Reclassification of the Sporobolomyces roseus - International ...

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2309–2314 DOI: 10.1099/ijs.0.02297-0 

1 Systematic Mycology and 

Lichenology Laboratory, 

Institute of Microbiology, 

Chinese Academy of 

Sciences, Beijing 100080, 

China 

2 Japan Collection of 

Microorganisms, RIKEN 

(The Institute of Physical 

and Chemical Research), 

Wako, Saitama, 351-0198, 

Japan 

3 Yeast Division, 

Centraalbureau voor 

Schimmelcultures, 

Uppsalalaan 8, 3594 CT 

Utrecht, The Netherlands 

4 Yothi Research Unit, 

National Center for 

Genetic Engineering and 

Biotechnology (BIOTEC), 

National Science and 

Technology Development 

Agency (NSTDA), 

73/1 Rama VI Road, 

Bangkok 10400, Thailand 

INTRODUCTION 

Sporobolomyces roseus Kluyver & van Niel is a 

common ballistoconidium-forming yeast species and 

occurs in many different habitats, but most frequently 

in the phyllosphere (Derx, 1930; Tubaki, 1953; Last, 

1955; Nakase, 2000). According to the recent taxo- 

................................................................................................................................................. 

Published online ahead of print on 27 May 2002 as DOI 10.1099/ 

ijs.0.02297-0. 

Abbreviation: ITS, internal transcribed spacer. 

The GenBank accession numbers for the sequences determined in this study 

are AY069991–AY070006 (ITS region) and AY070007–AY070018 (26S rDNA 

D1/D2 domain) as indicated in Fig. 1. 

Reclassification of the Sporobolomyces roseus 

and Sporidiobolus pararoseus complexes, with 

the description of Sporobolomyces phaffii 

sp. nov. 

Feng-Yan Bai, 1 Jian-Hua Zhao, 1 Masako Takashima, 2 Jian-Hua Jia, 1 

Teun Boekhout 3 and Takashi Nakase 2,4 

Author for correspondence: Feng-Yan Bai. Tel: 86 10 6255 5692. Fax: 86 10 6256 0912. 

e-mail: baifysun.im.ac.cn 

More than 50 ballistoconidium-forming yeast strains, isolated from plant 

leaves collected in Yunnan, China, were identified as Sporobolomyces roseus 

Kluyver & van Niel by conventional methods. However, comparison of the 

internal transcribed spacer (ITS) region and 26S rDNA D1/D2 domain sequences 

indicated that these strains represented more than one species. Type or 

authentic strains of the synonyms of Sporobolomyces roseus and the closely 

related species Sporidiobolus pararoseus Fell & Tallman were employed in the 

rDNA sequence comparison. Sporobolomyces boleticola Ramırez, 

Sporobolomyces pollaccii Verona & Ciferri, Sporobolomyces roseus var. 

madurae Janke and Torulopsis somala Verona were confirmed to be conspecific 

with Sporobolomyces roseus. Another synonym of this species, 

Sporobolomyces salmoneus Derx, was located together with Sporobolomyces 

marcillae Santa Maria in a separate clade. Two synonyms of Sporidiobolus 

pararoseus, Sporobolomyces carnicolor Yamasaki & Fujii (nom. inval.) and 

Sporobolomyces japonicus Iizuka & Goto, were revealed to represent two 

distinct species. The name Sporobolomyces carnicolor is validated, with strain 

CBS 4215 T as the type strain. A novel species represented by five of the 

selected Yunnan strains was confirmed, for which the name Sporobolomyces 

phaffii sp. nov. is proposed (type strain CH 2.052 T AS 2.2137 T JCM 11491 T 

CBS 9129 T ). This study also indicates that yeast species with similar ITS 

sequences may have quite different D1/D2 sequences. 

Keywords: Sporobolomyces phaffii sp. nov., Sporobolomyces roseus, Sporidiobolus 

pararoseus, Sporobolomyces carnicolor, Sporobolomyces japonicus 

nomic system of basidiomycetous yeasts (Boekhout & 

Nakase, 1998), more than 50 ballistoconidiumforming 

yeast strains isolated from various wilting 

leaves collected in Yunnan, China, in 1996 were 

identified as Sporobolomyces roseus by conventional 

methods. However, these strains varied to different 

degrees in carbon and nitrogen compound assimilation 

patterns. Comparison of the internal transcribed 

spacer (ITS) region and 26S rDNA D1D2 domain 

sequences of representatives of these strains showed 

that more than one species existed among them. 

Therefore, the species circumscription of Sporobolomyces 

roseus should be redefined. 

The authentic strains of two synonyms of Sporobolo- 

02297 2002 IUMS Printed in Great Britain 2309

F.-Y. Bai and others 

myces roseus, Sporobolomyces ruberrimus Yamasaki & 

Fujii var. ruberrimus (CBS 7500) and Sporobolomyces 

ruberrimus var. albus Yamasaki & Fujii (CBS 7253, 

CBS 7501), have been shown to represent a distinct 

species by D1D2 domain sequence analysis (Fell et 

al., 2000). The type or authentic strains of the 

remaining synonyms of Sporobolomyces roseus were 

employed in the present study, when available from 

culture collections. Since Sporobolomyces roseus is 

phenotypically similar and phylogenetically closely 

related to Sporobolomyces shibatanus (Okunuki) 

Verona & Ciferri, the anamorph of Sporidiobolus 

pararoseus Fell & Tallman (Boekhout, 1991; Boekhout 

& Nakase, 1998; Hamamoto & Nakase, 2000), the 

type strains of the synonyms of the latter species were 

also included. The taxonomic status of these taxa was 

clarified by ITS region and D1D2 domain sequence 

analysis. The taxonomic positions of representative 

Yunnan strains were determined and a hitherto undescribed 

yeast species was found among them. We 

describe the latter as Sporobolomyces phaffii sp. nov., 

in honour of the late Herman J. Phaff. 

METHODS 

Yeast strains and characterization. The yeast strains 

examined are listed in Table 1. The strains from Yunnan 

Table 1. Yeast strains employed 

Strain Source/notes 

Sporidiobolus pararoseus 

CBS 491T Soil 

CBS 484 Type of Sporobolomyces pararoseus 

Sporobolomyces blumeae JCM 10212T Blumea sp. 

Sporobolomyces carnicolor CBS 4215T Unknown 

Sporobolomyces japonicus CBS 5744T Oil brine 

Sporobolomyces marcillae CBS 4217T Air 

Sporobolomyces phaffii sp. nov. 

CH 2.049 Wilting leaf of Ehretia corylifolia 

CH 2.052T Wilting leaf of Nerium indicum 

CH 2.083 Wilting leaf of Oxytenanthera sp. 

CH 2.091 Wilting leaf of Eriobotrya japonica 

CH 2.304 Wilting leaf of Oryza sativa 

Sporobolomyces roseus 

CBS 485 Type of Sporobolomyces pollaccii 

CBS 486T Type of Sporobolomyces roseus 

CBS 993 Type of Torulopsis somala 

CBS 2646 Type of Sporobolomyces roseus var. madurae 

CBS 2840 Authentic strain of Sporobolomyces boleticola 

CBS 2841 Authentic strain of Sporobolomyces boleticola 

CH 2.053 Wilting leaf of Nerium indicum 

CH 2.116 Wilting leaf of Sapindus delavayi 

CH 2.332 Wilting leaf of Nicotiana tabacum 

Sporobolomyces ruberrimus CBS 7500T Air 

Sporobolomyces salmoneus CBS 488T Etiolated grass 

Sporobolomyces sp. CH 2.500 Wilting leaf of Parthenocissus sp. 

were isolated by the improved ballistoconidium-fall method 

(Nakase & Takashima, 1993). Type and authentic strains 

were obtained from the Centraalbureau voor Schimmelcultures 

(CBS), The Netherlands, and the Japan Collection 

of Microorganisms (JCM), Japan. 

Most of the morphological, physiological and biochemical 

characteristics were examined according to standard 

methods commonly employed in yeast taxonomy (Yarrow, 

1998). Assimilation of nitrogen compounds was investigated 

on solid media with starved inocula as described by Nakase 

& Suzuki (1986). Vitamin requirement tests were performed 

according to Komagata & Nakase (1967). 

Extraction, purification and identification of ubiquinones 

were carried out according to Nakase & Suzuki (1986). 

Xylose in the cell hydrolysate was analysed by HPLC as 

described by Suzuki & Nakase (1988). 

ITS region and 26S rDNA D1/D2 domain sequencing. Nuclear 

DNA was extracted using the method of Makimura et al. 

(1994). The DNA fragment covering the ITS region and 26S 

rDNA D1D2 domain was amplified with the primers ITS1 

(5-GTCGTAACAAGGTTTCCGTAGGTG-3) and NL4 

(5-GGTCCGTGTTTCAAGACGG-3). PCR was performed 

for 36 cycles of denaturation at 94 C for 1 min, 

annealing at 52 C for 1 min and extension at 72 C for 

15 min. Cycle sequencing was performed with the forward 

primers ITS1 and NL1 (5-GCATATCAATAAGCGGA- 

GGAAAAG-3) and the reverse primers ITS4 (5-TCCTC- 

CGCTTATTGATATGC-3) and NL4 using the ABI 

2310 International Journal of Systematic and Evolutionary Microbiology 52

BigDye cycle sequencing kit. Electrophoresis was done on 

an ABI PRISM 377 DNA sequencer. 

Molecular phylogenetic analysis. The sequences of the ITS 

regions or 26S rDNA D1D2 domains of the strains 

determined in this study and the reference sequences were 

aligned with the program clustal x (Thompson et al., 1997) 

and adjusted manually. Reference sequences were obtained 

from DDBJEMBLGenBank, where they had been deposited 

by other authors (Fell et al., 2000; Takashima & 

Nakase, 2000). Phylogenetic trees were constructed from the 

evolutionary distance data calculated from Kimura’s twoparameter 

model (Kimura, 1980) using the neighbourjoining 

method (Saitou & Nei, 1987). Bootstrap analyses 

(Felsenstein, 1985) were performed on 1000 random 

resamplings. 

RESULTS AND DISCUSSION 

Taxonomic status of the synonyms of 

Sporobolomyces roseus and Sporidiobolus 

pararoseus 

Boekhout (1991) and Boekhout & Nakase (1998) listed 

15 taxonomic synonyms under Sporobolomyces roseus. 

The type or authentic strains are available from culture 

collections for only eight of them. The sequences of the 

ITS regions and D1D2 domains of these strains were 

determined in the present study, except for the D1D2 

sequence of the type strain of Sporobolomyces 

ruberrimus, which had been determined by Fell et al. 

(2000). 

Among the taxonomic synonyms of Sporidiobolus 

pararoseus listed by Boekhout (1991) and Boekhout & 

Nakase (1998), Sporobolomyces pararoseus Olson & 

Hammer (CBS 484T, mt A1) and Sporobolomyces 

ruber Yamasaki & Fujii (nom. inval., CBS 4216, mt 

A2) have been confirmed to be conspecific with the 

former by mating compatibility and D1D2 

sequencing (Boekhout, 1991; Statzell-Tallman & Fell, 

1998; Fell et al., 2000). Sporobolomyces marcillae 

Santa Maria was shown to be a distinct species by 

D1D2 sequencing (Fell et al., 2000). The type or 

authentic strains of the remaining two synonyms of 

Sporidiobolus pararoseus, Sporobolomyces carnicolor 

Yamasaki & Fujii (nom. inval.) and Sporobolomyces 

japonicus Iizuka & Goto, were used in this study for 

ITS and D1D2 sequencing. In addition, the ITS 

sequence of the type strain of Sporobolomyces 

marcillae was determined. 

The relationships among the taxa studied are depicted 

by the phylogenetic trees drawn from the sequences of 

the ITS region (Fig. 1a) and the D1D2 domain (Fig. 

1b). Sporidiobolus johnsonii and Sporidiobolus salmonicolor 

were used as outgroups. The recently described 

species Sporobolomyces blumeae Takashima & Nakase 

(2000) formed a basal branch in both trees. The other 

taxa clustered around Sporobolomyces roseus and 

Sporidiobolus pararoseus. In the ITS tree (Fig. 1a), 

both clades were supported strongly (99–100%) by 

bootstrap analysis. In the D1D2 tree (Fig. 1b), the 

Sporobolomyces roseus clade was strongly supported 


(bootstrap value 100%), but the Sporidiobolus 

pararoseus clade was not (bootstrap value 50%). 

Among the synonyms of Sporobolomyces roseus, 

Sporobolomyces boleticola Ramırez, Sporobolomyces 

pollaccii Verona & Ciferri, Sporobolomyces roseus var. 

madurae Janke and Torulopsis somala Verona were 

confirmed to be conspecific with Sporobolomyces 

roseus, because they have ITS and D1D2 sequences 

that are identical or very similar (only one nucleotide 

substitution) to those of the type strain of Sporobolomyces 

roseus. 

Sporobolomyces salmoneus Derx, a synonym of Sporobolomyces 

roseus, was clearly separated from the 

Sporobolomyces roseus clade in both trees, and 

clustered together with Sporobolomyces marcillae in 

the Sporidiobolus pararoseus clade. The type strains of 

Sporobolomyces salmoneus and Sporobolomyces 

marcillae have identical D1D2 sequences and differ 

by only 1 nt in their ITS regions. 

Two synonyms of Sporidiobolus pararoseus, Sporobolomyces 

carnicolor and Sporobolomyces japonicus, 

respectively differed from the type strain by 19–24 and 

7–8 nt in the ITS region and D1D2 domain. They also 

differed from one another remarkably (Fig. 1). Sporobolomyces 

carnicolor and Sporobolomyces japonicus 

should therefore be reinstalled as two distinct species. 

Sporobolomyces carnicolor was proposed by Yamasaki 

& Fujii (1950) without a Latin description or type 

designation, and therefore needs to be validated. 

Taxonomic status of the Sporobolomyces roseus 

strains from Yunnan 

A total of 670 yeast strains were isolated from 43 

wilting leaf samples collected in Yunnan, China, in 

1996 using the improved ballistoconidium-fall method 

(Nakase & Takashima, 1993). Approximately 100 

ballistoconidium-forming yeast strains were initially 

selected for phenotypic characterization. Of them, a 

total of 55 strains were identified as Sporobolomyces 

roseus according to Boekhout (1991) and Boekhout & 

Nakase (1998). Of nine representative strains (Table 1) 

selected for ITS sequencing, strains CH 2.053, CH 

2.116 and CH 2.332 were confirmed to belong to 

Sporobolomyces roseus (Fig. 1a). The sequence of 

strain CH 2.500 differed from that of the type by 5 nt 

(14%). 

The other five strains, CH 2.049, CH 2.052, CH 2.083, 

CH 2.091 and CH 2.304, had identical ITS sequences 

and the sequence differed from that of the type strain 

of Sporobolomyces ruberrimus by 3 nt (10%). Previous 

studies on basidiomycetous yeasts have indicated that 

conspecific strains usually have less than 1% 

nucleotide divergence in the ITS1 and ITS2 regions 

overall (Sugita et al., 1999a, b; Takashima & Nakase, 

2000). However, Bai et al. (2001a, b) found that 

conspecific strains might have up to 5–7 nt differences 

(approx. 2%) in the ITS regions. Therefore, the 

taxonomic relationships of these five strains with 

http://ijs.sgmjournals.org 2311


(a) 

(b) 

Sporobolomyces ruberrimus and that of strain CH 

2.500 with Sporobolomyces roseus were examined 

further by D1D2 sequencing. 

Strains CH 2.052, CH 2.083, CH 2.091 and CH 2.304 

had identical D1D2 sequences and differed from CH 

2049 by only 1 nt. They differed from Sporobolomyces 

ruberrimus in as many as 18–19 nt (30%) in the 

D1D2 domain (Fig. 1b). These results indicate that 

these strains represent a distinct species, for which the 

name Sporobolomyces phaffii sp. nov. is proposed. 

Strain CH 2.500 differed from the type strain of 

Sporobolomyces roseus by 3 nt in the D1D2 domain, 

suggesting that this strain probably represents a 

distinct species. A definite taxonomic decision for 

strain CH 2.500 should be supported by additional 

data, for example, DNA–DNA reassociation, and 

perhaps by the identification of additional strains. 

..................................................................................................... 

Fig. 1. Phylogenetic trees drawn from 

neighbour-joining analysis based on 

sequences of (a) the ITS region (including 

58S rDNA) and (b) the D1/D2 domain, 

depicting the relationships of taxa in the 

Sporobolomyces roseus and Sporidiobolus 

pararoseus complexes, as well as the novel 

isolates from Yunnan, China. Bootstrap 

percentages over 50% from 1000 bootstrap 

replicates are shown. 

The significance of phenotypic and phylogenetic 

comparison 

The novel species Sporobolomyces phaffii is indistinguishable 

from Sporobolomyces roseus by conventional 

characterization. The three species Sporobolomyces 

carnicolor, Sporobolomyces japonicus and 

Sporidiobolus pararoseus also have almost identical 

phenotypes. Sporobolomyces roseus and Sporobolomyces 

phaffii can be differentiated from the latter 

three species in their ability to assimilate nitrate and 

nitrite. Though the type strains of Sporobolomyces 

salmoneus and Sporobolomyces marcillae have identical 

D1D2 sequences and very similar ITS sequences, 

their nitrogen assimilation patterns are completely 

different. Sporobolomyces salmoneus can utilize nitrate, 

nitrite and ethylamine hydrochloride as sole sources of 

nitrogen, whereas Sporobolomyces marcillae can not. 

This explains why the former was previously regarded 


as a synonym of Sporobolomyces roseus and the latter 

a synonym of Sporidiobolus pararoseus (Boekhout, 

1991). The taxonomic relationship between Sporobolomyces 

salmoneus and Sporobolomyces marcillae 

should be studied further. 

The relationships among the taxa in the Sporidiobolus 

pararoseus complex revealed by the ITS sequences are 

discordant with those revealed by the D1D2 sequence 

data, especially for the relationship between Sporobolomyces 

phaffii sp. nov. and Sporobolomyces 

ruberrimus (Fig. 1). Analysis of 18S rDNA sequences 

may be helpful to confirm the phylogenetic relationships 

among these species. Fell et al. (2000) found 

that some basidiomycetous yeast species with identical 

or similar D1D2 sequences could be separated 

by ITS sequence data. On the other hand, the present 

study indicates that species with similar ITS sequences 

may have quite different D1D2 sequence data. 

Latin diagnosis of Sporobolomyces carnicolor 

Yamasaki & Fujii ex Bai & Boekhout 

In YM liquido post dies 3 ad 25 C, cellulae vegetativae 

ovoideae, ellipsoideae vel elongatae, 25–6235– 

17 µm, singulae, binae vel aggregatae. Post unum 

mensem ad 20 C, annulus, pelliculum et sedimentum 

formantur. In agaro YM post unum mensem ad 20 C, 

cultura rubro-aurantiaca, rugosa, margine erosa. Mycelium 

et pseudomycelium non formantur. Ballistosporae 

ovoideae vel ellipsoideae, 25–3762–102 µm. 

Fermentatio nulla. Glucosum, galactosum (lente), lsorbosum 

(lente), saccharosum, maltosum, cellobiosum, 

trehalosum, raffinosum, melezitosum, inulinum (lente), 

amylum solubile (lente), d-xylosum (lente), d-arabinosum, 

d-ribosum, ethanolum, glycerolum, ribitolum 

(lente), d-mannitolum, glucitolum, methyl α-d-glucosidum 

(lente), salicinum, glucono-δ-lactonum, acidum 

succinicum et acidum citricum assimilantur at non lactosum, 

melibiosum, l-arabinosum, l-rhamnosum, erithritolum, 

galactitolum, acidum 2-ketogluconicum, acidum 

5-ketogluconicum, acidum dl-lacticum, inositolum, 

acidum d-glucuronicum nec acidum d-galacturonicum. 

Ammonium sulfatum, l-lysinum et cadaverinum assimilantur 

at non kalium nitricum, natrum nitrosum nec 

ethylaminum. Ad crescentiam vitaminum non necessarium 

est. Materia amyloidea iodophila non formantur. 

Urea finditur. Diazonium caeruleum B positivum. Ubiquinonum 

majus: Q-10. Typus: CBS 4215T, depositus in 

collectione Centraalbureau voor Schimmelcultures, 

Utrecht, The Netherlands. 

Latin diagnosis of Sporobolomyces phaffii Bai, 

Takashima & Nakase sp. nov. 

In YM (Difco) liquido post dies 3 ad 25 C, cellulae 

vegetativae ovoideae vel ellipsoideae, 20–4040– 

48 µm, singulae aut binae. Post unum mensem ad 20 C, 

annulus, pelliculum et sedimentum formantur. In agaro 

YM post unum mensem ad 20 C, cultura rubroaurantiaca, 

glabra, nitida, et margine glabra. Mycelium 

et pseudomycelium non formantur. Ballistosporae ovoi- 


deae vel ellipsoideae,20–2280–90 µm. Fermentatio 

nulla. Glucosum, saccharosum, maltosum, cellobiosum, 

trehalosum, melibiosum, raffinosum, melezitosum, amylum 

solubile, ethanolum (lente), d-mannitolum (lente), 

methyl α-d-glucosidum, salicinum, glucono-δ-lactonum 

et acidum succinicum assimilantur at non galactosum 

(vel exigue et lente), l-sorbosum, lactosum, inulinum, dxylosum, 

l-arabinosum, d-arabinosum, d-ribosum, lrhamnosum, 

glycerolum, erithritolum, ribitolum, galactitolum, 

glucitolum (vel exigue et lente), acidum 2ketogluconicum, 

acidum 5-ketogluconicum, acidum dllacticum, 

acidum citricum, inositolum, acidum d-glucuronicum 

nec acidum d-galacturonicum. Ammonium 

sulfatum, kalium nitricum, natrum nitrosum assmilantur, 

ethylaminum (variabile) et l-lysinum (fortasse 

exigue) assimilantur at non cadaverinum (vel exigue). 

Ad crescentiam vitaminum non necessarium est. 

Maxima temperatura crescentiae: 32–33 C. Materia 

amyloidea iodophila non formantur. Urea finditur. 

Diazonium caeruleum B positivum. Ubiquinonum 

majus: Q-10. Xylosum in cellulis absens. 

Typus: Isolatus ex folio Nerii indici Mill., AS 2.2137T 

(originaliter ut CH 2.052T) depositus in collectione 

China General Microbiological Culture Collection 

Center, Academia Sinica, Beijing. 

Description of Sporobolomyces phaffii Bai, 

Takashima & Nakase sp. nov. 

Sporobolomyces phaffii (phaffi.i. N.L. gen. n. phaffii in 

honour of the late Herman J. Phaff, USA). 

In YM broth, after 3 days at 25 C, the cells are ovoid 

to ellipsoidal, 20–4040–80 µm (Fig. 2a). A ring, 

pellicle and sediment are formed. After 1 month at 

17 C, a ring, pellicle and sediment are present. On YM 

agar, after 3 days at 25 C, the streak culture is 

butyrous, smooth, glistening with orange to orangered 

colour. After 1 month at 20 C, the culture is 

butyrous and becomes mucoid or reticulate in some 

areas, orange-red, with the margin entire to eroded. 

Mycelia and pseudomycelia are not formed on Dalmau 

plate culture on corn meal agar. On corn meal agar, 

ballistoconidia are formed on short sterigmata, ellipsoidal 

or ovoid, 20–2280–90 µm (Fig. 2b). 

Glucose is not fermented. The following carbon 

compounds are assimilated: glucose, sucrose, maltose, 

cellobiose, trehalose, melibiose, raffinose, melezitose, 

soluble starch, ethanol (delayed), d-mannitol 

(delayed), methyl α-d-glucoside, salicin, glucono-δlactone, 

succinic acid and citric acid. The following are 

not assimilated: galactose (or weak and delayed), lsorbose, 

lactose, inulin, d-xylose, l-arabinose, darabinose, 

d-ribose, l-rhamnose, glycerol, erythritol, 

ribitol, galactitol, glucitol (or weak and delayed), 2ketogluconic 

acid, 5-ketogluconic acid, dl-lactic acid, 

citric acid, inositol, d-glucuronic acid and dgalacturonic 

acid. KNO , NaNO , ethylamine (variable) 

and l-lysine (or weak) 

 

are utilized 

 

as sole sources 

of nitrogen; cadaverine is not utilized or is utilized 

weakly. Growth in vitamin-free medium is positive. 

http://ijs.sgmjournals.org 2313


(a) 

(b) 

................................................................................................................................................. 

Fig. 2. Sporobolomyces phaffii sp. nov. CH 2.052 T . (a) 

Vegetative cells grown in YM broth for 5 days at 17 C. (b) 

Ballistoconidia produced on corn meal agar after 5 days at 

17 C. Bars, 10 µm. 

Maximum growth temperature is 32–33 C. Starchlike 

compounds are not produced. Urease activity is 

positive. Diazonium blue B reaction is positive. The 

major ubiquinone is Q-10. Xylose is absent in the 

whole-cell hydrolysate. 

The type strain, strain CH 2.052T ( JCM 11491T 

CBS 9129T), was isolated in 1996 from a wilting leaf of 

Nerium indicum Mill. collected in Yunnan, China. This 

strain has also been deposited in the China General 

Microbiological Culture Collection Center 

(CGMCC), Institute of Microbiology, Chinese Academy 

of Sciences, Beijing, China, as strain AS 2.2137T. 

ACKNOWLEDGEMENTS 

This study was supported by grants no. 30170002 from the 

National Natural Science Foundation of China (NSFC) and 

no. KSCX2-SW-101C from the Chinese Academy of 

Sciences. 

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