Introduction

The genus Udeniomyces was proposed for threes species, Udeniomyces megalosporus, Udeniomyces puniceus and Udeniomyces pyricola (Nakase and Takematsu 1992), which were classified previously in the ‘pyricola group’ of the genus Bullera (Nakase 1987) and characterized by forming large bilaterally symmetrical ballistoconidia and pinkish-white to pale pink colonies (Nakase 1989). Niwata et al. (2002) described Udeniomyces pannonicus on the basis of its morphological and chemotaxonomic characteristics. However, this species is phylogenetically more closely related to Itersonilia perplexans than to other species of Udeniomyces (Niwata et al. 2002; Takashima and Nakase 2011) and will be excluded from the genus in the future.

An investigation of the diversity of the ballistoconidium-forming phyllosphere yeasts in Kanas Nature Reserve (coordinates: 48°49′N, 87°2′E; altitude: 1,340 m) located in the Altai Mountains in Xinjiang province of China was carried out in July 2004. Among the strains isolated, five with pinkish-white colonies and large bilaterally symmetrical ballistoconidia were classified into one group. Molecular analyses based on 26S rRNA D1/D2 and ITS sequences indicated that the five strains represent a new Udeniomyces species, for which the name U. kanasensis sp. nov. is proposed.

Materials and methods

Fifteen wilting plant leaf samples were collected along the downstream river of Kanas Lake within a distance of approximately 10 km. Yeasts were isolated from the wilting leaves using the ballistoconidia-fall method described by Nakase and Takashima (1993). Morphological, physiological and biochemical characteristics were examined according to standard methods (Kurtzman et al. 2011). Assimilation of nitrogen compounds was investigated on solid media with starved inoculums as described by Nakase and Suzuki (1986). Extraction, purification and identification of ubiquinones were carried out according to Yamada and Kondo (1973).

Nuclear DNA was extracted using the method of Makimura et al. (1994). The DNA fragments covering the 26S rRNA D1/D2 domains and ITS region were amplified and sequenced as described by Bai et al. (2002). The sequences determined in this study and the reference sequences retrieved from GenBank were aligned with the Clustal X program (Thompson et al. 1997). The phylogenetic trees were constructed from the evolutionary distance data calculated from Kimura’s two-parameter model (Kimura 1980) using the neighbor-joining method (Saitou and Nei 1987). Bootstrap analyses (Felsenstein 1985) were performed on 1,000 random resampling.

Results and discussion

Phenotypic characterization

A total of 112 strains were isolated from the fifteen wilting leaf samples. Based on phenotypic properties including the color and texture of colonies, the ability to form ballistoconidia and the shape of vegetative cells and ballistoconidia if produced, the strains were classified into six groups (Table 1). Group 1 included 35 isolates characterized by forming red or orange-red, butyrous or mucoid colonies. Group 2 contained 41 strains characterized by forming white or cream, butyrous or mucoid colonies and subglobose or ellipsoidal cells. Group 3 had 12 strains characterized by forming orange-yellow colonies. Group 4, containing eight isolates, was characterized by forming cream colonies and bilaterally symmetrical ballistoconidia. Group 5, comprising 11 strains, was characterized by forming whitish or brownish-yellow colonies and fusiform cells. Group 6, consisting of five strains, was characterized by forming pinkish-white colonies, long ellipsoidal or ovoid cells and large bilaterally symmetrical ballistoconidia with a size range of (5.5–10) × (10–18) μm (Fig. 1b). The ballistoconidia of this group are morphologically similar to, but somewhat larger than, those formed by Bensingtonia and Sporobolomyces species (Nakase et al. 2011; Hamamoto et al. 2011). Tilletiopsis species also produce bilaterally symmetrical ballistoconidia, which, however, are usually thinner than those formed by the group 6 strains (Boekhout 2011). The ballistoconidia of Bullera species are usually rotationally symmetrical and nearly globose and smaller than those formed by the group 6 strains (Boekhout et al. 2011). Further characterization showed that the major ubiquinone of the strains in group 6 was Q-10. Hyphae, teliospore-like thick-walled cells and sexual structures were not observed in the cultures of single strains or pairwise mixtures of the five strains on corn meal agar, malt extract agar and sucrose-yeast extract agar media at 17°C for 2 months. The results suggest that the five strains belong to the genus Udeniomyces (Takashima and Nakase 2011).

Table 1 Yeast strains from wilting leaves selected for rRNA gene sequence analysis
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Fig. 1
figure 1

U. kanasensis sp. nov. (CGMCC 2.02627T=CBS 12488T) vegetative cells grown in YM broth for 7 days at 17°C (a) and ballistoconidia produced on corn meal agar after 5 days at 17°C (b). Bars, 10 μm

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Molecular phylogeny

Nineteen strains were selected from the six morphological groups for 26S rRNA gene D1/D2 domain and ITS region sequence analyses, resulting in the identification of 13 species distributed in eight genera (Table 1). The five Udeniomyces strains exhibited identical sequences in the D1/D2 domain and ITS region, suggesting they are conspecific. They were clustered in a clade together with U. megalosporus, U. puniceus, U. pyricola and several unpublished strains with strong bootstrap supports in the trees drawn from the D1/D2 domain and ITS region sequences (Fig. 2). In the D1/D2 domain, they differed from the described species U. puniceus, U. megalosporus and U. pyricola by 4, 4 and 11 mismatches, respectively. In the ITS region, they differed from the most closely related species U. puniceus by 38 substitutions and 13 indels and from the other two described Udeniomyces species by more than 15% mismatches in the ITS region. The above data indicated that the five strains represent a distinct novel species closely related to U. puniceus. The name U. kanasensis sp. nov. is therefore proposed.

Fig. 2
figure 2

Phylogenetic trees drawn from neighbor-joining analysis based on sequences of a the 26S rDNA D1/D2 domain and b the ITS region (including 5.8S rDNA), depicting the relationships of the species U. kanasensis sp. nov. with closely related taxa. Bootstrap percentages over 50% from 1,000 bootstrap replicates are shown. Reference sequences were retrieved from GenBank under the accession numbers indicated. The scale bar indicates 1 and 5% sequence divergence for D1/D2 and ITS tree, respectively. Xanthophyllomyces dendrorhous was set as the outgroup

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Four undescribed strains from France, PDD-38b-3 (JN176617), PDD-32b-40 (JF706582), PDD-32b-21 (JF706580) and PDD-32b-71 (JN176594) isolated from cloud water (puy de Dome summit, 1465 m a.s.l., France), were closely related with the five Chinese strain in the D1/D2 tree (Fig. 2a). They differed from the Chinese strains by only 1–3 indels in the D1/D2 domain, suggesting that these French strains may be conspecific with the Chinese strains. However, their taxonomic relationship need be confirmed by ITS sequence analysis. Strains with similar D1/D2 sequences may have quite different ITS sequences in this group. For example, another Chinese strain isolated by us, HS 11.1 (AY841862), possessed identical D1/D2 sequence with that of the type strain of U. pyricola (Fig. 2a); however, it differed from the latter by 15 mismatches (nine substitutions and six indels) in the ITS region (Fig. 2b), indicating strain HS 11.1 may not belong to U. pyricola.

Ecological distribution

The strains of U. kanasensis sp. nov. studied were isolated from wilting leaves of herbaceous and woody plants (Table 1), implying that this species may occur in the phyllosphere of various plants. Number of this species or closely related species may occur in other types of substrates. The four undescribed France strains conspecific or closely related with U. kanasensis sp. nov. were isolated from cloud water. U. puniceus, the close relative of the new species, was first isolated from a frozen fish in Japan and was also isolated from seawater of the Pacific Ocean off the west coast of Baja California, Mexico (Takashima and Nakase 2011). The other described species of the genus usually occur in plant material (Takashima and Nakase 2011). Though Udeniomyces species may occur in different substrates, they are usually psychrophilic. The Kanas Nature Reserve from where the new species was isolated is located in the northern temperate climate zone in China, with the average annual temperature and annual precipitation of −1.0°C and 550–600 mm, respectively. July is the hottest month of 1 year with an average temperature of 16.5°C (Wang et al. 2007). U. kanasensis sp. nov. does not grow at the temperature above 22°C, being consistent with the psychrophilic nature of other four described Udeniomyces species (Nakase 2000; Takashima and Nakase 2011). This indicates that members of the genus Udeniomyces may usually be found in low temperature environments.

Latin diagnosis of U. kanasensis Wang, Bai, Qiu and Han sp. nov

In YM (Difco) liquido post dies 7 ad 17°C, cellulae vegetativae ellipsoideae elongatae aut ovoideae (4.5–8) × (10–16) μm, singulae aut binae. Sedimentum formatur. In agaro YM post unum mensem ad 17°C, cultura roseus-album, glabra, butyracea, margine glabra. Pseudomycelium non formatur. Ballistosporae ellipsoideae vel pyriformes (5.5–10) × (10–18) μm. Fermentatio nulla. Glucosum, saccharosum, maltosum, cellobiosum, trehalosum, raffinosum, melezitosum, amylum solubile (exigue), d-xylosum (exigue), l-arabinosum, d-mannitolum et glucitolum (exigue) assimilantur at non galactosum, l-sorbosum, lactosum, melibiosum, inulin, l-rhamnosum, d-glucosaminum, methanolum, ethanolum, glycerolum, erythritolum, galactitolum, methyl α-d-glucosidum, acidum citricum, acidum dl-lacticum, acidum succinicum, inositolum nec hexadecanum at variabile d-arabinosum, d-ribosum, ribitolum nec salicinum. Ammonium sulfatum, kalium nitricum et natrium nitrosum assimilantur at non l-lysinum, cadaverinum et ethylaminum. Vitaminae externae ad crescentiam necessaria snut. Maxima temperatura crescentiae: 22°C. Materia amyloidea iodophila non formatur. Urea finditur. Diazonium caeruleum B positivum. Ubiquinonum majus: Q-10. Typus: Isolatus ex folio Cotoneaster melanocarpus Lodd., XJ 6E2T, depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (CGMCC 2.02627T=CBS 12488 T).

Description of U. kanasensis Wang, Bai, Qiu and Han sp. nov

In YM broth, after 7 days at 17°C, cells are long ellipsoidal or ovoid (4.5–8) × (10–16) μm (Fig. 1), single or in pairs. Budding is polar. Sediment is formed. After 1 month at 17°C, a ring and sediment are present. On YM agar, after 1 month at 17°C, the streak culture is pinkish-white, smooth. The margin is entire. In Dalmau plate culture on corn meal agar, pseudomycelium is not formed. Ballistoconidia are ellipsoidal or pyriform (5.5–10) × (10–18) μm. Fermentation of glucose is negative. Glucose, sucrose, maltose, cellobiose, trehalose, raffinose, melezitose, soluble starch (weak), d-xylose (weak), l-arabinose, d-mannitol and d-glucitol (weak) are assimilated. Galactose, l-sorbose, lactose, melibiose, inulin, l-rhamnose, d-glucosamine, methanol, ethanol, glycerol, erythritol, galactitol, methyl α-d-glucoside, critic acid, dl-lactic acid, succinic acid, inositol and hexadecane are not assimilated. d-arabinose, d-ribose, ribitol and salicin are variably assimilated. Ammonium sulfate, potassium nitrate and sodium nitrite are assimilated. l-Lysine, ethylamine hydrochloride and cadaverine dihydrochloride are not assimilated. Maximum growth temperature is 22°C. Growth in vitamin-free medium is negative. Starch-like substances are not produced. Growth on 50% (w/w) glucose-yeast extract agar is negative. Urease activity is positive. Diazonium Blue B reaction is positive. The major ubiquinone is Q-10. The type strain, XJ 6E2 T, was isolated from a leaf of Cotoneaster melanocarpus Lodd. collected from Kanas Lake in Xinjiang, China in July, 2004. This strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Academia Sinica, Beijing, China, as CGMCC 2.02627T, and in the Centraalbureau voor Schimmelcultures, Uppsalalaan 83584 CT Utrecht, The Netherlands, as CBS 12488 T. The Mycobank deposit number is MB 563659.

Physiologically, U. kanasensis differs from the four previously described Udeniomyces species by being unable to produce starch-like substances. In addition, U. kanasensis differs from the closely related species U. puniceus in its negative assimilation reactions of ethanol, glycerol, galactitol, succinic acid and citric acid (Table 2).

Table 2 Physiological characteristics that distinguish U. kanasensis sp. nov. from described Udeniomyces species
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Etymology

The specific epithet kanasensis (ka.nas.en’sis N.L. fem. adj.) refers to the geographic origin of the type strain of the species.