J. Gen. Appl. Microbiol., 37, 423-429 (1991)

CANDIDA FRACI, A NEW SPECIES OF ANAMORPHIC YEAST ISOLATED FROM FERMENTING STRAWBERRY

MOTOFUMI SUZUKI,* TAKASHI NAKASE, ANDYOSHIMURA FUKAZAWA'

Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako 351-01, Japan 'Department of Microbiology, Yamanashi Medical College, Tamaho-cho, Yamanashi 409-38, Japan

(Received August 7, 1991)

A new anamorphic yeast species, Candida fragi Suzuki, Nakase et Fukazawa, is proposed for a strain isolated from fermenting strawberry that was formerly identified as C. sake, or C. natalensis. Candida fragi resembles Cnatalensis. C. oleophila, and C. sake but is clearly differen- tiated from these species by DNA-DNA relatedness, electrophoretic enzyme patterns, and the proton magnetic resonance spectra of cell wall . Practically, C. fragi is distinguished from C. natalensis by its inability to assimilate , mannoheptulose, and DL-; from C. sake by its inability to assimilate trehalose and a-methyl-D- glucoside and its ability to assimilate 5-ketogluconic acid and xylitol; and from C. oleophila by its inability to assimilate trehalose, to ferment , and to grow in 100 ppm of cycloheximide.

In 1962, one of the authors (T. Nakase) isolated a strain of anamorphic yeast from fermenting strawberry collected at a market in Tokyo. This strain showed characteristics similar to those of Torulopsis sake Saito et Oda, which was recog- nized in "The Yeasts, a Taxonomic Study," published in 1952 (4) but it could not be assigned to this species because it produced pseudomycelia. Later, with the transfer of TTsake into the genus Candida by van Uden and Buckley (10), it could be identified as Candida sake (Saito et Oda) van Uden et Buckley. In the 2nd edition of "The Yeasts, a Taxonomic Study," published in 1970, van Uden and Buckley (10) regarded Candida natalensis van der Walt et Tscheuschner, Candida vanriji Capriotti, Candida cloacae Komagata, Nakase et Katsuya, and Candida maltosa Komagata, Nakase et Katsuya as synonyms for C. sake because

* Address reprint requests to: Dr . Motofumi Suzuki, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-l, Wako 351-01, Japan.

423 424 SUZUKI, NAKASE, and FUKAZAWA VOL. 37 they showed similar physiological characteristics to one another. Meyer et al. (5), however, separated C. maltosa from C. sake on the basis of DNA-DNA reasso- ciation experiments. They regarded C. cloacae and Candida subtropicalis Nakase, Fukazawa et Tsuchiya as synonyms for C. maltosa; and C. natalensis, Candida tropicalis (Castellani) Berkhout var. lambica (Harrison) Diddens et Lodder and CC vanriji as those for C. sake. In 1975, Fukazawa et al. (1) carried out extensive taxonomic studies on C. sake and related species, including C. tropicalis var. lambica, C. maltosa, C. vanriji, Candida salmonicola Komagata et Nakase, C. cloacae, Candida parapsilosis (Ashford) Langeron et Talice var. intermedia van Rij et Verona, and C. natalensis. They classified these yeasts into four groups, I, II, III, and IV, based on the proton magnetic resonance (PMR) spectra of purified cell wall mannans and the sero- logical analysis of cell surface antigens. Groups I, II, III, and IV were represented by C. sake, C. maltosa, C. parapsilosis var. intermedia (=C: oleophila Montrocher), and C. natalensis, respectively. They suggested that the four groupings represented at least fourr species. According to them, C. natalensis should form an independent species comprising the two strains, the type strain and a strain isolated from strawberry as mentioned above. In 1980, Montrocher (6) suggested the validity of C. natalensis based on the serological characteristics of cytoplasmic protein antigens. This observation sup- ports the classification by Fukazawa et al. (1) but not that by Meyer et al. (5). In a previous paper (9), we confirmed the distinctness of C. natalensis from C. sake on the basis of DNA-DNA reassociation experiments, electrophoretic patterns of seven enzymes, and the proton magnetic resonance (PMR) spectra of cell wall mannans. Further, we suggested the distinctness of a strain isolated from straw- berry from C. natalensis. This strain showed little DNA relatedness to the type strains of C. natalensis and C. sake. The PMR spectra of mannans and the electrophoretic enzyme patterns correlated well with DNA relatedness. This paper proposes a new species, Candida fragi, for a strain isolated from strawberry that was formerly assigned to C. sake or C. natalensis, with several supplemental data confirming the validity of a new species.

MATERIALS AND METHODS

Strains examined. A strain (JCM 1791) to be described as a new species was isolated from fermenting strawberry by one of the authors (T. Nakase) at a market at Yodobashi in Tokyo, in May 1962, and has been maintained at the Central Research Laboratories, Ajinomoto, Co., Inc., Kawasaki, Japan, as AJ 4616. Candida natalensis JCM 1445T,C. oleophila JCM 1620T,C, sake JCM 2951T, and C. parapsilosis JCM 1785Tare employed for comparative study. Strains marked with a superscript "T" represent the type strain. Examination of taxonomic characteristics. Most of methods employed for the examination of morphological, physiological, and biochemical characteristics were 1991 Candida fragi sp. n. 425 those described by van der Walt and Yarrow (11). The maximum growth tem- perature was determined in YM broth (Difco Labs) using metal block baths. Vitamin requirements were investigated according to Komagata and Nakase (3). Assimilation of nitrogen compounds was investigated on solid media with starved inoculum as described in a previous paper (8). Chemotaxonomic characteristics were examined by the method previously reported (9). DNAs were extracted and purified according to the method described in the previous papers (7, 9). DNA-DNA hybridization was carried out using the membrane filter technique of Kaneko (2), as described by Nakase and Suzuki (7).

RESULTS AND DISCUSSION

Candida fragi Suzuki, Nakase et Fukazawa, sp. nov. In liquido "YM," post dies 3 ad 25°C, cellulae rotundae, ovales vel elong- atae, 2.0-5.2 X 4.4-11.2 ,am, singulae, binae, aut in catenis simplicibus aut ramosis. Pellicula et sedimentum formantur. In agaro "YM," post unum mensem ad 17°C, cultura cremea, butyracea, non nitida, elevata et rugosa, margine pilosa. Pseudo- mycelium bene formatur. Glucosum, sucrosum (tardum et exiguum) et maltosum (tardum et exiguum) fermentantur at non galactosum, lactosum, raffinosum nec melibiosum. Glucosum, galactosum, L-sorbosum, sucrosum, maltosum, cellobi- osum, melezitosum, amylum solubile (tardum et exiguum), D-xylosum, D-ribosum (lente), ethanolum (lente), glycerolum, ribitolum, D-mannotolum, D-glucitolum, salicinum (lente), glucono-b-D-lactonum, acidum 2-ketogluconicum, acidum 5 -ketogluconicum, acidum succinicum et acidum citricum assimilantur at non tre- halosum, lactosum, melibiosum, rafpinosum, inulinum, L-arabinosum, D- arabinosum, L-rhamnosum, erythritolum, galactitolum, a-methyl-D-glucosidum, acidum DL-lacticum, inositolum, mannoheptulosum, DL-glyceraldehydum nec xy- litolum. Kalium nitricum non assimilatur. Maxima temperatura crescentiae: 31- 32°C. Ad crescentiam biotinum necessarium est. Diazonium caeruleum B nega- tivum. Proportio molaris guanini + cytosini in acido deoxyribonucleinico: 37.1 mol% (ex Tm). Ubiquinonum majus: Q-9. Teleomorphosis ignota. Holotypus: Isolatus ex frago (Fragaria chiloensi var. ananassa), Tokyo, v. 1962, T. Nakase, JCM 1791 (originaliter ut AJ 4616) conservatur in collectionibus culturarum quas `Japan Collection of Microorganisms, Wako, Saitama 351-01,' sustentat.

Growth in YM broth: After 3 days at 25°C, cells are round, oval, or elongate, 2.0-5.2 X 4.4-11.2 ,um, single, in pairs or in simple or branched chains (Fig. 1). A dull creeping pellicle and a sediment are formed. Growth on YM agar: After one month at 17°C, the streak culture is cream-colored, butyrous, dull, raised, and rugose. The margin is fringed with pseudomycelia. Dalmau plate culture on corn meal agar: Well developed pseudomycelia are 426 SUZUKI, NAKASE, and FUKAZAWA VoL. 37

Fig. 1. Vegetative cells of Candid a fragi grown in YM broth for 3 days at 25°C. A, cells in pellicle; B, cells in sediment. Scale= l0,um.

Fig. 2. Pseudomycelia of Candida fragi produced on corn meal agar after 7 days at 25°C. Scale= 20,um. produced (Fig. 2). Fermentation: + Galactose + (slow and weak) Melibiose + (slow and weak) Assimilation of carbon compounds: Glucose + Glycerol + Galactose + Erythritol L- + Ribitol + 1991 Candida fragi sp, n. 427

Sucrose + Galactitol Maltose + D-Mannitol + + D-Glucitol + Trehalose a-Methyl-D-glucoside Lactose Salicin + (latent) Melibiose Glucono-o-D-lactone + Rafpinose 2-Ketogluconic acid + + 5-Ketogluconic acid + DL-Lactic acid Soluble + (slow and weak) Succinic acid + D- + Citric acid + L- Inositol D-Arabinose Mannoheptulose D- + (latent) DL-Glyceraldehyde L- Xylitol Ethanol + (latent) Assimilation of nitrogen compounds: Ammonium sulfate + Ethylamine hydrochloride + Potassium nitrate - L-Lysine hydrochloride + Sodium nitrite - Cadaverine dihydrochloride + Maximum growth temperature: 31-32°C. Vitamin required at 25°C: Biotin. Production of starch-like substances: Negative. Growth on 50% glucose-yeast extract agar: Negative. Liquefaction of gelatin: Negative. Hydrolysis of fat: Negative. Acid formation on chalk agar: Weakly positive. Growth in 10% sodium chloride plus 5 % glucose in yeast nitrogen base. Negative. Growth in the presence of 100 ppm of cycloheximide: Negative. Splitting of arbutin: Positive. Hydrolysis of urea: Negative. Color reaction with DBB: Negative. G + C content in nuclear DNA: 37.1 mol% (from Tm). Major ubiquinone: Q-9. composition of alkali-extracted purified via copper complex: . Type strain: JCM 1791 is the type strain of the species. This strain was isolated by T. Nakase from fermenting strawberry (Fragaria chiloensis var. ana- nassa) collected at a market at Yodobashi in Tokyo in May 1962. It has been maintained in the collection of Ajinomoto Co., Inc., Kawasaki, Japan, as Candida sp. AJ 4616 and sent to the Japan Collection of Microorganisms, Wako, Saitama 351-01. 428 SUZUKI, NAKASF, and FUKAZAWA Voi.. 37

Table 1. DNA relatedness among Candida oleophila, Candida natalensis, ('andida sake, and Candida ,fragi.

Etymology: The specific epithet of fragi (fra'gi. L. gen. ni fragi of strawberry, a common name for Fragaria chiloensis var. ananassa) refers to its habitat. In the previous paper (9), we clearly demonstrated the difference of C. sake, C, natalensis, and the strain JCM 1791, which was formerly identified as C sake or C. natalensis, on the basis of a DNA-DNA reassociation experiment, electrophoretic comparison of seven enzymes, and PMR spectra of cell wall mannans. In the present study, we carried out a DNA-DNA reassociation experiment between the strain JCM 1791 and the type strain of C. oleophila, because Montrocher (6) placed C. natalensis and C. oleophila in the same group based on serological studies on cytoplasmic proteins. As shown in Table 1, DNAs from C. natalensis and the strain JCM 1791 showed little relatedness to DNA from C. oleophila. Undoubtedly, the strain JCM 1791 represents a distinct species from C. sake, C. natalensis, and C. oleophila, for which a new species Candida fragi Suzuki, Nakase et Fukazawa is proposed. Practically, C. fragi is distinguished from C. natalensis by its inability to assimilate trehalose, mannoheptulose, and DL-glyceraldehyde, from C. sake by its inability to assimilate trehalose and a-methyl-D-glucoside and by its ability to assimilate 5-ketogluconic acid and xylitol, and from C. oleophila by its inability to assimilate trehalose, to ferment ga,lactose, and to grow in 100 ppm of cyclohexi- mide.

We express our sincere thanks to Dr. K. Yamada, Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan, who kindly supplied us the strain AJ 4616, to Prof. J. Sugiyama, Institute of Applied Microbiology, The University of Tokyo, for his help in the preparation of Latin diagnosis, and to Prof. K. Komagata, Tokyo University of Agriculture, for his invaluable suggestions for the present study.

REFERENCES

1) Fukazawa, Y., Nakase, T., Shinoda, T., Nishikawa, A., Kagaya, K., and Tsuchiya, T., Significance of cell wall structures on yeast classification: Proton magnetic resonance and serological and deoxyribonucleic acid characterization of Candida sake and related species. Int. J. Syst. Bacteriol., 1991 Candida fragi sp. n. 429

25, 304-314 (1975). 2) Kaneko, T., Kakusan. In Biseibutsu no Kagaku Bunrui Zikkenhou (Nucleic acids. In Methods in Chemotaxonomy of Microorganisms) (in Japanese), ed. by Komagata, K., Gakkai Shuppan Center, Tokyo (1982), p. 225-282. 3) Komagata, K. and Nakase, T., Reitoshokuhin no Biseibutsu ni kansuru kenkyu. V. Shihan reitoshokuhin yori bunri shita kobo no seijo (Microbiological studies on frozen foods. V. General properties of yeasts isolated from frozen foods) (in Japanese). Shokuhin Eiseigaku Zasshi, 8, 53- 57 (1967). 4) Lodder, J. and Kreger-van Rij, N. J. W., The Yeasts, a Taxonomic Study, North-Holland Publ. Co., Amsterdam (1952), p. 425-426. 5) Meyer, S. A., Anderson, K., Brown, R. E., Smith, M. Th., Yarrow, D., Mitchell, G., and Ahearn, D. G., Physiological and DNA characterization of Candida maltosa, a hydrocarbon-utilizing yeast. Arch. Microbiol., 104, 225-231 (1975). 6) Montrocher, R., Significance of immunoprecipitation in yeast taxonomy: Antigenic analysis of some species within the genus Candida. Cell. Mol. Biol., 26, 293-302 (1980). 7) Nakase, T. and Suzuki, M., Taxonomic studies on Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and related species. 1. Chemotaxonomic investigations. J. Gen. Appl. Microbiol., 31,49 -69 (1985). 8) Nakase, T. and Suzuki, M., Bullera megalospora, a new species of yeast forming large ballisto- spores isolated from dead leaves of Oryza sativa, Miscanthus sinensis, and Sasa sp. in Japan. J. Gen. Appl. Microbiol., 32, 225-240 (1986). 9) Suzuki, M. and Nakase, T., Validity of Candida natalensis van der Walt et Tscheuscher. Yeast, 5, s361-s365 (1989). 10) van Uden, N. and Buckley, H., Genus 2. Candida. In The Yeasts, a Taxonomic Study, 2nd ed., ed. by Lodder, J., North-Holland Pub!. Co., Amsterdam (1984), p. 893-1087. 11) van der Walt, J. P. and Yarrow, D., Methods for the isolation, maintenance, classification and identification of yeasts. In The Yeasts, a Taxonomic Study, 3rd ed., ed. by Kreger-van Rij, N. J. W., Elsevier Sci. Publ., Amsterdam (1984), p. 45-105.