Antonie van Leeuwenhoek (2010) 98:475–481 DOI 10.1007/s10482-010-9463-z ORIGINAL PAPER Candida asiatica sp. nov., an anamorphic ascomycetous yeast species isolated from natural samples from Thailand, Taiwan, and Japan Savitree Limtong • Rungluk Kaewwichian • Somjit Am-In • Takashi Nakase • Ching-Fu Lee Wichien Yongmanitchai • Received: 2 February 2010 / Accepted: 26 May 2010 / Published online: 12 June 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Six yeast strains of a novel anamorphic yeast species were isolated from natural samples collected in Thailand (RV60T and LYSM9), Taiwan (SC5L04 and GE19S05), and Japan (JCM 11058 and JCM 11059). Analysis of the D1/D2 domain of the large subunit rRNA gene sequences revealed that the sequences of five strains (LYSM9, SC5L04, GE19S05, JCM 11058, and JCM 11059) were identical and differed from the sequence of strain RV60T by only one nucleotide substitution. The closest recognized species in terms of pairwise sequence similarity was Candida abiesophila, but S. Limtong (&)
R. Kaewwichian
S. Am-In
W. Yongmanitchai Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand e-mail: fscistl@ku.ac.th S. Am-In Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand T. Nakase NITE Biological Resource Center, Department of Biotechnology, National Institute of Technology and Evolution, Chiba, Japan C.-F. Lee Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan the level of nucleotide substitution (14.2–14.3%) was sufficient to justify the description of a separate species. Phenotypic characteristics of the six strains were almost the same except for two strains from Japan, which showed the ability to ferment glucose and grew in the presence of 10% NaCl and 5% glucose while the others did not. The phenotypic characteristics of the six strains included proliferation by multilateral budding, absence of ascospores, arthrospores, and ballistospores, and negative Diazonium blue B and urease tests. The major ubiquinone was Q-7. On the basis of the above findings, the six strains were assigned to a single novel species of Candida, for which the name Candida asiatica sp. nov. is proposed. The type strain is RV60T (BCC 25966T = NBRC 103863T = CBS 10863T). Keywords Candida asiatica sp. nov.
Ascomycetous yeast
New yeast species
Thailand
Taiwan
Japan Introduction Yeasts of the genus Candida, one of the largest genera in terms of number of species, are widely distributed in nature and are polyphyletic. Species of this genus have been isolated from various sources in terrestrial and aquatic habitats. In recent years, many novel Candida species have been described from 123 476 Antonie van Leeuwenhoek (2010) 98:475–481 strains isolated in Thailand, Taiwan, and Japan from various natural samples, e.g., soils, estuarine waters, sediments in mangrove forests, insect frasses, and beetle galleries (Boonmak et al. 2009; Endo et al. 2008; Limtong et al. 2007b, 2008, 2010; Liu et al. 2008; Nakase et al. 2009). Phylogenetic analysis showed that these novel species are distributed in various clades, e.g., Ambrosiozyma, Metschnikowia, Pichia, Saturnispora, Wickerhamomyces, and Yarrowia. In the course of investigations of yeasts from estuarine water of a mangrove forest in Laem Son National Park, Ranong Province, Thailand, various yeast species, both known and novel species (e.g., Candida andamanensis, C. laemsonensis, and C. ranongensis), were found (Am-In et al. 2010). A strain of one novel species was found to be the same as a strain obtained during an investigation of yeasts in soil from Phu Ruea National Park, Loei Province, from where one novel species was previously described as Geotrichum phurueaensis (Kaewwichian et al. 2010). These two strains of the novel species were found to belong to the same species as two strains each from Taiwan and Japan. The two strains from Taiwan were obtained during investigations of yeasts from soils and plants in Taiwan, in which various novel strains were discovered, e.g., C. dajiaensis, C. jianshihensis, C. sanyiensis, and C. yuanshanicus (Liu et al. 2008). The two strains isolated from Japan were from two samples of the same kind of fruit. In the present study, a novel species of the genus Candida, Candida asiatica sp. nov., is proposed for these six strains. Materials and methods Yeast strains Six strains of the proposed novel species are shown in Table 1. Two strains (RV60T and LYSM9) were isolated from Thailand. Strain RV60T was isolated from estuarine water of a mangrove forest by membrane filtration as described previously by Limtong et al. (2007b). Two hundred milliliters of estuarine water was filtered through a 0.8-lm-pore-size membrane filter, and the filter was placed on acidified yeast extract malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, pH 3.7– 3.8) supplemented with 0.025% sodium propionate and 200 mg l-1 chloramphenicol, and incubated at 28–30°C until colonies appeared. Strain LYSM9 was obtained from forest soil by an enrichment technique following the method of Limtong et al. (2007a) using acidified YM broth supplemented with 0.025% sodium propionate and 200 mg l-1 chloramphenicol, and shake flask cultivation at 28–30°C. The enriched culture was streaked onto acidified YM agar and Table 1 Strains of Candida asiatica sp. nov. investigated in this study Strain Sample collection GenBank accession no. of Source D1/D2 Locality Month and year RV60T (BCC 25966T = NBRC 103863T = CBS 10863T) AB334112 Estuarine water Mangrove forest, Laem Son National Park, Ranong Province, Thailand April 1999 LYSM9 AB498994 Soil Forest, Phu Ruea National Park, Loei Province, Thailand July 2007 GE19S05 FJ527125 Soil SC5L04 EF653946 Leaf of Angiopteris lygodiifolia Rosenst, family Marattiaceae Sea coast mountain, Fongbin, Hualein, Taiwan Mountain, Dasi, Taoyuan, Taiwan June 2008 October 2006 JCM 11058 AB543318 Souring fruit of Ficus carica, Fig orchard, Mitsuguchi, Yasuura-cho, family Moraceae (sample 1) Toyota-gun, Hiroshima Prefecture, Japan October 1993 JCM 11059 AB543319 Souring fruit of Ficus carica, Fig orchard, Mitsuguchi, Yasuura-cho, family Moraceae (sample 2) Toyota-gun, Hiroshima Prefecture, Japan October 1993 123 Antonie van Leeuwenhoek (2010) 98:475–481 incubated at 28–30°C until colonies appeared. Colonies were picked on the basis of different colony morphology and purified by streaking on YM agar. Two strains from Taiwan consisted of strain SG5L04, which was isolated from leaf of Angiopteris lygodiifolia Rosenst of the family Marattiaceae, and strain GE19S05, which was obtained from soil. They were isolated following the method described by Lee et al. (2008) as follows. Approximately 1 g soil or pieces of leaves was diluted in 9 ml sterilized water, 0.1 mL of appropriate dilution was spread on an acidified (pH 3.5) YM agar plate, and the plate was incubated at 24°C for 3 days. Representative colonies were picked and purified by streaking on YM agar. Strains JCM 11058 and JCM 11059 were isolated from two samples of two souring fruits of Ficus carica of the family Moraceae in Japan. Samples obtained from natural openings at the top of fruit after ripening were streaked on YM agar supplanted with 100 mg l-1 chloramphenicol and incubated at 25°C for 3–4 days. Among the many colonies that appeared, representative colonies were picked and purified by streaking on YM agar. DNA sequencing and phylogenetic analysis The sequence of the D1/D2 domain of the large subunit (LSU) rRNA gene was determined from polymerase chain reaction (PCR) products of genomic DNA. Methods for DNA isolation, amplification of the D1/ D2 domain of the LSU rRNA gene by PCR, and purification of PCR product were described previously (Limtong et al. 2007a). The PCR product was submitted to Macrogen Inc. (Korea) for sequencing with primers NL1 and NL4. The sequences were compared pairwise using a BLASTN homology search program (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 maximum-parsimony and neighbor-joining methods using MEGA version 4 (Tamura et al. 2007). 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 477 standard methods described by Yarrow (1998). Ascospore formation was determined by cultivation of individual strains or strains paired on YM agar, 5% malt extract agar, Fowell’s acetate agar, corn meal agar, and Gorodkowa agar at 15°C or 25°C and examination at 3- to 7-day intervals for 6 weeks. Assimilation of nitrogen compounds was investigated on solid media with starved inocula following the method of Nakase and Suzuki (1986). Vitamin requirement was determined according to the method of Komagata and Nakase (1967). Growth at various temperatures was determined by cultivation in yeast extract malt extract (YM) broth (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 1% glucose). Ubiquinones were extracted from cells cultivated in 500-mL Erlenmeyer flasks containing 250 mL 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 purification according to the methods described by Yamada and Kondo (1973) and Kuraishi et al. (1985). Isoprenologues were identified by high-performance liquid chromatography (HPLC) as described previously (Limtong et al. 2007a). Results and discussion Sequences and phylogenetic analysis Analysis of the D1/D2 domain of the large subunit rRNA gene sequences revealed that the sequences of five strains (LYSM9, SC5L04, GE19S05, JCM 11058, and JCM 11059) were identical and differed from the sequence of strain RV60T by only 1 nucleotide substitution in 545 nucleotides (nt). The species closest to them in terms of pairwise sequence similarity was Candida abiesophila, but with 14.2– 14.3% nucleotide substitution (78–79 nucleotide substitutions and 6 gaps out of 551 nt). According to Kurtzman and Robnett (1998), yeast strains showing nucleotide substitutions of greater than 1% in the D1/D2 domain of the LSU rRNA gene are usually different species. Therefore, we concluded that the six strains (RV60T, LYSM9, SC5L04, GE19S05, JCM 11058, and JCM 11059) represented a single, novel species. The phylogenetic trees based on the sequence of the D1/D2 domain of the LSU rRNA gene constructed by both maximum-parsimony 123 478 and neighbor-joining methods further demonstrated that all six strains clustered together in a branch separated from the other described related species (data not shown). However, the trees obtained by maximum-parsimony and by neighbor-joining programs using various outgroup species, i.e., Kregervanrija delftensis, Saccharomyces cerevisiae, and Saturnispora ahearinii, were not congruent. This may be due to the fact that the relationship of the novel species with its closest relatives is very distant and sequences with large divergences were included in the phylogenetic analysis. Therefore, phylogenetic placement of the novel species was not possible. Antonie van Leeuwenhoek (2010) 98:475–481 On the basis of morphological, biochemical, physiological, and chemotaxonomic characteristics, and the sequence analysis of the D1/D2 domain of the LSU rRNA gene, we concluded that the six strains represent a single novel species of Candida, for which the name C. asiatica sp. nov. is proposed (MB 518136). C. asiatica can be distinguished from C. abiesophila, its closest relative in terms of pairwise sequence similarity, by only a few characteristics, including its ability to assimilate ribitol and citric acid and its growth in vitamin-free medium. Members of the genus Candida have been isolated from various habitats. The six strains of the novel species proposed in this study were discovered from four habitats including water, soil, leaf, and fruit, in both aquatic and terrestrial environments. Also, they were found in three countries in Asia. This implies that the novel species is distributed in various habitats and geographic zones. fermentatur (variabile) at non sucrosum, maltosum, lactosum, trehalosum nec raffinosum. Assimilantur D-glucosum, D-xylosum, ethanolum, glycerolum, ribitolum, D-mannitolum, D-glucitolum, D-glucono-d-lactonum, acidium DL-lacticum, acidum succinicum, acidum citricum, ethylaminum, L-lysinum et cadaverinum. Non assimilantur D-galactosum, L-sorbosum, sucrosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, N-acetyl-D-gluosaminum, methanolum, erytritolum, galactitolum, a-methyl-D-glucosidum, salicinum, acidum D-gluconicum, acidum D-glucuronicum, acidum D-galacturonicum, acidum 2-keto-D-gluconicum, acidum 5-keto-D-gluconicum, inositolum, nitrosum nec nitricum. Vitamina externa ad crescentiam necessaria non sunt. Crescit in 50% glucosum et 10% NaCl/5% glucosum (variabile). Non crescit in 60% glucosum, 16% NaCl/5% glucosum et 0.01% cycloheximido. Crescere potest in temperatura 20, 25 et 30°C et non crescit in temperatura 35°C. Amylum et acidum non formatur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur. Ubiquinonum majus: Q-7. Holotypus: Stirps RV60T isolatus aqua, Ranong Provincia, Thailandia. Cultura et conservatus in Collectionie Culturarum in BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailandia ut BCC 25966T; NITE Biological Resources Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Chiba, Japonia conservatus ut NBRC 103863T et Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands ut CBS 10863T. Latin diagnosis of C. asiatica Limtong, Kaewwichian, Am-In, Nakase and Lee sp. nov. Description of C. asiatica Limtong, Kaewwichian, Am-In, Nakase and Lee sp. nov. In medio liquido: ‘YM’, post dies 3 ad 25°C cellulae ovoideae, ellipsoideae, aut cylindricae (2–6 9 3–9 lm), singulae aut binae, per germinationem multipolarem reproducentes. In agaro ‘YM’, post dies 3 ad 25°C, cultura butyrosa, cremea, semi-nitens, glabra et margo glabra vel undulata. Pseudohyphae formantur nec hyphae non formantur. Ascosporae non formantur. Growth in YM broth: After 3 days at 25°C, cells are ovoidal, ellipsoidal, and cylindrical (2–6 9 3–9 lm), and occur singly or in pairs (Fig. 1a). Budding is multilateral. Growth on YM agar: After 3 days at 25°C, the streak culture is butyrous, cream-colored, semi-glistening, with a smooth surface, and has an entire to undulate margin. Pseudohyphae are formed (rare in the case of strains JCM 11058 and JCM 11059 Species identification and ecology 123 D-Glucosum D-galactosum, Antonie van Leeuwenhoek (2010) 98:475–481 479 Assimilation D-Glucose T Fig. 1 Candida asiatica sp. nov. (RV60 ). a Budding cells in YM broth after 3 days at 25°C. b Pseudohyphae formed on potato dextrose agar after 7 days at 25°C. Scale bar = 10 lm and not well developed in the case of strains LYSM9, SC5L04, GE19S05, JCM 11058, and JCM 11059), but true hyphae are not formed in slide culture on potato dextrose agar after 7 days at 25°C (Fig. 1b). No ascospores are produced from individual strains or strains paired on YM agar, 5% malt extract agar, Fowell’s acetate agar, corn meal agar, or Gorodkowa agar after 6 weeks at 15°C or 25°C. D-Galactose ? - L-Sorbose - Sucrose - Maltose - Cellobiose - Trehalose - Lactose - Melibiose - Raffinose - Melezitose - Inulin - Soluble starch - D-Xylose ? L-Arabinose - D-Arabinose D-Ribose - L-Rhamnose - N-Acetyl-D-glucosamine - Methanol - Ethanol ? Glycerol ? Erythritol - Ribitol ? Galactitol - D-Mannitol ? D-Glucitol ? Methyl-a-D-glucoside - Salicin - D-Gluconic acid D-Glucuronic acid - D-Galacturonic acid D-Glucono-d-lactone ? 2-Keto-D-gluconic acid - 5-Keto-D-gluconic acid - DL-Lactic ? acid Succinic acid ? Citric acid ? Inositol - Nitrate - D-Galactose ?/- Nitrite - Sucrose - Ethylamine ? Maltose - L-Lysine ? Lactose - Cadaverine ? Trehalose - Additional growth tests Raffinose - Vitamin-free medium Fermentation D-Glucose ? 123 480 Antonie van Leeuwenhoek (2010) 98:475–481 0.01% Cycloheximide - 50% Glucose 60% Glucose ? - 10% NaCl/5% glucose ?/- 16% NaCl/5% glucose - Growth at 20°C ? Growth at 25°C ? Growth at 30°C ? Growth at 35°C - Acid formation from glucose - Diazonium blue B color reaction - Urease - Amyloid production - Major ubiquinone Q-7 Holotype: RV60T is the holotype of C. asiatica. The strain was isolated from estuarine water collected from a mangrove forest in Laem Son National Park, Ranong Province, Thailand. 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 25966T; NITE Biological Resources Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Chiba, Japan, as NBRC 103863T and Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands as CBS 10863T. Etymology: The species epithet asiatica (a.sia.ti.ca N. L. fem. adj.) was chosen for this novel species, referring to Asia, from where the six strains of the novel species were isolated. Acknowledgements Authors would like to thank Dr. T. Sato, Mr. H. Nitta, and Dr. Suzuki for isolation of the two strains from Japan. Thanks are given to the students in the laboratory of Professor S. Limtong for their assistance in sample collection and carrying out some experiments. 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