DSpace at VNU: Four new species of Kockovaella isolated from plant leaves collected in Vietnam

DSpace at VNU: Four new species of Kockovaella isolated from plant leaves collected in Vietnam tài liệu, giáo án, bài gi...

J Gen Appl Microbiol., 46, 297–310 (2000) Full Paper Four new species of Kockovaella isolated from plant leaves collected in Vietnam Dao Thi Luong,1,2,* Masako Takashima,1 Pham Van Ty,2 Nguyen Lan Dung,2 and Takashi Nakase1,† Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351–0198, Japan Vietnam Type Culture Collection, Centre of Applied Microbiology, University of Science, Vietnam National University, Hanoi, Vietnam (Received June 26, 2000; Accepted November 9, 2000) Five ballistoconidiogenous yeast strains, isolated from plant leaves at Cuc Phuong National Forest of Ninh Binh, Vietnam, were assigned to the genus Kockovaella based on morphological and chemotaxonomical characteristics They represent four new species based on analyses of 18S rDNA sequence, sequences of internal transcribed spacer regions, and DNA-DNA reassociation experiments Four new species, Kockovaella calophylli (1 strain), Kockovaella cucphuongensis (2 strains), Kockovaella litseae (1 strain), and Kockovaella vietnamensis (1 strain) are proposed for these strains Key Words——Kockovaella calophylli; Kockovaella cucphuongensis; Kockovaella litseae; Kockovaella vietnamensis; systematics Introduction Kockovaella, a ballistoconidiogenous anamorphic yeast genus, was described by Nakase et al (1991) This genus is characterized by the reproduction of ballistoconidia, nonballistosporous stalked conidia and budding cells, Q-10 as the major ubiquinone, and the presence of xylose in the cells Six species are recognized in the genus: K imperatae, K thailandica (Nakase et al., 1991), K sacchari (Takashima and Nakase, 1998), K machilophila, K phaffii, and K schi* Address reprint requests to: Ms Dao Thi Luong, Vietnam Type Culture Collection, Centre of Applied Microbiology, University of Science, Vietnam National University, Hanoi, Vietnam † Present Address: Yothi Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 73/1 Rama VI Road, Bangkok 10400, Thailand mae (Cañete-Gibas et al., 1998) During a survey of ballistoconidiogenous yeasts on plant materials of Asia, five yeasts isolated from Vietnam were assigned to the genus Kockovaella Four new species are proposed for these five strains based on analyses of 18S rDNA sequence, sequences of internal transcribed spacer (ITS) regions, and DNA-DNA reassociation experiments Materials and Methods Yeast strains The yeast strains used in this study were isolated from plant leaves collected in Cuc Phuong National Forest of Ninh Binh, Vietnam (Table 1), using the ballistoconidium-fall method on YM agar as reported by Nakase and Takashima (1993) Morphological, physiological, and biochemical characteristics Most methods used for the examination of morphological, physiological, and biochemical char- 298 LUONG et al Table Vol 46 Strains used in this study DDBJ accession numbers Species Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp K schimae Fellomyces chinensis F distylii F lichenicola F sichuanensis T Strain VY-61 VY-67 VY-125 VY-127 VY-137 JCM 10051T JCM 10164T JCM 9862T JCM 10165T JCM 10166T Source Litsea euosma Schimastoglottis calyptrata Smilax glabra Bambusa schijostachyoides Calophyllum membranaceum Schima mertensiana Physcia atrostriata, Heterodermia albicans, or Phlyctis argena Distylium lepidotum Physcia atrostriata, Heterodermia albicans, or Phlyctis argena Physcia atrostriata, Heterodermia albicans, or Phlyctis argena 18S rDNA ITS & 5.8S AB042218 AB042219 AB042220 AB042221 AB042222 AB042223 AB042224 AB042225 AB042226 AB042227 AB042228 AB042229 AB042230 AB042231 AB042232 Type strain acteristics were described by Yarrow (1998) The determination of maximum growth temperature was made in YM broth, using metal block baths The assimilation of nitrogen compounds was determined by the method of Nakase and Suzuki (1986) The vitamin requirement followed the method by Komagata and Nakase (1967) Chemotaxonomic characteristics Extraction, purification, and identification of ubiquinones were carried out according to the method of Nakase and Suzuki (1986) The presence or absence of xylose in the cells was analyzed by thin-layer chromatography (Nakase et al., 1976) after hydrolizing the cells with trifluoroacetic acid (Suzuki and Nakase, 1988) Sequencing and phylogenetic analysis The sequences of 18S rDNA and ITS regions, including 5.8S rDNA, was determined after amplifying the DNA by using PCR Both strands were sequenced directly (Takashima and Nakase, 1999) Generated sequences were aligned with related species by using the CLUSTAL W ver 1.74 computer program (Thompson et al., 1994) Reference sequences used for the phylogenetic study were obtained from the database (Table 2) The phylogenetic tree was constructed from the evolutionary distance data according to Kimura (1980) by use of the neighbor-joining method (Saitou and Nei, 1987) Sites where gaps existed in any sequences were excluded Bootstrap analyses (Felsenstein, 1985) were performed from 1,000 random resamplings For a comparison of ITS regions among closely related species, pairwise sequences were aligned by sight, and the sequence similarity including gaps was calculated DNA-DNA relatedness Isolation and purification of DNA used the methods of Takashima and Nakase (2000) The DNA base composition was determined by HPLC after the enzymatic digestion of DNA to deoxyribonucleosides (Tamaoka and Komagata, 1984) The DNA-GC Kit (Yamasa Shoyu Co Ltd., Chiba, Japan) was used as a quantitative standard DNA-DNA reassociation experiments used a membrane-filter method (Hamamoto and Nakase, 1995) Results and Discussion Five isolates (VY-61, VY-67, VY-I25, VY-127, and VY-137) were characterized by the presence of xylose in the cells, Q-10 as a major ubiquinone, the production of symmetrical ballistoconidia, the proliferation by stalked conidia, and budding cells The isolates were identified in the genus Kockovaella (Nakase et al., 1991) The 18S rDNA sequences of five Kockovaella isolates were determined and a phylogenetic tree for these isolates and 45 species of the genera Bullera, Cryptococcus, Fellomyces, and Kockovaella was constructed by the neighbor-joining method based on 1,674 bases (Fig 1) The result showed that five isolates were in a cluster and closely related with Fellomyces chinensis (Prillinger et al., 1997), F distylii (Hamamoto et al., 1998), F lichenicola, F sichuanensis (Prillinger et al., 1997), and Kockovaella schimae The sequences of ITS regions were determined and the sequence similarity was calculated between five 2000 Four new species of Kockovaella Table 299 18S rDNA sequence data obtained from the databank for constructing the phylogenetic tree Scientific name Bullera armeniaca Bullera coprosmaensis Bullera crosea Bullera dendrophila Bullera globispora Bullera hannae Bullera huiaensis Bullera miyagiana Bullera mrakii Bullera oryzae Bullera penniseticola Bullera pseudoalba Bullera sinensis Bullera unica Bullera variabilis Bulleromyces albus Cryptococcus amylolentus Cryptococcus cellulolyticus Cryptococcus dimennae Cryptococcus flavus Cryptococcus heveanensis Cryptococcus hungaricus Cryptococcus laurentii DDBJ/EMBL/ GenBank No D78323 D78326 D31648 D31649 D31650 D78327 D78331 D31651 D78325 D31652 AB005452 D31660 D78328 D78330 D31654 X60179 AB032619 AB032624 AB032627 AB032629 AB032635 AB032638 AB032640 isolates of the genus Kockovaella and phylogenetically closely related species (Table 3) The results showed that those of VY-67 and VY-125 were identical and showed 97.6–99.1% sequence similarity for ITS1 and 97.1–99.1% for ITS2 between other isolates Those of VY-61, VY-127, and VY-137 showed 99.1–100%, 97.1–99.1%, and 97.1–100% sequence similarity for ITS1 and 96.5–98.2%, 97.1–99.2%, and 99.1–99.2% for ITS2, respectively, between other isolates They showed less than 90% similarity for ITS1 and 86.8– 96.4% for ITS2 between closely related species These results indicated that Kockovaella strains isolated from plants of Vietnam were distinct from known Fellomyces and Kockovaella species, and they were closely related to each other (Sugita et al., 1999) ITS sequence data also indicated that F chinensis and F lichenicola were phylogenetically closely related For the two species, we will discuss another paper DNA-DNA reassociation experiments were carried out among five isolates and phylogenetically related species (Table 4) Two strains VY-67 and VY-125 showed a high DNA relatedness with each other Scientific name Cryptococcus luteolus Cryptococcus podzolicus Fellomyces borneensis Fellomyces chinensis Fellomyces distylii Fellomyces fuzhouensis Fellomyces horovitziae Fellomyces lichenicola Fellomyces ogasawarensis Fellomyces penicillatus Fellomyces polyborus Fellomyces sichuanensis Fellomyces thailandicus Kockovaella imperatae Kockovaella machilophila Kockovaella phaffii Kockovaella sacchari Kockovaella schimae Kockovaella thailandica Udeniomyces megalosporus Udeniomyces pyricola Udeniomyces puniceus DDBJ/EMBL/ GenBank No AB032641 AB032645 AB032659 AB032660 AB001036 AB001032 AB001033 AB032661 AB001035 AB001034 D64117 AB032662 AB044804 AB005561 AB005479 AB005480 AB005453 AB005482 D64133 D31657 D31659 D31658 (84–93%) and low reassociation values between other isolates (33–47%) and to reference species (2–17%) Strains VY-61 and VY-127 showed low reassociation values between other isolates (31–35% and 15–28%, respectively) and to reference species (2–16% and 2–10%, respectively) Strain VY-137 showed 52–73% relatedness with other isolates at 67°C, when DNA of VY-137 was employed as a probe, whereas it showed 25–47% relatedness when those of other isolates were employed as probes When the experiments were carried out at 69°C, DNA relatedness between VY-137 and other Kockovaella isolates were a little lower than those at 67°C (31–56%) The experiments performed again at 74°C, using the same filters, showed the low reassociation values between other isolates (33–44%) These results indicated that the loosely bound DNA on the filters at 67°C or 69°C was released at an elevated temperature Based on the facts, two strains, VY-67 and VY-125, were considered to represent a single new species, and strains VY-61, VY-127, and VY-137 represented three different new species Because these four new species are closely related 300 LUONG et al Vol 46 Fig Phylogenetic tree of five isolates of the genus Kockovaella and related species based on the 18S rDNA sequences The tree was constructed from the evolutionary distance data according to Kimura (1980), using the neighbor-joining method (Saitou and Nei, 1987) with bootstrapping (Felsenstein, 1985) The numerals represent results from 1,000 replicate bootstrap samplings (a frequency of less than 50% is not shown) as described above and shown in Table 5, however, they can distinguish from each other by the combination of assimilation ability of glycerol, erythritol, ribitol, 2-ketogluconic acid, DL-lactic acid, and inositol They are also discriminated from each other by ITS sequences (Fig 2, A and B) 2000 Four new species of Kockovaella a 98.2 99.1 100 — 98.3 88.2 86.7 86.7 86.6 84.2 96.5 99.2 97.1 97.1 — 86.6 85.8 85.8 85.7 84.3 93.6 91.8 94.1 94.1 91.3 — 86.7 86.7 84.0 81.8 96.4 94.7 95.9 95.9 94.2 94.2 — 100 92.4 78.5 JCM 9862 98.2 99.1 — 100 98.3 88.2 86.7 86.7 86.6 84.2 JCM 10166 97.0 — 97.6 97.6 97.1 87.4 86.7 86.7 86.6 83.4 JCM 10165 — 100 99.1 99.1 99.1 87.4 86.7 86.7 86.6 83.4 JCM 10164 JCM 10051 VY-61 VY-137 VY-67 VY-125 VY-127 JCM 10051 JCM 10164 JCM 10165 JCM 10166 JCM 9862 VY-127 Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp K shimae Fellomyces chinensis F lichenicola F sichuanensis F distylii VY-125 Strain VY-67 Species VY-137 Sequence similarities of ITS region among five Kockovaella isolates and related species.a VY-61 Table 301 95.9 94.1 96.4 96.4 93.6 94.3 98.2 — 92.4 90.0 95.9 94.7 95.3 95.3 94.2 94.2 95.9 95.6 — 86.5 87.8 89.0 87.2 87.2 86.8 87.8 90.6 78.5 81.8 — Lower left triangle shows sequence similarities in ITS1 and upper right triangle shows them in ITS2 Table DNA-DNA reassociation experiment among five Kockovaella isolates and related species Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp Kockovaella sp K schimae Fellomyces chinensis F distylii F lichenicola F sichuanensis VY-61 VY-67 VY-125 VY-127 VY-137 JCM 10051 JCM 10164 JCM 9862 JCM 10165 JCM 10166 53.2 53.6 53.6 54.1 53.5 52.8 54.8 50.2 53.3 54.3 100 ND 35 33 31 16 ND 100 93 ND ND ND ND ND ND ND 33 84 100 47 47 17 15 ND 28 100 25 10 3 52 ND 69 73 100 26 11 6 ND 10 100 VY-137 67°C VY-137 JCM 10051 VY-137 VY-127 Mol% GϩC VY-125 Strain VY-67 Species VY-61 % relative binding of DNA from 69°C 74°C 31 56 51 44 100 3 4 30 44 44 30 100 ND, not determined Description of New Taxa Kockovaella calophylli Luong, Takashima, Ty, Dung & Nakase, sp nov In liquido “YM” post dies ad 17°C, cellulae vegetativae sphaericae vel ovoideae aut ogiveae, 2.0–4.0ϫ 2.0–6.0 mm, singulae, binae, in catenis aut in fasciculis, propagantes formatione conidiorum stipitatorum et gemmarum blasticarum Post unum mensem ad 17°C, pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-flava, rugosa, non nitida et margine erosa Pseudomycelium formatur Ballistoconidia apiculata, 2.0–3.0ϫ3.0–6.0 mm Fermentatio nulla Glucosum, galactosum, L-sorbosum (lente et exiguum), sucrosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum, raffinosum, melezitosum, amylum solubile, D-xylosum, Larabinosum (lente), D-arabinosum (exiguum), D-ribo- 302 LUONG et al sum, L-rhamnosum (lente), galactitolum (lente et exiguum), D-mannitolum (lente), D-glucitolum (exiguum), salicinum (lente et exiguum), glucuno-d-lactonum (lente et exiguum), acidum 5-ketogluconicum (lente et exiguum), acidum D-glucuronicum (lente), acidum Dgalacturonicum (lente), acidum succinicum (lente) et acidum citricum (lente) assimilantur at non inulinum, ethanolum, glycerolum, erythritolum, ribitolum, amethyl-D-glucosidum, acidum 2-ketogluconicum, acidum DL-lacticum nec inositolum Ammonium sulfatum et L-lysinum assimilantur at non kalium nitricum, natrium nitrosum, cadaverinum nec ethylaminum Maxima temperatura crescentiae: 31–32°C Ad crescentiam thiaminum necessarium est et niacinum stimulare Materia amyloidea iodophila non formantur Ureum hydrolysatur Diazonium caeruleum B: Positivum Proportio molaris guaniniϩcytosini in acido deoxyribonucleico: 53.5 mol% per HPLC Systema Vol 46 ubiquini: Q-10 Xylosum in cellulis presens Holotypus: Isolatus ex folio Calophylli membranacei, Vietnam, JCM 10842/VTCC1 0180 (originaliter ut VY-137) conservatur in collectionibus culturarum quas “Japan Collection of Microorganisms,” Wako, Saitama et “Vietnam Type Culture Collection,” Hanoi, Vietnam sustentat Growth in YM broth: After days at 17°C, the vegetative cells are spherical to ovoidal with a few ogival ones and measure 2.0–4.0ϫ2.0–6.0 mm They occur singly, in pairs or in groups, and reproduce by budding and stalked conidia (Fig 3) Stalked conidia are observed in both pellicle and sediment The conidia are separated at the distal end of the stalks from parent cells A sediment and a dull, wrinkled climbing pellicle are formed after one month at 17°C Growth in YM agar: After one month at 17°C, the streak culture is light yellow, wrinkled, dull, and rough (A) 2000 Four new species of Kockovaella 303 (B) Fig Primary structure of the ITS1 (A) and ITS2 (B) sequences among five Kockovaella isolates and related species K_shim_10051, Kockovaella shimae JCM 10051; F_chin_10164, Fellomyces chinensis JCM 10164; F_lich_10165, F lichenicola JCM 10165; F_sich_10166, F sichuanensis JCM 10166; F_dist_9862, F distylii JCM 9862 netted, and it has an erose margin Stalked conidia and ballistoconidia are produced Dalmau plate culture on corn meal agar: Pseudomycelium is formed after weeks of incubation at 17°C Formation of ballistoconidia: Ballistoconidia are formed abundantly on corn meal agar after days incubation at 17°C (Fig 3) They are symmetrical and apiculate, measuring 2.0–3.0ϫ3.0–6.0 mm Fermentation: Absent Assimilation of carbon compounds: Glucose ϩ Galactose ϩ L-Sorbose ϩ (latent and weak) Sucrose ϩ Maltose Cellobiose Trehalose Lactose Melibiose Raffinose Melezitose Inulin Soluble starch D-Xylose L-Arabinose D-Arabinose D-Ribose L-Rhamnose Ethanol ϩ ϩ ϩ ϩ ϩ ϩ ϩ Ϫ ϩ ϩ ϩ (slow) ϩ (weak) ϩ ϩ (slow) Ϫ JCM 10842T JCM 10840T JCM 10839 JCM 10838T JCM 10841T JCM 10051T JCM 10164T JCM 9862T JCM 10165T JCM 10166T Kockovaella calophylli Kockovaella cucphuongensis Kockovaella cucphuongensis Kockovaella listeae Kockovaella vietnamensis K schimae b Fellomyces chinensis F distylii c F lichenicola F sichuanensis ϩ ϩ ϩ ϩ ϩ ϩ Ϫ Ϫ Ϫ Ϫ Formation of ballistoconidia lw w w w lw Ϫ Ϫ ϩ Ϫ Ϫ ϩ ϩ ϩ l ϩ lw ϩ ϩ ϩ l ϩ s l l l Ϫ ϩ ϩ w w ϩ l l ϩ l l ϩ ϩ s s s l l l s ϩ ϩ ϩ s ϩ w l s l s l w ϩ lw s ϩ ϩ l ϩ ϩ lw lw ϩ lw w a Type strain ϩ, positive; Ϫ, negative; l, latent; s, slow; w, weak; lw, latent and weak b Canete-Gibas et al (1998) c Hamamoto et al (1998) T Strain Species s l s l l l s Ϫ w w Ϫ Ϫ lw lw lw Ϫ Ϫ ϩ Ϫ Ϫ Ϫ lw w lw lw Ϫ lw ϩ lw lw Ϫ s w lw Ϫ lw w ϩ lw lw lw l s s w ϩ w ϩ w lw s l s ϩ w ϩ ϩ ϩ l lw w s lw w lw l s ϩ lw Ϫ lw w w w w Ϫ l ϩ s w lw s w w w lw ϩ ϩ l ϩ Ϫ s s Ϫ lw Ϫ l ϩ s w lw w lw w w Ϫ l ϩ w w Ϫ Ϫ lw lw Ϫ Ϫ lw Ϫ lw lw l l s ϩ ϩ lw l ϩ s s s s w w w lw l ϩ s w Ϫ Ϫ Ϫ s w Ϫ ϩ Ϫ w l L-Sorbose Lactose Melibiose Raffinose L-Arabinose D-Arabinose D-Ribose L-Rhamnose Glycerol Erythritol Ribitol Galactitol D-Mannitol D-Glucitol Salicin Glucono-d-lactone 2-Ketogluconic acid 5-Ketogluconic acid DL-Lactic acid Succinic acid Citric acid Inositol Carbon source s ϩ l ϩ ϩ l ϩ ϩ l l D-Galacturonic s ϩ s ϩ ϩ l ϩ ϩ l s acid acid D-Glucuronic Assimilation of a Nitrogen source Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ lw Ϫ lw lw Potassium nitrate Salient characteristics of Kockovaella isolates and related species Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ lw lw Vitamin requirements and vitamin stimulation Ϫ Thiamine, niacin Ϫ Thiamine Ϫ Thiamine Ϫ Thiamine Ϫ Thiamine Ϫ Thiamine lw Thiamine Ϫ Thiamine lw Thiamine lw Thiamine Ethylamine hydrochloride Cadaverine dihydrochloride Table Maximum growth temperature (°C) 31–32 29–30 29–30 29–30 31–32 28–29 29–30 27Ϫ28 28–29 31–32 304 LUONG et al Vol 46 2000 Four new species of Kockovaella Fig Kockovaella calophylli JCM 10842 (A) Vegetative cells grown in YM broth for days at 17°C (B) Ballistoconidia produced on corn meal agar after days at 17°C Scale bars indicate 10 mm Glycerol Ϫ Erythritol Ϫ Ribitol Ϫ Galactitol ϩ (latent and weak) D-Mannitol ϩ (slow) D-Glucitol ϩ (weak) a-Methyl-D-glucoside Ϫ Salicin ϩ (latent and weak) Glucono-d-lactone ϩ (latent and weak) 2-Ketogluconic acid Ϫ 5-Ketogluconic acid ϩ (latent and weak) D-Glucuronic acid ϩ (slow) D-Galacturonic acid ϩ (slow) DL-Lactic acid Ϫ Succinic acid ϩ (latent) Citric acid ϩ (latent) Inositol Ϫ Assimilation of nitrogen compounds: Ammonium sulfate ϩ Potassium nitrate Ϫ Sodium nitrite Ϫ Ethylamine hydrochloride Ϫ L-Lysine hydrochloride ϩ Cadaverine dihydrochloride Ϫ Maximum growth temperature: 31–32°C Vitamins: Thiamine required and niacin stimulated Production of starch-like substances: Negative Diazonium blue B color reaction: Positive Urease: Positive Liquefaction of gelatin: Negative Acid production on chalk agar: Negative GϩC content of nuclear DNA: 53.5 mol% (by 305 HPLC) Ubiquinone system: Q-10 Xylose in the cells: Present Strain examined: Strain VY-137 was isolated by Dao Thi Luong in February 1999 from a leaf of Calophyllum membranaceum Gard (Guttiferae), collected by Dao Thi Luong and Takashi Nakase at Cuc Phuong National Forest of Ninh Binh, Vietnam This strain has been deposited in Japan Collection of Microorganisms (JCM), RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama, Japan, and at the Vietnam Type Culture Collection (VTCC), Centre of Applied Microbiology, University of Science, Vietnam National University, Hanoi, Vietnam, with the accession numbers JCM 10842 and VTCC1 0180, respectively Kockovaella cucphuongensis Luong, Takashima, Ty, Dung & Nakase, sp nov In liquido “YM” post dies ad 17°C, cellulae vegetativae sphaericae vel ovoideae aut ogiveae, 3.0–5.0ϫ 3.0–7.0 mm, singulae, binae, in catenis aut in fasciculis, propagantes formatione conidiorum stipitatorum et gemmarum blasticarum Post unum mensem ad 17°C, pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-flava, rugosa, non nitida et margine erosa Pseudomycelium formatur Ballistoconidia apiculata, 2.5–3.5ϫ2.5–7.0 mm Fermentatio nulla Glucosum, galactosum, L-sorbosum (exiguum), sucrosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum (lente), raffinosum (lente), melezitosum, amylum solubile, D-xylosum, Larabinosum (lente), D-arabinosum (lente), D-ribosum, L-rhamnosum (lente), erythritolum (lente et exiguum), ribitolum (lente), galactitolum (lente), D-mannitolum (lente), D-glucitolum (lente), salicinum (exiguum), glucono-d-lactonum (lente), acidum 2-ketogluconicum (lente), acidum 5-ketogluconicum (exiguum), acidum D-glucuronicum, acidum D-galacturonicum, acidum succinicum (lente) et acidum citricum (lente), assimilantur at non inulinum, ethanolum, glycerolum, amethyl-D-glucosidum, acidum DL-lacticum nec inositolum Ammonium sulfatum et L-lysinum assimilantur at non kalium nitricum, natrium nitrosum, cadaverinum nec ethylaminum Maxima temperatura crescentiae: 29–30°C Ad crescentiam thiaminum necessarium est Materia amyloidea iodophila non formantur Ureum hydrolysatur Diazonium caeruleum B: Positivum Proportio molaris guaniniϩcytosini in acido deoxyribonucleico: 53.7 mol% per HPLC Systema ubiquini: Q-10 Xy- 306 LUONG et al losum in cellulis presens Holotypus: Isolatus ex folio Smilacis glabrae, Vietnam, JCM 10840/VTCC1 0177 (originaliter ut VY-125) conservatur in collectionibus culturarum quas “Japan Collection of Microorganisms,” Wako, Saitama et “Vietnam Type Culture Collection,” Hanoi, Vietnam sustentat Growth in YM broth: After days at 17°C, the vegetative cells are spherical to ovoidal with a few ogival ones and measure 3.0–5.0ϫ3.0–7.0 mm They occur singly, in pairs or in groups, and reproduce by budding and stalked conidia (Fig 4) Stalked conidia are observed in both pellicle and sediment The conidia are separated at the distal end of the stalks from parent cells A sediment and a dull, wrinkled climbing pellicle are formed after one month at 17°C Growth in YM agar: After one month at 17°C, the streak culture is light yellow, wrinkled, dull, and has an erose margin Dalmau plate culture on corn meal agar: Pseudomycelium is formed after weeks of incubation at 17°C Formation of ballistoconidia: Ballistoconidia are formed abundantly on corn meal agar after days incubation at 17°C (Fig 4) They are symmetrical and apiculate, measuring 2.5–3.5ϫ2.5–7.0 mm Fermentation: Absent Assimilation of carbon compounds: Glucose ϩ Galactose ϩ L-Sorbose ϩ (weak) Sucrose ϩ Maltose ϩ Cellobiose ϩ Trehalose ϩ Lactose ϩ Melibiose ϩ (slow) Raffinose ϩ (latent) Melezitose ϩ Inulin Ϫ Soluble starch ϩ D-Xylose ϩ L-Arabinose ϩ (latent) D-Arabinose ϩ (latent) D-Ribose ϩ L-Rhamnose ϩ (latent) Ethanol Ϫ Glycerol Ϫ Erythritol ϩ (latent and weak) Vol 46 Fig Kockovaella cucphuongensis (A) Vegetative cells grown in YM broth for days at 17°C of JCM 10840 (B) Ballistoconidia produced on corn meal agar after days at 17°C of JCM 10840 (C) Vegetative cells grown in YM broth for days at 17°C of JCM 10839 (D) Ballistoconidia produced on corn meal agar after days at 17°C of JCM 10839 Scale bars indicate 10 mm Ribitol ϩ (slow) Galactitol ϩ (slow) D-Mannitol ϩ (latent) D-Glucitol ϩ (slow) a-Methyl-D-glucoside Ϫ Salicin ϩ (weak) Glucono-d-lactone ϩ (latent) 2-Ketogluconic acid ϩ (latent) 5-Ketogluconic acid ϩ (weak) D-Glucuronic acid ϩ D-Galacturonic acid ϩ DL-Lactic acid Ϫ Succinic acid ϩ (latent) Citric acid ϩ (slow) Inositol Ϫ Assimilation of nitrogen compounds: Ammonium sulfate ϩ Potassium nitrate Ϫ 2000 Four new species of Kockovaella Sodium nitrite Ϫ Ethylamine hydrochloride Ϫ L-Lysine hydrochloride ϩ Cadaverine dihydrochloride Ϫ Maximum growth temperature: 29–30°C Vitamin required: Thiamine Production of starch-like substances: Negative Diazonium blue B color reaction: Positive Urease: Positive Liquefaction of gelatin: Negative Acid production on chalk agar: Negative GϩC content of nuclear DNA: 53.7 mol% (by HPLC) Ubiquinone system: Q-10 Xylose in the cells: Present Strain examined: Type strain VY-125 was isolated by Dao Thi Luong in February 1999 from leaves of a green brier, Smilax glabra Roxb, and strain VY-67 was isolated from leaves of an aroid, Schimastoglottis calyptrata Zoll & Mor collected by Dao Thi Luong and Takashi Nakase at Cuc Phuong National Forest of Ninh Binh, Vietnam These strains have been deposited in JCM and VTCC with the accession numbers JCM 10840 and VTCC1 0177 for VY-125 and JCM 10839 and VTCC1 0178 for VY-67, respectively Kockovaella litseae Luong, Takashima, Ty, Dung & Nakase, sp nov In liquido “YM” post dies ad 17°C, cellulae vegetativae sphaericae vel ovoideae aut ogiveae, 3.0–6.0ϫ 3.5–8.0 mm, singulae, binae, in catenis aut in fasciculis, propagantes formatione conidiorum stipitatorum et gemmarum blasticarum Post unum mensem ad 17°C, pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-flava vel gilvus-aurantiaca, rugosa, hebes, aspera et margine erosa Pseudomycelium formatur Ballistoconidia apiculata, 2.5–4.0ϫ2.5–6.0 mm Fermentatio nulla Glucosum, galactosum, L-sorbosum (exiguum), sucrosum, maltosum, cellobiosum, trehalosum, lactosum (lente), melibiosum (lente), raffinosum, melezitosum, amylum solubile, D-xylosum, Larabinosum (lente), D-arabinosum (lente), D-ribosum, L-rhamnosum (lente), glycerolum (lente et exiguum), erythritolum (lente et exiguum), ribitolum (lente et exiguum), galactitolum, D-mannitolum, D-glucitolum (exiguum), salicinum (exiguum), glucuno-d-lactonum (exiguum), acidum 5-ketogluconicum (exiguum), acidum D-glucuronicum, acidum D-galacturonicum, acidum DLlacticum (lente et exiguum), acidum succinicum, 307 Fig Kockovaella litseae JCM 10838 (A) Vegetative cells grown in YM broth for days at 17°C (B) Ballistoconidia produced on corn meal agar after days at 17°C Scale bars indicate 10 mm acidum citricum (exiguum) et inositolum (lente) assimilantur at non inulinum, ethanolum, a-methyl-D-glucosidum nec acidum 2-ketogluconicum Ammonium sulfatum et L-lysinum assimilantur at non kalium nitricum, natrium nitrosum, cadaverinum nec ethylaminum Maxima temperatura crescentiae: 29–30°C Ad crescentiam thiaminum necessarium est Materia amyloidea iodophila non formantur Ureum hydrolysatur Diazonium caeruleum B: Positivum Proportio molaris guaniniϩcytosini in acido deoxyribonucleico: 53.2 mol% per HPLC Systema ubiquini: Q-10 Xylosum in cellulis presens Holotypus: Isolatus ex folio Litseae euosmae, Vietnam, JCM 10838/VTCC1 0176 (originaliter ut VY-61) conservatur in collectionibus culturarum quas “Japan Collection of Microorganisms,” Wako, Saitama et “Vietnam Type Culture Collection,” Hanoi, Vietnam sustentat Growth in YM broth: After days at 17°C, the vegetative cells are spherical to ovoidal with a few ogival ones and measure 3.0–6.0ϫ3.5–8.0 mm They occur singly, in pairs or in groups, and reproduce by budding and stalked conidia (Fig 5) Stalked conidia are observed in both pellicle and sediment The conidia are separated at the distal end of the stalks from parent cells A sediment and a dull, wrinkled climbing pellicle are formed after one month at 17°C Growth in YM agar: After one month at 17°C, the streak culture is light yellow to light orange, wrinkled, dull, or semishining near the bottom, and rough netted, and it has an erose margin Satellite colonies of ballistospores are observed on the agar 308 LUONG et al Dalmau plate culture on corn meal agar: Pseudomycelium is formed after weeks of incubation at 17°C Formation of ballistoconidia: Ballistoconidia are formed abundantly on corn meal agar after days at 17°C (Fig 5) They are symmetrical and apiculate, measuring 2.5–4.0ϫ2.5–6.0 mm Fermentation: Absent Assimilation of carbon compounds: Glucose ϩ Galactose ϩ L-Sorbose ϩ (weak ) Sucrose ϩ Maltose ϩ Cellobiose ϩ Trehalose ϩ Lactose ϩ (latent) Melibiose ϩ (latent) Raffinose ϩ Melezitose ϩ Inulin Ϫ Soluble starch ϩ D-Xylose ϩ L-Arabinose ϩ (latent) D-Arabinose ϩ (latent) D-Ribose ϩ L-Rhamnose ϩ (latent) Ethanol Ϫ Glycerol ϩ (latent and weak) Erythritol ϩ (latent and weak) Ribitol ϩ (latent and weak) Galactitol ϩ (slow) D-Mannitol ϩ D-Glucitol ϩ (weak) a-Methyl-D-glucoside Ϫ Salicin ϩ (weak) Glucono-d-lactone ϩ (weak) 2-Ketogluconic acid Ϫ 5-Ketogluconic acid ϩ (weak) D-Glucuronic acid ϩ D-Galacturonic acid ϩ DL-Lactic acid ϩ (latent and weak) Succinic acid ϩ Citric acid ϩ (weak) Inositol ϩ (slow) Assimilation of nitrogen compounds: Ammonium sulfate ϩ Potassium nitrate Ϫ Sodium nitrite Ϫ Vol 46 Ethylamine hydrochloride Ϫ L-Lysine hydrochloride ϩ Cadaverine dihydrochloride Ϫ Maximum growth temperature: 29–30°C Vitamin required: Thiamine Production of starch-like substances: Negative Diazonium blue B color reaction: Positive Urease: Positive Liquefaction of gelatin: Negative Acid production on chalk agar: Negative GϩC content of nuclear DNA: 53.2 mol% (by HPLC) Ubiquinone system: Q-10 Xylose in the cells: Present Strain examined: Strain VY-61 was isolated by Dao Thi Luong in February 1999 from a leaf of Litsea euosma J J Sm (Lauraceae), collected by Dao Thi Luong and Takashi Nakase at Cuc Phuong National Forest of Ninh Binh, Vietnam This strain has been deposited in JCM and VTCC with the accession numbers JCM 10838 and VTCC1 0176, respectively Kockovaella vietnamensis Luong, Takashima, Ty, Dung & Nakase, sp nov In liquido “YM” post dies ad 17°C, cellulae vegetativae sphaericae vel ovoideae aut ogiveae, 3.0–4.0ϫ 3.0–6.0 m m, singulae, binae, in catenis aut in fasciculis, propagantes formatione conidiorum stipitatorum et gemmarum blasticarum Post unum mensem ad 17°C, pellicula fragilis et sedimentum formantur Cultura in agaro “YM,” gilvus-aurantiaca, rugosa, non nitida et margine erosa Pseudomycelium formatur Ballistoconidia apiculata, 2.5–6.0ϫ2.5–8.0 mm Fermentatio nulla Glucosum, galactosum, L-sorbosum (lente et exiguum), sucrosum, maltosum, cellobiosum, trehalosum, lactosum, melibiosum (lente), raffinosum (lente), melezitosum, amylum solubile, Dxylosum, L-arabinosum (lente), D-arabinosum (lente), D-ribosum, L-rhamnosum (lente), glycerolum (lente et exiguum), erythritolum (lente et exiguum), galactitolum (exiguum), D-mannitolum (exiguum), D-glucitolum (lente et exiguum), salicinum (exiguum), glucuno-d-lactonum (exiguum), acidum 2-ketogluconicum (lente et exiguum), acidum 5-ketogluconicum (exiguum), acidum D-glucuronicum, acidum D-galacturonicum, acidum succinicum, acidum citricum (exiguum) et inositolum (exiguum) assimilantur at non inulinum, ethanolum, ribitolum, a-methyl-D-glucosidum nec acidum DL-lacticum Ammonium sulfatum et L-lysinum 2000 Four new species of Kockovaella assimilantur at non kalium nitricum, natrium nitrosum, cadaverinum nec ethylaminum Maxima temperatura crescentiae: 31–32°C Ad crescentiam thiaminum necessarium est Materia amyloidea iodophila non formantur Ureum hydrolysatur Diazonium caeruleum B: Positivum Proportio molaris guaniniϩcytosini in acido deoxyribonucleico: 54.1 mol% per HPLC Systema ubiquini: Q-10 Xylosum in cellulis presens Holotypus: Isolatus ex folio Bambusae schijostachyoidis, Vietnam, JCM 10841/VTCC1 0179 (originaliter ut VY-127) conservatur in collectionibus culturarum quas “Japan Collection of Microorganisms,” Wako, Saitama et “Vietnam Type Culture Collection,” Hanoi, Vietnam sustentat Growth in YM broth: After days at 17°C, the vegetative cells are spherical to ovoidal with a few ogival ones and measure 3.0–4.0ϫ3.0–6.0 mm They occur singly, in pairs or in groups, and reproduce by budding and stalked conidia (Fig 6) Stalked conidia are observed in both pellicle and sediment The conidia are separated at the distal end of the stalks from the parent cells A sediment and a dull, wrinkled climbing pellicle are formed after one month at 17°C Growth in YM agar: After one month at 17°C, the streak culture is light orange, wrinkled, dull, and rough netted, and it has an erose margin Satellite colonies of ballistoconidia are observed on the agar Dalmau plate culture on corn meal agar: Pseudomycelium is formed after weeks of incubation at 17°C Formation of ballistoconidia: Ballistoconidia are formed abundantly on corn meal agar after days incubation at 17°C (Fig 6) They are symmetrical and apiculate, measuring 2.5–6.0ϫ2.5–8.0 mm Fermentation: Absent Assimilation of carbon compounds: Glucose ϩ Galactose ϩ L-Sorbose ϩ (latent and weak) Sucrose ϩ Maltose ϩ Cellobiose ϩ Trehalose ϩ Lactose ϩ Melibiose ϩ (latent) Raffinose ϩ (latent) Melezitose ϩ Inulin Ϫ Soluble starch ϩ 309 Fig Kockovaella vietnamensis JCM 10841 (A) Vegetative cells grown in YM broth for days at 17°C (B) Ballistoconidia produced on corn meal agar after days at 17°C Scale bars indicate 10 mm ϩ ϩ (slow) D-Arabinose ϩ (slow) D-Ribose ϩ L-Rhamnose ϩ (latent) Ethanol Ϫ Glycerol ϩ (latent and weak) Erythritol ϩ (latent and weak) Ribitol Ϫ Galactitol ϩ (weak) D-Mannitol ϩ (weak) D-Glucitol ϩ (latent and weak) a-Methyl-D-glucoside Ϫ Salicin ϩ (weak) Glucono-d-lactone ϩ (weak) 2-Ketogluconic acid ϩ (latent and weak) 5-Ketogluconic acid ϩ (weak) D-Glucuronic acid ϩ D-Galacturonic acid ϩ DL-Lactic acid Ϫ Succinic acid ϩ Citric acid ϩ (weak) Inositol ϩ (weak) Assimilation of nitrogen compounds: Ammonium sulfate ϩ Potassium nitrate Ϫ Sodium nitrite Ϫ Ethylamine hydrochloride Ϫ L-Lysine hydrochloride ϩ Cadaverine dihydrochloride Ϫ Maximum growth temperature: 31–32°C Vitamin required: Thiamine D-Xylose L-Arabinose 310 LUONG et al Production of starch-like substances: Negative Diazonium blue B color reaction: Positive Urease: Positive Liquefaction of gelatin: Negative Acid production on chalk agar: Negative GϩC content of nuclear DNA: 54.1 mol% (by HPLC) Ubiquinone system: Q-10 Xylose in the cells: Present Strain examined: Strain VY-127 was isolated by Dao Thi Luong in February 1999 from a leaf of bamboo, Bambusa schijostachyoides Kurz, collected by Dao Thi Luong and Takashi Nakase at Cuc Phuong National Forest of Ninh Binh, Vietnam This strain has been deposited in JCM and VTCC with the accession numbers JCM 10841 and VTCC1 0179, respectively Acknowledgments This study was supported in part by special coordination funds for promoting science and technology of the Science and Technology Agency of the Japanese Government References Cañete-Gibas, C F., Takashima, M., Sugita, T., and Nakase, T (1998) Three new species of Kockovaella isolated from plants collected in the Ogasawara Islands J Gen Appl Microbiol., 44, 11–18 Felsenstein, J (1985) Confidence 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Nakase, T (2000) Four new species of the genus Sporobolomyces isolated from leaves in Thailand Mycoscience, 41, 357–369 Tamaoka, J and Komagata, K (1984) Determination of DNA base composition by reversed-phase high-performance liquid chromatography FEMS Lett., 25, 125–128 Thompson, J D., Higgins, D G., and Gibson, T J (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res., 22, 4673–4680 Yarrow, D (1998) Methods for the isolation, maintenance and identification of yeasts In The Yeasts, a Taxonomic Study, 4th ed., ed by Kurtzman, C P and Fell, J W., Elsevier, Amsterdam, pp 77–100  ... other isolates They showed less than 90% similarity for ITS1 and 86.8– 96.4% for ITS2 between closely related species These results indicated that Kockovaella strains isolated from plants of Vietnam. .. nitrate Salient characteristics of Kockovaella isolates and related species Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ lw lw Vitamin requirements and vitamin stimulation Ϫ Thiamine, niacin Ϫ Thiamine Ϫ Thiamine Ϫ Thiamine... was isolated by Dao Thi Luong in February 1999 from leaves of a green brier, Smilax glabra Roxb, and strain VY-67 was isolated from leaves of an aroid, Schimastoglottis calyptrata Zoll & Mor collected