Bot. Bull. Acad. Sin. (2005) 46: 223-229

WEI et al. — A new record of Pseudozyma from Taiwan

Pseudozyma antarctica in Taiwan: a description based on morphological, physiological and molecular characteristics

Yu-Hui WEI, Fwu-Ling LEE, Wen-Haw HSU, Shyue-Ru CHEN, Chien-Cho CHEN, Chiou-Yen WEN, Shie-Jea LIN, Wen-Shen CHU, Gwo-Fang YUAN, and Guey-Yuh LIOU*

Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, P.O. Box 246, Hsinchu, Taiwan 300, Republic of China

(Received June 14, 2004; Accepted January 6, 2005)

Abstract. The genus Pseudozyma is an anamorph that belongs to the Ustilaginales. Pseudozyma species are unusual yeast-like fungi and are most frequently isolated from plant materials. The Pseudozyma strain, BCRC 33871, was isolated from the flower of Albizia julibrissin in Taiwan. BCRC 33871 was identified as P. antarctica and described based on morphological, physiological, and molecular data. This discovery marked the first finding of this species in Taiwan. To ensure correct identification, the intraspecific variations of P. antartica were illustrated based on physiological characteristics and phylogenetic analysis of ITS1 and ITS2 rDNA sequences. Additionally, distinguishing characteristics of P. antartica and other related species were discussed.

Keywords: Morphology; New Record; Physiological characteristics; Pseudozyma; rDNA sequencing; Taiwan.

Introduction

Ustilaginales are economically important fungi. Ustilago is the largest genus, and most of its species parasitize monocotyledonous hosts. A number of anamorphs of Ustilaginales have been classified in diverse genera, for example Candida, Pseudozyma, Sporobolomyces, Sterigmatomyces, Stephanoascus, and Trichosporon (Boekhout et al., 1998). These species have been reclassified into Pseudozyma Bandoni emend. Boekhout by morphological, physiological, biochemical, and molecular data (Boekhout, 1995). Phylogenetic analysis inferred from partial sequences of the 26S rDNA indicated that Pseudozyma species and Ustilaginales parasitizing grasses form a monophyletic group. Furthermore, the type species of Pseudozyma, P. prolifica, is most closely related to Ustilago maydis. Accordingly, Pseudozyma species are anamorphs of Ustilaginales that parasitize grasses (Boekhout, 1995; Boekhout et al., 1995; Begerow et al., 2000).

Pseudozyma species are unusual yeast-like fungi and are most frequently isolated from plant materials, such as leaves, flowers, and fruits (Boekhout and Fell, 1998; Trindade et al., 2002). Seven species are listed in the genus Pseudozyma by Boekhout and Fell (1998): P. antarctica, P. aphidis, P. flocculosa, P. fusiformata, P. prolifica, P. rugulosa, and P. tsukubaensis. Recently, Sugita et al. (2003) isolated Pseudozyma strains from patient blood in Thailand and named two new species, P. parantarctica and P. thailandica.

A project involving the screening of osmophilic yeast-like fungi in Taiwan isolated one species of Pseudozyma which was identified as P. antarctica. This discovery marked the first finding of this species in Taiwan. Assessing identity and diversity is extremely crucial in identifying of P. antarctica for industrial procedures. This investigation aimed to (i) describe the local strain, and (ii) analyze the intraspecific and interspecific variation of P. antarctica using a polyphasic approach.

Materials and Methods

Isolation of BCRC 33871

The Pseudozyma isolate, No. 176, was isolated from a flower of Albizia julibrissin in Taiwan using a slight modification of the method developed by Hajny et al. (1964). The collected samples were inoculated into 20-ml flasks containing 5 ml of a medium comprising 40% glucose and 1% yeast extract and were incubated at 30°C under shaking for six days. The cultures were streaked on plate medium containing 20% glucose, 1% yeast extract, and 2% agar. Pure cultures were established by picking and transferring individual colonies to the same medium. The culture was deposited as BCRC 33871 in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute.

Morphological, Physiological and Biochemical Characteristics

The morphological and physiological characteristics were examined using the methods described by Yarrow (1998). Moreover, BCRC 33871 was compared with the type

*Corresponding author. Tel: 886-3-5223191-763; Fax: 886-3-5214016; E-mail: gyl@firdi.org.tw

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

strains of P. antarctica, BCRC 33858, and published descriptions of related species (Boekhout and Fell, 1998; Sugita et al., 2003).

Preparation of Genomic DNA

The culture was inoculated into YM broth (DIFCO 0712) and harvested after incubation at 20°C for ten days. Additionally, DNA for PCR was extracted using the Chelex method (Chen, 1998).

PCR Amplification and Direct DNA Sequencing of rDNA

The internal transcribed spacer (ITS) regions of the rRNA gene were defined using primers ITS5 (5´-GGAAGTAAAAGTCGTAACAAGG) and ITS4 (5´-TCCTCCGCTTATTGATATGC) (White et al., 1990). The reaction was performed in a GeneAmp PCR system 9700 (Applied Biosystems) using 30 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min with an initial denaturation at 94°C for 5 min before a cycling and a final extension at 72°C for 7 min. Sequencing reactions were performed employing the ABI PRISM^TM BigDye^TM Terminator, v 3.0 Ready Reaction Cycle Sequencing Kit as directed by the manufacturer. Finally, the PCR products were sequenced using the ABI PRISM^TM 3700 DNA analyzer.

Molecular Phylogenetic Analysis

The BCRC 33871 sequence was compared with those of Pseudozyma antarctica and other related species (Table 1). Sequences were aligned using CLUSTAL W (Thompson et al., 1994). Additionally, the alignment of all sequences was visually checked and optimized, and alignment gaps were treated as an additional character state. A phylogenetic analysis was conducted, using a neighbor joining program from the package PHYLIP 3.5C

(Felsenstein, 1993). The parsimony program implemented in PHYLIP was used to compare the tree topologies. For neighbor-joining analysis, the distances between sequences were calculated using the Maximum Likelihood model (Kishino and Hasegawa, 1989). Finally, a bootstrap analysis was performed with 1,000 replications. Trees were viewed using TreeView (Page, 1996).

Results and Discussion

Morphological and Physiological Description of BCRC 33871

Pseudozyma antarctica (S. Goto, Sugiyama & Iizuka) Boekhout, 1995, J. Gen. Appl. icrobiol. 41: 359-366. (Figure 1)

=Sporobolomyces antarcticus S. Goto, Sugiyama & Iizuka, 1969, Mycologia 61: 759.

Growth on 5% malt extract agar: After five days at 20°C, cells are cylindrical to fusiform, with variable size, 5.0-8.1 × 1.8-2.4 µm. Conidiogenesis is polar on short denticles and has sympodial proliferation. Hyphae are abundant, 1.5-2.4 µm width and with sterigmata on which fusiform blastoconidia are formed. Colonies are dimorphic, smooth to somewhat irregularly furrowed, pale cream-white, and with the margin fringed.

Slide culture on 5% malt extract agar: After five days at 20°C, cells are cylindrical to fusiform, with variable size, 3.6-16.9 × 1.5-3.1 µm. Conidiogenesis is polar on short denticles and with sympodial proliferation. Hyphae are abundant, 1.5-2.2 µm wide and with sterigmata on which fusiform blastoconidia are formed.

Habitat. flower of Albizia julibrissin.

Specimen examined. TAIWAN. Chanhua, Teinwei, April 17, 1998. (BCRC 33871= isolation no. 176)

Fermentation of carbon compounds: see Table 2

Figure 1. Pseudozyma antarctica BCRC 33871 growth on 5% malt extract agar at 20°C for 5 days. A, hyphae and chains of blastoconidia; B, blastoconidia, scale bar = 5 µm.

WEI et al. — A new record of Pseudozyma from Taiwan

Assimilation of carbon compounds and nitrogen compounds: see Table 2.

rDNA Sequence Analysis

The phylogenetic tree presented here indicated a close relationship between Pseudozyma and Ustilago. This result was in accordance with Boekhout (1995) and Begerow et al. (2000). On the other hand, one Sporisorium species, S. aegypticum, was assigned to the Ustilago-Pseudozyma clade. However, Stoll et al. (2003) illustrated the basal position of S. aegypticum to Ustilago.

In our phylogram, BCRC 33871 was clustered in the clade of Pseudozyma antartica (Figure 2). The sequences of ITS regions of BCRC 33871 were identical to the type strain of P. antarctica, JCM 10317^T (CBS 214.83), and patients' blood isolates, M9935 & M9954. BCRC 33871 and P. antarctica CBS 516.83, isolated from unpolished rice in Japan, displayed 99.8% similarity. Sugita et al. (1999) found that conspecific strains have a less than 1% nucleotide difference in their ITS regions, after comparing the nuclear DNA-DNA hybridization. The overall ITS sequence similarity between strains of P. antarctica was more than 99%.

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

WEI et al. — A new record of Pseudozyma from Taiwan

Figure 2. Neighbor-joining tree of strains of Pseudozyma antarctica and related species inferred from 587 nucleotides from ITS1 and ITS2 rDNA. Topology was rooted with Sporisorium scitamineum. Numbers given on branches indicate the confidence level from a 1,000-replicate bootstrap sampling. (Frequencies below 50% are not indicated.)

On the other hand, the ITS sequences displayed a less than 97% similarity between P. antarctica and known Pseudozyma species.

The type strain of P. antarctica was isolated from lake sediment in Antarctica and was initially classified in the genus Sporobolomyces based on morphological and physiological properties. However, morphologically it differs from other Sporobolomyces species by lacking ballistospores (Goto et al., 1969; Boekhout, 1995). Recent studies have suggested a closer relationship to the genus Pseudozyma (Boekhout, 1995; Begerow et al., 2000). More recently, the strains of P. antarctica were isolated from unpolished new crop rice in Japan (Boekhout and Fell, 1998), various fruits in Brazil (Trindade et al., 2002), blood from a patient in Thailand (Sugita et al., 2003), and the

Albizia julibrissin flower in Taiwan. These strains from Antarctica, Japan, Thailand, and Taiwan display highly similar physiological characteristics (Table 2) and sequences in the ITS regions of their rDNA (Figure 2). Therefore, based on the morphological, physiological, and molecular data, BCRC 33871 was identified as P. antarctica.

Based on the phylogenetic analysis inferred from partial sequences of the 18S rDNA (Avis et al., 2001) and D1/D2 26S rDNA sequences (Sugita et al., 2003), P. antarctica is closely related to P. aphidis and P. rugulosa. Pseudozyma aphidis was proposed to be conspecific with P. antarctica (Kurtzman, 1990). However, based on DNA-DNA hybridization, partial 26S rDNA sequences and physiological differences, P. aphidis was considered a separate species (Boekhout and Fell, 1998). Furthermore, analysis

Botanical Bulletin of Academia Sinica, Vol. 46, 2005

analysis is useful for the identification of Pseudozyma species.

Acknowledgements. The authors would like to thank the Ministry of Economic Affairs of the Republic of China for financially supporting this research under Contract No. MOEA92-EC-17-A-17-R7-0525. Dr Li-Ling Liaw and Ms Ing-Er Hwang are appreciated for their assistance in DNA sequencing.

Literature Cited

Avis, T.J., S.J. Caron, T. Boekhout, R.C. Hamelin, and R.R. Belanger. 2001. Molecular and physiological analysis of the powdery mildew antagonist Pseudozyma flocculosa and related fungi. Phytopathology 91: 249-254.

Begerow, D., R. Bauer, and T. Boekhout. 2000. Phylogenetic placements of ustilaginomycetous anamorphs as deduced from nuclear LSU rDNA sequences. Mycol. Res. 104: 53-60.

Boekhout, T., R.J. Bandoni, J.W. Fell, and K.J. Kwon-Chung. 1998. Discussion of teleomorphic and anamorphic genera of heterobasidiomycetous yeasts. In C.P. Kurtzman, and J.W. Fell (eds.), The Yeasts: a Taxonomic Study. Elsevier Science Publish, Amsterdam, pp. 609-625.

Boekhout, T. and J.W. Fell. 1998. Pseudozyma Bandoni emend. Boekhout and a comparison with the yeast state of Ustilago maydis (de Candolle) Corda. In C.P. Kurtzman, and J.W. Fell (eds.), The Yeasts: a Taxonomic Study. Elsevier Science Publish, Amsterdam, pp. 790-797.

Boekhout, T. 1995. Pseudozyma Bandone emend. Boekhout, a genus for yeast-like anamorphs of Ustilaginales. J. Gen. Appl. Microbiol. 41: 359-366.

Boekhout, T., J.W. Fell, and K. O'Donnell. 1995. Molecular systematics of some yeast-like anamorphs belonging to the Ustilaginales and Tilletiales. Stud. Mycol. 38: 175-183.

Chen, C.J. 1998. Morphological and Molecular Studies in the Genus Tremella. Bibliotheca Mycologica Band 174. J. Cramer, Stuttgart, pp. 10.

Goto, S., J. Sugiyama, and H. Iizuka. 1969. A taxonomic study of antarctic yeasts. Mycologia 61: 748-774.

Felsenstein, J. 1993. PHYLIP (Phylogenetic Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, Washington.

of the ITS regions supported the conclusion of Boekhout and Fell (1998) as did the findings of Sugita et al. (2003).

Comparison of the Physiological Characteristics of P. antarctica and Related Species

Table 3 lists the physiological characteristics of P. antarctica and related species. Carbohydrates are not fermented in the genus Pseudozyma (Boekhout and Fell, 1998). Therefore, assimilation tests and other growth characteristics are important for the identification of Pseudozyma species. BCRC 33871 differs from P. antarctica JCM 10317^T (CBS 214.83) because it grows at 37°C and assimilates melibiose and rhamnose. For P. antarctica, growth at 37°C and assimilation of rhamnose are variable, based on the description of the type strain and on the isolate from Japan (Boekhout and Fell, 1998). Melibiose is not assimilated, based on the description of the type strain and other isolates from Thailand and Japan. However, the strain found in Taiwan, BCRC 33871, can assimilate melibiose, and therefore the character of melibiose assimilation in P. antarctica is variable.

Moreover, P. antarctica displays similar physiological characteristics in the assimilation to P. aphidis and P. rugulosa. However, P. antarctica differs from P. aphidis in assimilation of ethanol and saccharate, and differs from P. rugulosa in assimilation of lactose and saccharate. In comparing physiological characteristics, the assimilation of lactose, ethanol, and saccharate are key to circumscribing P. antarctica and closely related species.

Pseudozyma antarctica is a recently characterized yeast-like fungus capable of bioconverting n-alkalines into glycolipid biosurfactants (Kitamoto et al., 2001). It is also capable of b-glucosidase activity and mycocin production (Trindade et al., 2002). When selecting fungi for a specific function, it is essential to accurately classify and identify the isolates. In conclusion, this investigation indicates that P. antarctica can be genetically and physiologically differentiated from other closely related species. On the other hand, the conidial structures of Pseudozyma species exhibit poor differentiation and have a highly variable shape and size. Therefore, application of ITS rDNA sequence

WEI et al. — A new record of Pseudozyma from Taiwan

Hajny, G.J., J.H. Smith, and J.C. Garver. 1964. Erythritol production by yeast-like fungus. Appl. Microbiol. 12: 240-246.

Kishino, H. and M. Hasegawa. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from dna sequence data and the branching order in Hominoidea. J. Mol. Evol. 29: 170-179.

Kitamoto, D., T. Ikegami, G.T. Suzuki, A. Sasaki, Y. Takeyama, Y. Idemoto, N. Koura, and H. Yanagishita. 2001. Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma (Candida antarctica). Biotechnol. Lett. 23: 1709-1714.

Kurtzman, C.P. 1990. DNA relatedness among species of Sterigmatomyces and Fellomyces. Int. J. Syst. Bacteriol. 40: 56-59.

Page, R.D.M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: 357-358.

Roux, C., T. Almaraz, and G. Durrieu. 1998. Phylogeny of fungi responsible for smut of plants based on ITS sequence analysis. C. R. Acad. Sci. III, Sci. Vie 321: 603-609.

Stoll, M., M. Piepenbring, D. Begerow, and F. Oberwinkler. 2003. Molecular phylogeny of Ustilago and Sporisorium species (Basidiomycota, Ustilaginales) based on internal transcribed spacer (ITS) sequences. Can. J. Bot. 81: 976-984.

Sugita, T., M. Takashima, N. Poonwan, N. Mekha, K. Malaithao,

B. Thungmuthasawat, S. Prasarn, P. Luangsook, and T. Kudo. 2003. The first isolation of Ustilaginomycetous anamorphic yeasts, Pseudozyma species, from patients' blood and a description of two new species: P. parantarctica and P. thailandica. Microbiol. Immunol. 47: 183-190.

Sugita, T., A. Nishikawa, R. Ikeda, and T. Shinoda. 1999. Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J. Clin. Microbiol. 37: 1985-1993.

Thompson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.

Trindade, R.C., M.A. Resende, C.M. Silva, and C.A. Rosa. 2002. Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits. System. Appl. Microbiol. 25: 294-300.

White, T.J., T.D. Bruns, S.B. Lee, and J.W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White (eds.), PCR Protocols: a Guide to Methods and Applications. Academic Press, San Diego, California, pp. 315-322.

Yarrow, D. 1998. Methods for the isolation, maintenance, classification and identification of yeasts. In C.P. Kurtzman, and J.W. Fell (eds.), The Yeasts: a Taxonomic Study. Elsevier Science Publish, Amsterdam, pp. 75-100.