J. Gen. App!. Microbiol., 35, 53-58 (1989) BENSINGTONIA INGOLDH SP. NOV., A BAL- LISTOSPORE-FORMING YEAST ISOLATED FROM KNIGHTIA EXCELSA COLLECTED IN NEW ZEALAND TAKASHI NAKASE,* MUTSUMI ITOH, ANDJUNTA SUGIYAMA' Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-01, Japan The Institute of Applied Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan (Received February 8, 1989) A hitherto undescribed ballistospore-forming yeast was isolated from Knightia excelsa infected by sooty molds collected in New Zealand. It is characterized by ubiquinone-9 and the lack of xylose in the cells so that it is included in the genus Bensingtonia. This yeast resembles B. intermedia and B, yamatoana but is easily distinguished from these two species by the assimilation of nitrate and lactose and the requirement of pyridoxine. Bensingtonia ingoldii Nakase et Itoh sp. nov. is proposed for this yeast. In 1987, the authors isolated a ballistospore-forming yeast from a Rewarewa leaf, i.e. Knightia excelsa (Proteaceae) collected by one of the authors (J. Sugiyama). This strain contains no xylose in the whole cell hydrolyzate and has Q-9 as the major component of ubiquinones so that it is included in the "intermedius group," a group of species having Q-9 in the genus Sporobolomyces. Yamada and Nakagawa (15) found that this yeast is different from any known species of the Q-9- equipped species of Sporobolomyces in the electrophoretic pattern of seven enzymes. In 1988, Nakase and Boekhout (5) emended the diagnosis of the genus Bensingtonia and transferred all of the known Q-9-equipped species of Sporobolomyces into Bensingtonia. Therefore, we describe here this new isolate as a new species of Bensingtonia. MATERIALS AND METHODS Strain used. The strain designated as NZ-3 was isolated from Knightia excelsa * Address reprint requests to: Dr . T. Nakase, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-01, Japan. 53 54 NAKASE, ITOH, and SUGIYAMA VOL. 35 (Proteaceae) infected by sooty molds collected at Param Track, Huia (North Island), New Zealand by J. Sugiyama on May 3, 1987. The method for isolation followed the ballistospore-fall method reported by Derx (2), with slight modifications. Examination of morphological, physiological and biochemical characteristics. The morphological, physiological, and biochemical characteristics were examined according to the methods described in the 3rd edition of The Yeasts, a Taxo- nomic Study (12) with some exceptions. Vitamin requirement was tested by the method previously described (4). The maximum growth temperature was deter- mined in YM broth (Difco Labs., Detroit) using metal block baths (6). Assimilation of nitrogen compounds was examined on solid media with starved inoculum (8). Examination of chemo taxonomic characteristics. Isolation and purification of DNAs followed the procedures reported in the previous paper (8). DNA was hydrolyzed with nuclease Pl (Sigma, St. Louis) then the resulting deoxyribonucleo- tides were digested with alkaline phosphatase (Sigma). The molar ratio of de- oxyribonucleosides in the reaction mixture was analyzed by high performance liquid chromatography (HPLC). HPLC was performed on a LC-6A liquid chromato- graph (Shimadzu, Kyoto) fitted with a Cosmosil C18 column (Nakarai Chemicals, Kyoto). Deoxyribonucleosides were eluted with a mobile phase of 0.2 M NH4H2P04 solution supplemented with 1/20 volumes of acetonitrile. The standard equimolar deoxyribonucleoside mixture was prepared with a hydrolyzing DNA-GC Kit (Yamasa Shoyu, Choshi) with alkaline phosphatase. Ubiquinones were extracted with hexane after saponification of intact cells by the method of Yamada and Kondo (14) then purified by preparative thin layer chromatography using Merck Kieselgel 60 F254 plates and benzene as the solvent system. Ubiquinone isoprenologues were identified by HPLC as reported previously (9). Xylose in the cells was analyzed by HPLC as previously described using whole cell hydrolyzate (11). RESULTS AND DISCUSSION Bensingtonia ingoldii Nakase et Itoh, sp. nov. In liquido "YM," post 5 dies at 17°C, cellulae rotundae, ovoideae, ellipsoi- dales aut longae, 2.5-4 x 2.5-20µm, singulae aut binae. Annulus formatur. In agaro "YM," post unum mensem ad 17°C, cultura glauco-flava, glabra aut rugu- losa, butyracea, margine glabra. Pseudomyceium primitivum formatur. Ballisto- sporae reniformes, 2.5-3.5 x 5-7.5 ,um. Fermentatio nulla. Glucosum, galactosum (lente et exiguum), saccharosum, cellobiosum, trehalosum, lactosum, melibiosum (lente et exiguum), raffinosum (lente et exiguum), melezitosum, amylum solubile, D-xylosum, L-arabinosum, D-ribosum (lente et exiguum), ethanolum, glycero- lum, erythritolum, ribitolum, D-mannitolum, D-glucitolum (exiguum), salicinum 1989 Bensingtonia ingoldii sp. n. 55 Fig. 1. Vegetative cells and ballistospores of Bensingtonia ingoldii sp. nov. A: Vegetative cells grown in YM broth for 5 'days at 17°C. B-F: Ballistospores produced on YM agar after 9 days at 17°C. D: Germination of a ballistospore. (exiguum), glucono-b-lactonum, acidum 2-ketogluconicum, acidum 5-ketogluco- nicum, acidum D-glucuronicum, acidum succinicum (exiguum) et acidum citricum (exiguum) assimilantur at non L-sorbosum (vel lente et exiguum), maltosum, inulinum, D-arabinosum (vel lente et exiguum), L-rhamnosum, galactitolum, a- methyl-D-glucosidum, acidum DL-lacticum nec inositolum. Kalium nitricum assimi- latur. Maxima temperatura crescentiae: 27-28°C. Ad crescentiam acidum p- aminobenzonicum, pyridoxinum et thiaminum necessarium sunt. Diazonium cae- ruleum B positivum. Proportio molaris guanini + cytosini in acido deoxyribonuc- leico: 55.7 mol , (per HPLC). Ubiquinonum majus: Q-9. Xylosum in cellulis absens. Teleomorphosis ignota. Holotypus: Isolatus ex folio Knightiae excelsae, `Param Track, Huia,' Nova Zealandia, v. 1987, T. Nakase, JCM 7445 (originaliter ut NZ-3) conservatur in collectionibus culturarum quas `Japan Collection of Microorganisms,' Wako, Saitama sustentat. Growth in YM broth: After 5 days at 17°C, cells are round, oval, ellipsoidal, or sometimes elongate, 2.5-4 x 2.5-10µm, usually single or in pairs. Some cells bear r ballistospores (Fig. 1A). A ring and a sediment are formed. After one month, a trace of a ring and a sediment are present. Growth on YM agar: After one month at 17°C, the streak culture is greyish yellow (champagne), smooth or wrinkled especially near the bottom of the agar slant, dull, butyrous, and has an entire margin. 56 NAKASE, ITOH, and SUGIYAMA VOL. 35 Dalmau plate culture on corn meal agar: Primitive pseudomycelia are produced. Production of ballistospores: The production of ballistospores is moderate. They are kidney-shaped, 2.5-3.5 x 5-7.5 (Fig. l B-F). Fermentation: Absent. Assimilation of carbon compounds: Glucose + L-Rhamnose Galactose + (latent and weak) Ethanol + L-Sorbose or + (latent and Glycerol + weak) Erythritol + Sucrose + Ribitol + Maltose Galactitol Cellobiose + D-Mannitol + Trehalose + D-Glucitol + (weak) Lactose + a-Methyl-D-glucoside Melibiose + (latent and weak) Salicin + (weak) Raffinose + (latent and weak) Glucono-b-lactone + Melezitose + 2-Ketogluconic acid + Inulin 5-Ketogluconic acid + Soluble starch + D-Glucuronic acid + D-Xylose + DL-Lactic aicd L-Arabinose + Succinic acid + (weak) - or + (latent and D-Arabinose Citric acid + (weak) weak) Inositol D-Ribose + (latent and weak) Assimilation of nitrogen compounds: Ammonium sulfate + Ethylamine hydrochloride + Potassium nitrate + L-Lysine hydrochloride + Sodium nitrite - Cadaverine dihydrochloride + Maximum growth temperature: 27-28°C. Vitamins required: p-Aminobenzoic acid , pyridoxine, and thiamine. Production of starch-like substances: Negative . Growth on 5O0 (w/w) glucose yeast extract agar: Negative . Liquefaction of gelatin: Negative. Urease: Positive. Diazonium blue B reaction: Positive. Hydrolysis of fat: Negative. G+C content of nuclear DNA: 55.7mol 0 (by HPLC). Major ubiquinone: Q-9. Xylose in the cells: Absent. Type strain: NZ-3, isolated by T. Nakase from a leaf of Knightia excelsa infected by sooty molds, which was collected at Param Track, Huia (North Island), New Zealand, by 1. Sugiyama (Coll. no. NZ-87-2), on May 3, 1987. It is maintained 1989 Bensingtonia ingoldii sp. n. 57 in the Japan Collection of Microorganisms as JCM 7445. It has been also deposited as JAM 13923 in the Culture Collection of The Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan. The genus Bensingtonia Ingold was established based on a single species, Bensingtonia ciliata Ingold, which produced colorless yeast-like colonies and straight ballistospores (3). Later, Boekhout (1) reported that this fungus had Q-9 as the major component of ubiquinone and lacked xylose in the whole cell hydrolyzate. Nakase and Boekhout (5) emended the diagnosis of the genus Bensingtonia to permit the inclusion of species forming pigmented colonies and curved ballisto- spores. They characterized this genus by Q-9 as the major ubiquinone and the lack of xylose in the cells, and recognized the following seven species in the genus: B, ciliata Ingold, B. intermedia (Nakase et Suzuki) Nakase et Boekhout, B. miscanthi (Nakase et Suzuki) Nakase et Boekhout, B. naganoensis (Nakase et Suzuki) Nakase et Boekhout, B, subrosea (Nakase et Suzuki) Nakase et Boekhout, and B, yamatoana (Nakase, Suzuki et Itoh) Nakase et Boekhout. They did not recognize B. weUmanii