J. Microbiol. Biotechnol. (2013), 23(7), 905–912 http://dx.doi.org/10.4014/jmb.1301.01005 jmb New Species of the Genus Metschnikowia Isolated from Flowers in Indonesia, Metschnikowia cibodasensis sp. nov. Wellyzar Sjamsuridzal1*, Ariyanti Oetari1, Chiharu Nakashima2, Atit Kanti3, Rasti Saraswati4, Yantyati Widyastuti5, and Katsuhiko Ando6 1Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia 2Graduate School of Bioresources, Mie University, Tsu, Mie Prefecture 514-8507, Japan 3Research Center for Biology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia 4Indonesian Soil Research Institute, Ministry of Agriculture, Bogor 16123, Indonesia 5Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia 6Department of Biotechnology, National Institute of Technology and Evaluation, Chiba 292-0818, Japan Received: January 3, 2013 Revised: March 12, 2013 A novel species, Metschnikowia cibodasensis, is proposed to accommodate eight strains (ID03- Accepted: March 15, 2013 0093T, ID03-0094, ID03-0095, ID03-0096, ID03-0097, ID03-0098, ID03-0099, and ID03-0109) isolated from flowers of Saurauia pendula, Berberis nepalensis, and Brunfelsia americana in Cibodas Botanical Garden, West Java, Indonesia. The type strain of M. cibodasensis is ID03- T T T T First published online 0093 (= NBRC 101693 =UICC Y-335 = BTCC-Y25 ). The common features of M. cibodasensis May 27, 2013 are a spherical to ellipsoidopedunculate shaped ascus, which contains one or two needle- *Corresponding author shaped ascospores, and lyse at maturity. Asci generally develop directly from vegetative cells Phone: +62-21-7270163; but sometimes from chlamydospores. The neighbor-joining tree based on the D1/D2 domain Fax: +62-21-7270012; of nuclear large subunit (nLSU) ribosomal DNA sequences strongly supports that M. E-mail: [email protected]; [email protected] cibodasensis (eight strains) and its closest teleomorphic species, M. reukaufii, are different species by a 100% bootstrap value. The type strain of M. cibodasensis, ID03-0093T, differed from M. reukaufii NBRC 1679T by six nt (five substitutions and one deletion) in their D1/D2 region of nLSU rDNA, and by 18 nt (five deletions, four insertions, and nine substitutions) in their internal transcribed spacer regions of rDNA, respectively. Four strains representative of M. cibodasensis (ID03-0093T, ID03-0095, ID03-0096, and ID03-0099) showed a mol% G+C content of 44.05 ± 0.25%, whereas that of M. reukaufii NBRC 1679T was 41.3%. The low value of DNA- DNA homology (5-16%) in four strains of M. cibodasensis and M. reukaufii NBRC 1679T pISSN 1017-7825, eISSN 1738-8872 strongly supported that these strains represent a distinct species. Copyright© 2013 by The Korean Society for Microbiology Keywords: Ascomycetous yeast, flowers, Metschnikowia cibodasensis, taxonomy and Biotechnology Introduction terrestrial species of Metschnikowia are found in the nectar or corolla of flowers and in decaying fruit or plant tissue. Metschnikowia is a well-documented genus of teleomorphic They are transmitted to new niches by insects, such as bees ascomycetous yeasts. Since its first report by Metschnikoff and drosophilids [16, 17, 25, 32]. in 1884 as the genus Mosnospora, more than 40 species have Insects associated with ephemeral flowers harbor very been accommodated within the Metschnikowia lineage, specific yeast communities. The insects, principally nitidulid including several species of Candida, which is known as an beetles and drosophilid flies, vector a highly specific yeast anamorphic genus of Metschnikowia. Species of Metschnikowia community that may serve as food for the larvae of the are characterized by the formation of long needle-shaped insects [14, 17]. The proliferation of terrestrial flowering or acicular ascospores that lack appendages [25]. Most plants and associated insects has markedly affected the July 2013 ⎪ Vol. 23⎪ No. 7 906 Sjamsuridzal et al. genetic diversification in Metschnikowia [6]. In fact, several was put onto the YM agar medium. Incubation of the plates was newly described Metschnikowia and its anamorphic Candida done for 5 days at room temperature. species were isolated from a large numbers of fruit and The type strain ID03-0093T (NBRC 101693T = UICC Y-335T = T flowers-associated species. These included Metschnikowia BTCC-Y25 ) and another seven strains were deposited (living and koreensis [7], M. arizonensis, M. dekortorum [14], M. vanudenii, dried) at three culture collections: the NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation M. lachancei [6], Candida kunwiensis [8], M. santaceciliae, C. (NITE), Japan under the numbers NBRC 101693T–NBRC 101700; hawaiiana, C. kipukae [18], M. similis, M. colocasiae [15], M. University of Indonesia Culture Collection (UICC), Department of hamakuensis, M. kamakouana, M. mauinuiana [19], M. aberdeeniae Biology, Faculty of Mathematics and Natural Sciences, Universitas [20], M. orientalis [21], and M. shivogae [22], which were Indonesia, Depok, Indonesia under the numbers UICC Y-335T– recorded from flowers or their associated insects, and M. UICC Y-341 and UICC Y-351; and the Research Center for viticola, which was isolated from grapes [27]. Biotechnology Culture Collection (BTCC), Indonesian Institute of On the other hand, there is no report on Metschnikowia Sciences (LIPI), Cibinong, Indonesia under the numbers BTCC- isolated from Indonesia, although its prospect is likely Y25T–BTCC-Y32. The strains studied and their accession numbers because of the country’s diversity of hosts and vectors. In a of sequences of the D1/D2 domain of nuclear LSU ribosomal taxonomic study of fungi in Indonesia in July 2003, we took DNA and the ITS regions (including ITS1, 5.8S, and ITS2) are samples from flowers of several plants in the Cibodas shown in Table 1. Botanical Garden in West Java, Indonesia. Eight of the Morphological and Physiological Characterization strains represented a novel, sister species to M. reukaufii. The morphological and physiological characteristics of the We describe them as Metschnikowia cibodasensis sp. nov. eight isolates were determined by the standard method described by Yarrow [40]. The observations of asci and ascospores were Materials and Methods performed on diluted V8 agar (1:9 and 1:29), diluted Corn Meal agar (1:9), and Yeast Carbon Base agar at 15-20oC for up to 2 months. Yeast Strains The eight strains of M. cibodasensis were isolated from flowers Ultrastructure of Ascospores in Cibodas Botanical Garden in West Java, Indonesia. The flower The mode of ascogenesis was observed by scanning electron samples were collected by Chiharu Nakashima and Ariyanti microscopy (SEM) following the methods of Talens et al. [35, 36] Oetari on 23rd July 2003. Three strains (ID03-0093T, ID03-0094, and Mikata [24], with a slight modification. One-week-old cultures and ID03-0095) were isolated from Saurauia pendula Blume, four on diluted V8 agar (1:9) were scraped and suspended in 2 ml of strains (ID03-0096, ID03-0097, ID03-0098, and ID03-0099) were 67 mM phosphate buffer (pH 7.0). After the cells were collected by isolated from Berberis nepalensis Spreng., and strain ID03-0109 was centrifugation, they were washed with distilled water. The cells were isolated from Brunsfelsia americana L. All parts of the flower were then suspended in 0.5 ml of 67 mM phosphate buffer (pH 7.0) and weighed and cut into pieces. The flower pieces were washed with 0.25 ml of Zymolyase 100T (3 mg/ml) containing 2-mercaptoethanol, 30 ml of sterile distilled water and vortexed for 5 min. Washed and 10 µl of RNase (100 mg/ml). The samples were subsequently materials were placed directly onto YM agar (1% glucose, 0.3% washed with 67 mM phosphate buffer (pH 7.0) before they were yeast extract, 0.3% malt extract, and 1.5% agar) containing 0.05% dehydrated in a graded ethanol series (30 min each), which was chloramphenicol. The suspension was filtered using a 0.45 µm followed by washes in isoamyl acetate in absolute ethanol (1:1) membrane filter (Merck Millipore, USA), and the membrane filter (1 h) and then in absolute isoamyl acetate for 1 h. After treatment Table 1. Origin of the Metschnikowia cibodasensis strains sequenced in this study. Sample DDBJ/GenBank/EMBL Accession No. Strain Other designation(s) Isolation sources code (D1/D2 of LSU rDNA, ITS) ID03-0093T UICC Y-335 T, NBRC 101693 T, BTCC-Y25 T Flowers of Saurauia pendula 182 AB236910, AB236918 ID03-0094 UICC Y-336, NBRC 101694, BTCC-Y26 Flowers of Saurauia pendula 182 AB236911, AB236919 ID03-0095 UICC Y-337, NBRC 101695, BTCC-Y27 Flowers of Saurauia pendula 182 AB236912, AB236920 ID03-0096 UICC Y-338, NBRC 101696, BTCC-Y28 Flowers of Berberis nepalensis 183 AB236913, AB236921 ID03-0097 UICC Y-339, NBRC 101697, BTCC-Y29 Flowers of Berberis nepalensis 183 AB236914, AB236922 ID03-0098 UICC Y-340, NBRC 101698, BTCC-Y30 Flowers of Berberis nepalensis 183 AB236915, AB236923 ID03-0099 UICC Y-341, NBRC 101699, BTCC-Y31 Flowers of Berberis nepalensis 183 AB236916, AB236924 ID03-0109 UICC Y-351, NBRC 101700, BTCC-Y32 Flowers of Brunsfelsia americana 204 AB236917, AB236925 J. Microbiol. Biotechnol. Metschnikowia cibodasensis sp. nov. Isolated from Flowers in Indonesia 907 with a critical point dryer (Hitachi HCP-2), the materials were accession numbers for the D1/D2 domain of nLSU rDNA and ITS coated with Pt-Pd and examined at 20 kV under a JEOL scanning region sequences of the strains newly obtained in this study are electron microscope. shown in Table 1. Molecular Characterization Results and Discussion DNA preparation for DNA base composition and DNA- Phenotypic Affiliation of Strains DNA hybridization Morphologically, the strains of M. cibodasensis are similar T T Overnight cultures of strains ID03-0093 (NBRC 101693 ), ID03- to the teleomorphic genus Metschnikowia, which is classified 0095, ID03-0096, ID03-0099, and Metschnikowia reukaufii NBRC in the phylum Ascomycota, order Saccharomycetales, belonging 1679T on 50ml of YPD at 25oC with shaking at 180 rpm were to the Family Metschnikowiaceae.