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1 College of Life Science, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China
2 Department of Plant Pathology, China Agricultural University, Beijing 100094, China
3 Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, The Chinese Academy of Sciences, Beijing 100080, China
Correspondence
Li-Qun Zhang
zhanglq{at}cau.edu.cn
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the rRNA operon sequences of strains XY103T, XY201T and XY801T are DQ367881–DQ367883.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The variety of Metschnikowia species isolated from fruit surfaces has demonstrated a genetic diversification in the genus Metschnikowia under these specific environmental conditions (Davenport, 1976; Kurtzman & Droby, 2001; Péter et al., 2005). Sequence analyses of the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region among Metschnikowia species isolated from fruits have also indicated that further study on subgenus taxonomy for Metschnikowia is necessary (Giménez-Jurado et al., 2003). In the present study, 138 yeast strains were isolated from jujube fruits (Zizyphus jujuba cultivars ‘Dongzao’ and ‘Junzao’). Eight of them were distinguished from the other strains by conventional and chemotaxonomic characterization. On the basis of the D1/D2 domain and ITS region sequence comparisons, eight strains were classified into three groups representing three novel species in the genus Metschnikowia.
Jujube fruits used in this study were collected from several unmanaged orchards in Shanxi and Shandong Provinces, China, in September and October 2004. Yeast strains were isolated from the surfaces of jujube fruits by the method of Wilson et al. (1993). The isolates were purified by streaking and maintained on YM agar (Difco) at 4 °C. The strains studied are described in Table 1.
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). Assimilation of nitrogen compounds was investigated on solid media with starved inocula (Nakase & Suzuki, 1986). Sporulation was induced under the conditions recommended by Pitt & Miller (1968).
Nuclear DNA was extracted from yeast cells by using the method of Makimura et al. (1994). The ITS (including 5.8S rDNA) region and 26S rDNA D1/D2 domain sequences were determined according to the method described by Lu et al. (2004). Purified PCR products were ligated into the pBluescript SKII(+) vector (Stratagene). Recombined plasmid was transformed into Escherichia coli DH5
and the inserted RNA gene was sequenced by using the ABI BigDye Terminator cycle sequencing kit on an ABI PRISM 377 DNA sequencer. Sequences were aligned with the CLUSTAL X program (Thompson et al., 1997). A phylogenetic tree was constructed from evolutionary distance data calculated with Kimura's two-parameter model (Kimura, 1980) by using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap values were obtained from 1000 replications. Reference sequences were retrieved from GenBank.
Sequence analysis
Sequence analysis of the D1/D2 domain and ITS region is a useful tool for yeast species identification (Kurtzman & Robnett, 1998; Bai et al., 2002; Scorzetti et al., 2002; Dettman et al., 2003). Previous studies of ascomycetous yeasts have demonstrated that strains with more than 1 % substitution in the D1/D2 domain usually represent separate species (Kurtzman & Robnett, 1998).
In this study, three groups represented by strains XY103T, XY201T and XY801T were recognized among the eight strains by D1/D2 and ITS region sequence comparisons. Strains in the same group had identical sequences in the D1/D2 and ITS regions except for strain XY204, which differed from the other strains of the same group by only one nucleotide in the ITS region. Strain XY801T differed from strains XY103T and XY201T by 20 (4 %) and 19 (3.8 %) substitutions in the D1/D2 domain and by 4 (1.2 %) and 27 (7.7 %) substitutions in the ITS region, respectively. Strains XY103T and XY201T could be differentiated from each other by 9 (1.8 %) substitutions in the D1/D2 domain and 21 (6 %) substitutions in the ITS region.
A phylogenetic tree was generated from D1/D2 sequences. The phylogenetic positions of the three strains are depicted in Fig. 1. Strains XY103T, XY201T and XY801T formed a statistically well-supported clade with Metschnikowia chrysoperlae, Metschnikowia pulcherrima, Metschnikowia andauensis, Metschnikowia fructicola and two undescribed Metschnikowia species. Saccharomycete sp. HA1406 was related most closely to strain XY103T, its sequence differing by 4 bp. M. fructicola was the closest known species to strains XY103T and XY201T, differing from XY103T and XY201T by 15 (3.0 %) and 11 (2.2 %) substitutions, respectively, in the D1/D2 domain. Metschnikowia sp. NRRL Y-6148 was the closest yeast to strain XY801T based on its D1/D2 sequence, which was 5 bp different. The closest previously described species to strain XY801T was M. chrysoperlae, which differed from XY801T by 6 (1.2 %) substitutions in the D1/D2 domain and 17 (5.3 %) substitutions in the ITS region. Strains XY103T, XY201T and XY801T had approximately 10–19 (2–3.8 %) nucleotide substitutions from the other closely related known species, M. andauensis and M. pulcherrima, in their D1/D2 sequences (Fig. 1). In view of the number of nucleotide substitutions in the D1/D2 domain and ITS region, strains XY103T, XY201T and XY801T were distinct from each other and from any previously described Metschnikowia species. Although saccharomycete sp. HA1406 and Metschnikowia sp. NRRL Y-6148 were close to strains XY103T and XY801T, respectively, based on the D1/D2 domain, detailed morphological and physiological characteristics of HA1406 and Y-6148 are not available (Kurtzman & Robnett, 1998; Wuczkowski & Prillinger, 2004). It is difficult to determine whether they are conspecific with strains XY103T and XY801T.
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) produced needle-shaped ascospores on diluted (1 : 19) V8 agar after incubation for 14–20 days at 17 °C. Two needle-shaped ascospores formed in a sphaeropedunculate ascus were observed in strains XY103T, XY201T and XY801T (Fig. 2), which is a characteristic typical of some Metschnikowia species (Miller & Phaff, 1998).
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, 2003; Lachance & Bowles, 2002). However, some differential characters are still important. Strains XY103T, XY201T and XY801T were distinguished from each other by their assimilation reactions with D-xylose, salicin and methyl -D-glucoside and the fermentation of galactose (Table 2). Strains XY103T and XY201T could be differentiated from the closely related species M. fructicola by their assimilation of trehalose and their negative reactions for assimilation of hexadecane and fermentation of galactose and their ability to grow on medium without vitamins. Strain XY801T differed from the closely related species M. chrysoperlae by its ability to assimilate D-xylose, its inability to assimilate citrate and its growth on medium without vitamins. Strains XY103T, XY201T and XY801T differed from M. andauensis and M. pulcherrima in assimilation reactions with D-glucosamine and ethylamine and in growth on medium without vitamins.
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Latin diagnosis of Metschnikowia sinensis Xue et Zhang sp. nov.
In medio liquido YM (Difco) post dies 7 ad 25 °C, cellulae vegetativae globosae aut ovoidae (4.5–7.5x3.7–7.5 µm), singulae vel binae. Per gemmationem multipolarem reproducentes. Annulus et sedimentum formantur. Post unum mensem ad 25 °C, annulus et sedimentum formantur. In agaro YM post unum mensem ad 25 °C, butyrosa, brunneusa-cremea, glabra, convexa. In agaro farinae Zea mays post dies 7 ad 25 °C, pseudohyphae et hyphae verae non fiunt. Ascosporae fiunt in agaro V8 post dies 14–20 ad 17 °C. Glucosum et galactosum (infirme) fermentatur at non sucrosum, maltosum, lactosum nec raffinosum. Glucosum, galactosum, L-sorbosum, sucrosum, maltosum, cellobiosum, trehalosum, melezitosum, D-xylosum (lente et infirme), ethanolum, glycerolum, ribitolum, D-mannitolum, methyl -D-glucosidum, salicinum (infirme) et acidum succinicum (infirme) assimilantur at non lactosum, melibiosum, raffinosum, amylum solubile, L-arabinosum, erythritolum, D-arabinosum, D-ribosum, L-rhamnosum, inulinum, D-glucosaminum, methanolum, galactitolum, acidum DL-lacticum, acidum citricum, inositolum nec hexadecanum. Ammonium sulfatum, L-lysinum, ethylaminum et cadaverinum assimilantur at non kalium nitricum nec natrum nitrosum. Ad crescentiam vitaminum non necessarium est (infirme). Maxima temperatura crescentiae: 35 °C. Materia amyloidea iodophila non formantur. Ureum non hydrolysatur. Typus: isolatus ex jujuba, XY103T, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.3110T).
Description of Metschnikowia sinensis Xue & Zhang sp. nov.
Metschnikowia sinensis (sin.en'sis. N.L. fem. adj. sinensis pertaining to China, referring to the geographical origin of the type strain).
Growth in YM broth: after 7 days at 25 °C, cells are globose to oval, 4.5–7.5x3.7–7.5 µm, single or in pairs. Budding is multilateral (Fig. 2a). Spherical, heavy-walled chlamydospore-like ‘pulcherrima’ cells are abundant (Fig. 2b). Sediment and a ring are formed. After 1 month at 25 °C, sediment and a ring are present. Growth on YM agar: after 1 month at 25 °C, colonies are butyrous, brownish-cream, smooth and convex. Dalmau plate culture on cornmeal agar: after 7 days at 25 °C, pseudohyphae or true hyphae are not formed. Formation of ascospores: asci arising from chlamydospores are formed on diluted V8 agar (1 : 19) after incubation for 14–20 days at 17 °C. Asci are sphaeropedunculate, usually 19–33 µm in length, containing one or two needle-shaped ascospores (Fig. 2b). A summary of physiological and other characteristics is given in Table 2.
The type strain, XY103T (=AS 2.3110T=CBS 10357T), was isolated from jujube fruit (Zizyphus jujuba ‘Junzao’) collected in Jiaocheng, Shanxi Province, China, in September 2004.
Latin diagnosis of Metschnikowia zizyphicola Xue et Zhang sp. nov.
In medio liquido YM post dies 7 ad 25 °C, cellulae vegetativae globosae aut ovoidae (3.0–7.5x2.5–7.0 µm), singulae vel binae. Per gemmationem multipolarem reproducentes. Annulus et sedimentum formantur. Post unum mensem ad 25 °C, annulus et sedimentum formantur. In agaro YM post unum mensem ad 25 °C, butyrosa, brunneusa-cremea, glabra, convexa. In agaro farinae Zea mays post dies 7 ad 25 °C, pseudohyphae et hyphae verae non fiunt. Ascosporae fiunt in agaro V8 post dies 14–20 ad 17 °C. Glucosum fermentatur at non galactosum, sucrosum, maltosum, lactosum nec raffinosum. Glucosum, galactosum, L-sorbosum, sucrosum, maltosum, cellobiosum, trehalosum, melezitosum, D-xylosum, ribitolum, ethanolum, glycerolum, D-mannitolum, methyl -D-glucosidum, salicinum et acidum succinicum (infirme) assimilantur at non lactosum, melibiosum, raffinosum, amylum solubile, erythritolum, inulinum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, D-glucosaminum, methanolum, galactitolum, DL-acidum lacticum, acidum citricum, inositolum nec hexadecanum. Ammonium sulfatum, L-lysinum, ethylaminum et cadaverinum assimilantur at non kalium nitricum nec natrum nitrosum. Ad crescentiam vitaminum non necessarium est. Maxima temperatura crescentiae: 35 °C. Materia amyloidea iodophila non formantur. Ureum non hydrolysatur. Typus: isolatus ex jujuba, XY201T, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.3111T).
Description of Metschnikowia zizyphicola Xue & Zhang sp. nov.
Metschnikowia zizyphicola (zi.zy'phi.co.la. N.L. n. Zizyphus generic name of jujube; L. suff. -cola from L. n. incola inhabitant; N.L. n. zizyphicola inhabitant of Zizyphus, referring to the isolation of the type strain from jujube, Zizyphus jujuba).
Growth in YM broth: after 7 days at 25 °C, cells are globose to oval, 3.0–7.5x2.5–7.0 µm, single or in pairs. Budding is multilateral (Fig. 2c). Spherical, heavy-walled chlamydospore-like ‘pulcherrima’ cells are abundant (Fig. 2d). Sediment and a ring are formed. After 1 month at 25 °C, sediment and a ring are present. Growth on YM agar: after 1 month at 25 °C, colonies are butyrous, brownish-cream, smooth and convex. Dalmau plate culture on cornmeal agar: after 7 days at 25 °C, pseudohyphae or true hyphae are not formed. Formation of ascospores: asci arising from chlamydospores are formed on diluted V8 agar (1 : 19) after incubation for 14–20 days at 17 °C. Asci are sphaeropedunculate, usually 20–34 µm in length, containing one or two needle-shaped ascospores (Fig. 2d). A summary of physiological and other characteristics is given in Table 2.
The type strain, XY201T (=AS 2.3111T=CBS 10358T), was isolated from jujube fruit (Zizyphus jujuba ‘Junzao’) collected in Jiaocheng, Shanxi Province, China, in September 2004.
Latin diagnosis of Metschnikowia shanxiensis Xue et Zhang sp. nov.
In medio liquido YM post dies 7 ad 25 °C, cellulae vegetativae globosae aut ovoidae (3.7–7.5x2.5–7.5 µm), singulae vel binae. Per gemmationem multipolarem reproducentes. Annulus et sedimentum formantur. Post unum mensem ad 25 °C, annulus et sedimentum formantur. In agaro YM post unum mensem ad 25 °C, butyrosa, brunneusa-cremea, glabra, convexa. In agaro farinae Zea mays post dies 7 ad 25 °C, pseudohyphae et hyphae verae non fiunt. Ascosporae fiunt in agaro V8 post 14–20 dies ad 17 °C. Glucosum et galactosum (infirme) fermentatur at non sucrosum, maltosum, lactosum nec raffinosum. Glucosum, galactosum, L-sorbosum, sucrosum, ribitolum, maltosum, cellobiosum, trehalosum, melezitosum, D-xylosum, ethanolum, glycerolum, D-mannitolum, methyl -D-glucosidum (lente), salicinum (infirme) et acidum succinicum (infirme) assimilantur at non lactosum, erythritolum, melibiosum, raffinosum, amylum solubile, L-arabinosum, D-arabinosum, D-ribosum, inulinum, L-rhamnosum, D-glucosaminum, methanolum, galactitolum, acidum DL-lacticum, acidum citricum, inositolum nec hexadecanum. Ammonium sulfatum, L-lysinum, ethylaminum et cadaverinum assimilantur at non kalium nitricum nec natrum nitrosum. Ad crescentiam vitaminum non necessarium est (infirme). Maxima temperatura crescentiae: 35 °C. Materia amyloidea iodophila non formantur. Ureum non hydrolysatur. Typus: isolatus ex jujuba, XY801T, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.3112T).
Description of Metschnikowia shanxiensis Xue & Zhang sp. nov.
Metschnikowia shanxiensis (shan.xi.en'sis. N.L. fem. adj. shanxiensis pertaining to Shanxi, referring to the geographical origin of the type strain).
Growth in YM broth: after 7 days at 25 °C, cells are globose to oval, 3.7–7.5x2.5–7.5 µm, single or in pairs. Budding is multilateral (Fig. 2e). Spherical, heavy-walled chlamydospore-like ‘pulcherrima’ cells are abundant (Fig. 2f). Sediment and a ring are formed. After 1 month at 25 °C, sediment and a ring are present. Growth on YM agar: after 1 month at 25 °C, colonies are butyrous, brownish-cream, smooth and convex. Dalmau plate culture on cornmeal agar: after 7 days at 25 °C, pseudohyphae or true hyphae are not formed. Formation of ascospores: asci arising from chlamydospores are formed on diluted V8 agar (1 : 19) after incubation for 14–20 days at 17 °C. Asci are sphaeropedunculate, usually 22–35 µm in length, containing one or two needle-shaped ascospores (Fig. 2f). A summary of physiological and other characteristics is given in Table 2.
The type strain, XY801T (=AS 2.3112T=CBS 10359T), was isolated from jujube fruit (Zizyphus jujuba ‘Junzao’) collected in Jiaocheng, Shanxi Province, China, in September 2004.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Davenport, R. R. (1976). Distribution of yeast and yeast like organisms from aerial surfaces of developing apples and grapes. In Microbiology of Aerial Plant Surfaces, pp. 325–374. Edited by C. H. Dickinson & T. F. Preece. London: Academic Press.
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