Bensingtonia sakaguchii sp. nov. isolated from a leaf of Bischofia javanica in the Ogasawara Islands.

Bensingtonia Ingold is characterized by 0-9 as the major ubiquinone, the production of kidney-shaped ballistoconidia and the absence of xylose in the cells. Since Ingold (1986) established the genus Bensingtonia based on a single species, B. ciliata, ten species have been found. During a survey on ballistoconidium-forming yeasts from leaves of plants collected in the Ogasawara Islands, Japan, we isolated two strains of a hitherto undescribed yeast from a leaf of Bischofia javanica in Euphorbiaceae, which was collected in Chichi-jima, Ogasawara Islands, Japan. These isolates seemed to belong to the genus Bensingtonia because they have 0-9 as the major ubiquinone, lack xylose in the cells and produce kidney-shaped ballistoconidia. A new species, Bensingtonia sakaguchii Sugita, Takashima, Hamamoto et Nakase, is proposed for these two isolates. The specific epithet was chosen in honor of Prof. K. Sakaguchi who first studied yeasts in the Ogasawara Islands. The strains reported in this paper, OK-255 and OK259, were isolated using the ballistoconidium-fall method with YM agar (Difco Lab, MI, U.S.A.) plates. Most of the morphological, biochemical and physiological characteristics were examined using the methods described by van der Walt (1984). The assimilation of nitrogen compounds was investigated according to Nakase and Suzuki (1986). Vitamin requirements were examined using the method of Komagata and Nakase (1967). The maximum growth temperature was determined in YM broth (Difco Lab.) using metal block baths. The extraction, purification and identification of ubiquinones was carried out according to Nakase and Suzuki (1988). Xylose in the cells was analyzed as described by Suzuki and Nakase (1988). Isolation and purification of DNA was carried out according to Nakase and Suzuki (1986). The DNA base composition (mol% G+C) was determined by nucleotide analysis using high-performance liquid chromatography (HPLC) after DNA digestion with Alkalinephosphatase (Sigma, MO, U.S.A.) and Nuclease P1 (Yamasa Shoyu, Chiba, Japan) as described by Nakase et al. (1989). DNA-DNA hybridization experiments were performed using the membrane-filter method according to Nakase and Suzuki (1985). The small subunit ribosomal RNA coding region was amplified by polymerase chain reaction (PCR) as described by Suh and Nakase (1995). The PCR products were sequenced using a SequiTherm Long-Read Cycle Sequencing Kit (Epicentre Technologies Corp., WI, U.S.A.) with the primers as described in Suh et al. (1996). Analyses of DNA sequence reactions were performed with an ALFexpress DNA sequencer (Pharmacia Biotech, Uppsala, Sweden). The sequences were aligned using the Clustal W computer program (Thompson et al., 1994). Introns were excluded from the alignment. For phylogenetic analysis, the following reference small subunit ribosomal DNA (SSU rDNA) sequences were obtained from nucleotide sequence libraries (EMBL, GenBank and DDBJ): B. ciliata (1)38233), B. ingoldii (D38234), B. intermedia (1)38235), B. miscanthi (D38236), B. musae (1)43946), B. naganoensis (D38366), B. phylladus (1)38237), B. subrosea (D38238), B. yamatoana * Address reprint requests to: Dr. Takashi Sugita, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako 351-01, Japan. t Present address: Thailand Institute of Scientific and Technologi-