp. nov. and Bacillus altitudinis sp. nov., isolated from cryogenic tubes used for collecting air samples from high altitudes. Int J Syst Evol Microbiol 56: 1465–1473. Suh SO & Blackwell M (2004) Three new beetle-associated yeast species in the Pichia guilliermondii clade. FEMS Yeast Res 5: 87–95. Tavanti A, Davidson AD, Gow NAR, Maiden MCJ & Odds FC (2005) Candida orthopsilosis and Candida metapsilosis spp. Nov. to replace Candida parapsilosis group II and III. J Clin Microbiol 43: 284–292. Thanh VN, Hai DA & Lachance MA (2003) Issatchenkia hanoiensis, a new yeast species isolated from frass of the litchi fruit borer Conopomorpha cramerella Snellen. FEMS Yeast Res 4: 113–117. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F & Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882. Tournas VH & Katsoudas E (2005) Mould and yeast flora in fresh berries, grapes and citrus fruits. Int J Food Microbiol 105: 11–17. Yarrow D (1998) Methods for the isolation, maintenance and identification of yeasts. The Yeasts, A Taxonomic Study, 4th edn (Kurtzman CP & Fell JW, eds), pp. 77–100. Elsevier, Amsterdam. FEMS Yeast Res xx (2007) 000–000