INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Oct. 1994, p. 641-645 Copyright 0 1994, International Union of Microbiological Societies 0020-7713/94/$04.00 + 0 Vol. 44, No. 4 Candida caseinoEytica sp. nov., a New Species of Yeast Occurring in Necrotic Tissue of Opuntia and Stenocereus Species in the Southwestern United States and Baja California, Mexico HERMAN J. PHAFF,l* WILLIAM T. STARMER,2 MARC-ANDRE LACHANCE,3 AND PHILIP F. GANTER4 Department of Food Science and Technoloo, University of California, Davis, California 9561 6'; Department of Biology, Syracuse University, Syracuse, New York 1321 02; Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B73; and Department of Biology, Tennessee State University,Nashville, Tennessee 372044 We describe Candida caseinolytica, a new yeast species which occurs in rotting tissues of opuntias and other cacti in the North American Sonoran Desert and a few other localities. This small-celled, slowly growing yeast does not ferment any sugar and assimilates a limited number of carbon compounds, including 2- and 5-ketogluconicacids. It exhibits strong extracellular proteolytic activity on casein at pH 6.5, but gelatin is not hydrolyzed or is only weakly hydrolyzed by a few strains. The type strain of C. caseinolytica is strain UCD-FST 83-438.3 (= ATCC 90546 = CBS 7781). During extensive surveys oi more than 5,000 isolates in the yeast biota found in rotting tissues of various cactus species obtained from a wide variety of geographic areas (2,12,15), we isolated a small number of strains of a unique, slowly growing, small-celled yeast species; these strains were isolated mainly from various Opuntia and Stenocereus species (Table 1) grow- ing in a rather restricted region of the North American Sonoran Desert and a few other localities. The colonies of this yeast species were recognized on the isolation medium used (pH ca. 3.8) by their small size, late appearance compared with other cactus-specific species, very glossy smooth surfaces, and white color. The unusually small budding cells occurred mainly as single cells and in pairs. Subsequent physiological tests showed that these strains exhibited an exceptionally strong ability to hydrolyze casein at pH 6.5, a property that had not been observed previously in any of the other cactus-specific species described by us. As ascosporulation was not observed, the strains fit the description of the genus Candida sensu Yarrow and Meyer (17). A comparison of the phenotypic properties of this organism with the properties of other Candida species (3,8) and a recent up-to-date key to species of the genus (7) revealed that the cactus isolates represent a new species, for which we propose the name Candida caseinolytica. MATERIALS AND METHODS Samples of rotting cactus tissues collected during 1981,1983, 1984, 1985, 1986, 1987, and 1991 were obtained from host plants in the locations shown in Tnble 1. The technique used for yeast isolation and purification has been described in detail previously by Starmer and Phaff (13). The isolates were characterized phenotypically by methods currently used in yeast taxonomy (16), but additional carbon compounds were used in the assimilation tests. These addi- tional carbon compounds included hexadecane, glucosamine, N-acetylglucosamine, methanol, 2-propanol, acetone, and ethyl acetate. 2-Propanol, acetone, and ethyl acetate were tested by adding each compound at a concentration of 1% (vol/vol, based on medium volume) to the inverted cover of a flat-bottom glass petri dish after inoculation of the test cultures with a multipoint inoculator. The dishes were then sealed with * Corresponding author. Phone: (916) 752-1238. Fax: (916) 752- 4759. 64 1 Parafilm M (American Can Co., Greenwich, Conn.) and incubated upside down. The medium used for the casein hydrolysis test, which is not a routine test in yeast taxonomy, was a slightly modified version of the medium used by Ahearn et al. (1). First, 1.5 g of beef extract (Difco), 2.5 g of Bacto Tryptone, 0.5 g of glucose, and 8.5 g of agar were added to 440 ml of deionized water, and the preparation was autoclaved. Then 12 g of skim milk powder (Difco) was mixed with deionized water with a mortar and pestle, the volume was brought to 60 mi, and the preparation was autoclaved at 12 lb/in2 for 12 min. The two solutions were cooled to about 50°C and mixed together thoroughly before the final preparation was poured into petri dishes. The final pH was 6.5. The extent of clearing caused by casein hydrolysis was determined daily at 25°C for up to 1 week rather than 3 weeks, which was the time period used by Ahearn et al. (1). Susceptibility to triterpene glycosides was tested at 25 and 37°C in YM agar plates containing 1% dried, powdered Stenocereus gummosus (agria) tissue, as described by Starmer et al. (11). DNA was extracted, purified, and hybridized by the methods described by Price et al. (lo), except that purified DNA was concentrated by electrophoresis in a concentrator (ISCO, Lincoln, Nebr.) and the reference DNA was labeled with '1 as described by Holzschu et al. (5). The G+C contents of the nuclear DNAs were calculated from buoyant density values in cesium chloride gradients established in a Spinco model E analytical ultracentrifuge (lo). RESULTS Latin diagnosis of Candida caseinolytica sp. nov. In YM (Difco) liquid0 post dies 5 ad 30"C, cellulae ovoideae vel brevi-cylindricae, 1.3-2.5 X 2.5-5.0 pm, singulae, binae vel catenis brevis; sedimentum; pellicula nulla. Cultura in agaro malti post dies 21 ad 25°C cana-cremea, butyrosa vel mollis, glabra, nitida, margine glabro. In agaro farinae Zea mays post dies 10 pseudomycelium nullum. Fermentatio glucosi nullum. Glucosum, galactosum, sorbo- sum (lente), trehalosum (lente), xylosum, ethanolum, glucono- &lactonurn, gluconatum, 2-ketogluconatum, 5-ketoglucona- tum, acidum lacticum (lente), acidum succinicum (interdum exigue), ethyl acetas (exigue) assimilantur at non maltosum,