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Candida asparagi sp. nov., Candida diospyri
sp. nov. and Candida qinlingensis sp. nov., novel
anamorphic, ascomycetous yeast species
Hui-Zhong Lu, Jian-Hua Jia, Qi-Ming Wang and Feng-Yan Bai
Correspondence
Feng-Yan Bai
baify@sun.im.ac.cn
Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy
of Sciences, Beijing 100080, China
Among ascomycetous yeasts that were isolated from several nature reserve areas in China,
three anamorphic strains isolated from soil (QL 5-5
T
) and fruit (QL 21-2
T
and SN 15-1
T
) were
revealed, by conventional characterization and molecular phylogenetic analysis based on internal
transcribed spacer and large subunit (26S) rRNA gene D1/D2 region sequencing, to represent
three novel species in the genus Candida.Candida qinlingensis sp. nov. (type strain, QL 5-5
T
=AS
2.2524
T
=CBS 9768
T
) was related closely to a teleomorphic species, Williopsis pratensis. The
close relatives of Candida diospyri sp. nov. (type strain, QL 21-2
T
=AS 2.2525
T
=CBS 9769
T
) are
Candida friedrichii and Candida membranifaciens.Candida asparagi sp. nov. (type strain,
SN 15-1
T
=AS 2.2526
T
=CBS 9770
T
) forms a clade with Candida fructus.
In recent years, several hundred ascomycetous yeast strains
have been isolated from various substrates that were
collected in several nature reserve areas in China. After
morphological examination, representative strains were
selected for molecular characterization. The data revealed
that three taxa, represented by three anamorphic strains that
were isolated from the Qinling area, Shanxi Province, and
the Shennongjia area, Hubei Province, China, differed from
described yeast species in both internal transcribed spacer
(ITS) and large subunit (26S) rRNA gene D1/D2 domain
sequences. Phenotypic characterization also indicated that
the three taxa differed remarkably from their close relatives.
Therefore, three novel species are proposed for these strains.
The yeast strains examined were isolated by an enrichment
method using acidified malt extract medium (Yarrow,
1998). Strains QL 5-5
T
and QL 21-2
T
were isolated from soil
and the fruit of a persimmon (Diospyros kaki), respectively.
These samples were collected from Qinling Nature Reserve,
Shanxi Province, China. Strain SN 15-1
T
was isolated from
an Asparagus filicinus fruit that was collected from
Shennongjia Nature Reserve, Huber Province, China. The
type strains of Candida friedrichii (CBS 4114
T
), Candida
fructus (CBS 6380
T
), Candida membranifaciens (CBS
1952
T
), Candida musae (CBS 6381
T
) and Williopsis pratensis
(CBS 6870
T
), employed for ITS sequencing, were obtained
from the Centraalbureau voor Schimmelcultures (CBS),
Delft, The Netherlands.
Morphological, physiological and biochemical character-
istics were examined according to standard methods that are
employed in yeast taxonomy (Yarrow, 1998). Assimilation
of nitrogen compounds was investigated on solid media
with starved inocula (Nakase & Suzuki, 1986).
Nuclear DNA was extracted by the method of Makimura
et al. (1994). A DNA fragment covering the ITS region
(including the 5?8S rRNA gene) and 26S rRNA gene D1/
D2 domain was amplified with the primer pair ITS1 (59-
GTCGTAACAAGGTTTCCGTAGGTG-39) and NL4 (59-
GGTCCGTGTTTCAAGACGG-39). PCR was performed for
36 cycles with denaturation at 94 uC for 1 min, annealing at
55 uC for 1 min and extension at 72 uC for 2 min. After
purification, PCR products were sequenced directly with
the forward primers ITS1 and NL1 (59-GCATATCAATAA-
GCGGAGGAAAAG-39) and the reverse primers ITS4 (59-
TCCTCCGCTTATTGATATGC-39) and NL4, by using an
ABI BigDye Terminator cycle sequencing kit. Electropho-
resis was done on an ABI PRISM 377 DNA sequencer.
Sequences were aligned with the program CLUSTAL_X
(Thompson et al., 1997). A phylogenetic tree was con-
structed from evolutionary distance data that were
calculated with Kimura’s two-parameter model (Kimura,
1980) by using the neighbour-joining method (Saitou &
Nei, 1987). Bootstrap analysis (Felsenstein, 1985) was per-
formed on 1000 random resamplings. Reference sequences
were retrieved from GenBank under the accession numbers
indicated in the tree.
Abbreviation: ITS, internal transcribed spacer.
Published online ahead of print on 23 January 2004 as DOI 10.1099/
ijs.0.03055-0.
The GenBank/EMBL/DDBJ accession numbers for the 26S rRNA
gene D1/D2 and ITS region sequences determined in this study are
AY450916–AY450921 and AY452739–AY452742, respectively.
03055 G2004 IUMS Printed in Great Britain 1409
International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1409–1414 DOI 10.1099/ijs.0.03055-0
Sequence analysis
Databases of 26S rRNA gene D1/D2 sequences are now
available for all currently recognized yeast species
(Kurtzman & Robnett, 1997, 1998; Fell et al., 2000),
resulting in increasing use of this domain for yeast species
identification. Previous studies have shown that strains
with >1 % substitution in the D1/D2 domain usually
represent separate species. The ITS region has also been
proved to be useful for yeast taxonomy, with a similar
amount of intraspecific variation (James et al., 1996; Sugita
et al., 1999; Bai et al., 2001, 2002; Scorzetti et al., 2002;
Kurtzman & Robnett, 2003). Combined sequence analysis
of the D1/D2 and ITS regions for yeast species identifi-
cation has been recommended (Scorzetti et al., 2002).
D1/D2 sequence analysis showed that strain QL 5-5
T
is
related most closely to the ascogenous yeast species
W. pratensis (Fig. 1). However, the sequence of strain
QL 5-5
T
differed from that of W. pratensis by 2?4 % (five
substitutions, nine gaps) in the D1/D2 domain. The ITS
sequence of strain QL 5-5
T
(GenBank no. AY450917)
differed from that of W. pratensis (James et al., 1998) by
10?4 % (seven substitutions, eight gaps) and 5?8 % (nine
substitutions) in the ITS 1 and ITS 2 regions, respectively.
Strain QL 21-2
T
forms a clade with C. friedrichii and
C. membranifaciens (Fig. 1). This strain differed from each
of the latter two species by 1?5 % (six substitutions, two
gaps) in the D1/D2 domain. The ITS regions of strain QL 21-
2
T
(GenBank no. AY450919) and type strains of C. friedrichii
(GenBank no. AY452739) and C. membranifaciens (GenBank
no. AY452740) were sequenced and compared further. In
this region, the sequence of strain QL 21-2
T
differed from
those of C. friedrichii and C. membranifaciens by approxi-
mately 10 % (31–33 substitutions, five or six gaps); the latter
two species differed from each other by 1?8 % (four
substitutions, three gaps).
The close relationship between strain SN 15-1
T
and C. fructus
is depicted in Fig. 1. The D1/D2 region sequence of strain
SN 15-1
T
differed by 2?1 % (10 substitutions, one gap) from
that of C. fructus. The distinction of SN 15-1
T
was further
supported by ITS sequence comparison. The total lengths of
ITS 1 and ITS 2 regions of this group of species were only
about 170 bp. In this region, SN 15-1
T
(GenBank no.
Fig. 1. Phylogenetic tree drawn from neighbour-joining analysis of 26S rRNA gene D1/D2 domain sequences, depicting the
relationships of the three novel Candida species with closely related taxa. Bootstrap percentages >50 % from 1000
bootstrap replicates are shown. Reference sequences were retrieved from GenBank under the accession numbers indicated.
1410 International Journal of Systematic and Evolutionary Microbiology 54
H.-Z. Lu and others
AY450921) differed from C. fructus (GenBank no.
AY452741) by 11?4 % (13 substitutions, six gaps).
Kurtzman & Robnett (1997, 1998) predicted that C. musae
was a synonym of C. fructus, based on observation of their
identical D1/D2 sequences. The present study showed that
the ITS sequence of C. musae (GenBank no. AY452742) was
also identical to that of C. fructus, and thus confirmed the
conspecificity of these two taxa.
Morphology and physiology
As strain QL 5-5
T
was related most closely to the
teleomorphic species W. pratensis, special efforts have
been made to induce its sexual state. Most Williopsis species
form asci and ascospores on 5 % malt extract agar at 25 uC
after 1–3 weeks (Kurtzman, 1998a). However, a sexual state
was not observed in cultures of QL 5-5
T
on the same
medium or other media, including corn-meal agar and
potato dextrose agar. Ascospores of W. pratensis were also
no longer observed (Kurtzman, 1998a). Likewise, sexual
states were not observed in strain QL 21-2
T
or SN 15-1
T
.
Physiologically, strain QL 5-5
T
differed remarkably from
W. pratensis in the fermentation reactions of galactose,
sucrose and maltose and in the assimilation reactions of
L-sorbose and L-arabinose. Strain QL 21-2
T
differed from
C. friedrichii by its inability to assimilate melibiose, raffinose
and galactitol, its ability to assimilate L-rhamnose and
D-glucosamine and its higher maximum growth tempera-
ture. This strain differed from another closely related
species, C. membranifaciens, in the fermentation reactions of
sucrose and raffinose and the assimilation reactions of
melibiose, raffinose, inulin and galactitol. Strain SN 15-1
T
could be differentiated from its closest relative, C. fructus,
by galactose fermentation and galactose, cellobiose,
D-arabinose, methyl a-D-glucoside and salicin assimilation
reactions.
The molecular and physiological comparison made above
demonstrated that strains QL 5-5
T
, QL 21-2
T
and SN 15-1
T
represent three distinct, novel, ascomycetous yeast species.
According to the current taxonomy of yeasts, these species
can be assigned to the genus Candida Berkhout (Kurtzman,
1998b; Meyer et al., 1998). The names Candida qinlingensis
sp. nov., Candida diospyri sp. nov. and Candida asparagi
sp. nov. are therefore proposed for these three novel
anamorphic species.
Latin diagnosis of Candida qinlingensis Bai et
Lu sp. nov.
In medio liquido YM post dies 3ad 25 uC, cellulae globosae
(1?8–6?0mm) vel ellipsoideae (2?0–5?062?5–6?5mm),
cellulae singulae,binae,adhaerentes.per gemmationem
multipolarem reproducentes.Post 1mensem sedimentum
formatur.In agaro YM post 1mensem ad 25 uC, butyrosa,
candida vel cremea,glabra,pauro hebia,margo glabro vel
undulato.In agaro farinae Zea mays post dies 7, pseudohyphae
nullae.Ascomata nulla.Glucosum fermentatur at non
galactosum,sucrosum,maltosum,lactosum nec raffinosum.
Glucosum,galactosum,L-sorbosum,sucrosum,maltosum,
cellobiosum,trehalosum (lente), D-xylosum (lente),
L-arabinosum,ethanolum (lente), glycerolum,D-mannitolum
(lente), D-glucitolum (lente), methyl-a-D-glucosidum,
salicinum,acidum DL-lacticum,acidum succinicum et
acidum citricum (infirme)assimilantur at non lactosum,
melibiosum,raffinosum,melezitosum,inulinum,amylum
solubile,D-arabinosum,D-ribosum,L-rhamnosum,
D-glucosaminum,methanolum,erythritolum,ribitolum,
galactitolum,inositolum nec hexadecanum.Ammonium
sulfatum,ethylaminum,natrum nitrosum,L-lysinum et
cadaverinum assimilantur at non kalium nitricum.Ad
crescentiam vitaminae externae necessariae sunt.Maxima
temperatura crescentiae:30
uC. Materia amyloidea iodophila
non formantur.Diazonium caeruleum Bnon respondens.
Ureum non hydrolysatur.Typus:isolatus ex solis, QL 5-5
T
,
depositus in collectione China General Microbiological
Culture Collection Center, Academia Sinica (AS 2.2524
T
).
Description of Candida qinlingensis Bai & Lu
sp. nov.
Candida qinlingensis (qin.ling.en9sis N.L. fem. adj. qinling-
ensis pertaining to Qinling, the geographical origin of the
type strain of the species).
Growth in YM broth: after 3 days at 25 uC, cells are globose
(1?8–6?0mm) to ellipsoidal (2?0–5?062?5–6?5mm) and
occur singly, in pairs or in groups (Fig. 2a). Budding is
multilateral. After 1 month at 25 uC, sediment is present.
Growth on YM agar: after 1 month at 25 uC, the streak
culture is butyrous, white to cream, smooth and somewhat
dull, with an entire to undulating margin. Dalmau plate
culture on corn-meal agar: after 7 days at 25 uC, pseudo-
hyphae and ascospores are not formed. Glucose is
fermented; galactose, sucrose, maltose, lactose and raffinose
are not. Glucose, galactose, L-sorbose, sucrose, maltose,
cellobiose, trehalose (delayed), D-xylose (delayed),
L-arabinose, ethanol (delayed), glycerol, D-mannitol
(delayed), D-glucitol (delayed), methyl a-D-glucoside,
salicin, DL-lactic acid, succinic acid and citric acid (weak)
are assimilated; lactose, melibiose, raffinose, melezitose,
inulin, soluble starch, D-arabinose, D-ribose, L-rhamnose,
D-glucosamine, methanol, erythritol, ribitol, galactitol,
inositol and hexadecane are not. Ammonium sulfate,
ethylamine hydrochloride, sodium nitrite, L-lysine and
cadaverine dihydrochloride are assimilated; potassium
nitrate is not. Growth in vitamin-free medium is negative.
Maximum growth temperature is 30 uC. Starch-like com-
pounds are not produced. Urease activity is negative.
Diazonium blue B reaction is negative.
The type strain, QL 5-5
T
, was isolated from soil collected in
Qinling, Shanxi Province, China, in October, 2002. This
strain has been deposited in the China General
Microbiological Culture Collection Center (CGMCC),
Academia Sinica, Beijing, China, as AS 2.2524
T
(=CBS
9768
T
).
http://ijs.sgmjournals.org 1411
Three novel Candida species
Latin diagnosis of Candida diospyri Bai et Lu
sp. nov.
In medio liquido YM post dies 3ad 25 uC, cellulae ellipsoideae
vel elongatae (1?8–5?062?0–5?5mm), cellulae singulae,
binae et aggregatae.per gemmationem multipolarem repro-
ducentes.Post 1mensem sedimentum formatur.In agaro YM
post 1mensem ad 25 uC, butyrosa,candida vel cremea,glabra,
pauro hebia,margine glabra.In agaro farinae Zea mays post
dies 7, pseudohyphae nullae.Ascomata nulla.Glucosum et
galactosum fermentatur at non sucrosum,maltosum,lactosum
nec raffinosum.Glucosum,galactosum,L-sorbosum,sucrosum,
maltosum,cellobiosum,trehalosum,melezitosum,D-xylosum,
L-arabinosum,D-arabinosum,D-ribosum,L-rhamnosum,
D-glucosaminum (infirme), ethanolum,glycerolum,erythri-
tolum,ribitolum,D-mannitolum,D-glucitolum,methyl-a-D-
glucosidum,salicinum,acidum succinicum,acidum citricum
(lente)et hexadecanum assimilantur at non lactosum,
melibiosum,raffinosum,inulinum,amylum solubile,metha-
nolum,galactitolum,acidum DL-lacticum nec inositolum.
Ammonium sulfatum, ethylaminum,L-lysinum et cadaver-
inum assimilantur at non kalium nitricum nec natrum
nitrosum.Vitaminae externae ad crescentiam necessariae
sunt.Maxima temperatura crescentiae:35
uC. Materia
amyloidea iodophila non formantur.Diazonium caeruleum
Bnon respondens.Ureum non hydrolysatur.Typus:isolatus ex
fructu Diospyros kaki, QL 21-2
T
,depositus in collectione
China General Microbiological Culture Collection Center,
Academia Sinica (AS 2.2525
T
).
Description of Candida diospyri Bai & Lu
sp. nov.
Candida diospyri (di.os9py.ri. N.L. gen. n. diospyri of
Diospyros, referring to the genus name of Diospyros kaki,
the source of the type strain of the species).
Growth in YM broth: after 3 days at 25 uC, cells are
ellipsoidal to elongate (1?8–5?062?0–5?5mm) and occur
singly, in pairs or in groups (Fig. 2b). Budding is multi-
lateral. After 1 month at 25 uC, sediment is present. Growth
on YM agar medium: after 1 month at 25 uC, the streak
culture is butyrous, white to cream, smooth and somewhat
dull, with an entire margin. Dalmau plate culture on corn-
meal agar: after 7 days at 25 uC, pseudohyphae are not
formed. Ascospores are not formed. Glucose and galactose
are fermented; sucrose, maltose, lactose and raffinose are not
fermented. Glucose, galactose, L-sorbose, sucrose, maltose,
cellobiose, trehalose, melezitose, D-xylose, L-arabinose,
D-arabinose, D-ribose, L-rhamnose, D-glucosamine (weak),
ethanol, glycerol, erythritol, ribitol, D-mannitol, D-glucitol,
methyl a-D-glucoside, salicin, succinic acid, citric acid
(delayed) and hexadecane are assimilated; lactose, meli-
biose, raffinose, inulin, soluble starch, methanol, galactitol,
DL-lactic acid and inositol are not. Ammonium sulfate, L-
lysine, ethylamine hydrochloride and cadaverine dihy-
drochloride are assimilated. Potassium nitrate and sodium
nitrite are not assimilated. Growth in vitamin-free medium
is negative. Maximum growth temperature is 35 uC. Starch-
like compounds are not produced. Urease activity is
negative. Diazonium blue B reaction is negative.
The type strain, QL 21-2
T
, was isolated from Diospyros
kaki fruit collected in Qinling, Shanxi Province, China, in
October 2002. This strain has been deposited in the
CGMCC, Academia Sinica, Beijing, China, as AS 2.2525
T
(=CBS 9769
T
).
Latin diagnosis of Candida asparagi Bai et Lu
sp. nov.
In medio liquido YM post dies 3ad 25 uC, cellulae ellipsoideae
vel elongatae (1?2–4?561?8–5?5mm), cellulae singulae,
(a) (b) (c)
Fig. 2. Vegetative cells of (a) Candida qinlingensis QL 5-5
T
, (b) Candida diospyri QL 21-2
T
and (c) Candida asparagi SN
15-1
T
, grown in YM broth for 3 days at 25 6C. Bars, 10 mm.
1412 International Journal of Systematic and Evolutionary Microbiology 54
H.-Z. Lu and others
binae et adhaerentes.Per gemmationem multipolarem
reproducentes.Post 1mensem sedimentum formatur.In
agaro YM post 1mensem ad 25 uC, butyrosa,cremea,infimo-
convexa,seminitida,margo glabro vel undulato.In agaro
farinae Zea mays post dies 7, pseudohyphae nullae.Ascomata
nulla.Glucosum et galactosum fermentatur at non sucrosum,
maltosum,lactosum nec raffinosum.Glucosum,galactosum,
L-sorbosum,sucrosum,maltosum,cellobiosum,trehalosum,
melezitosum,D-xylosum,D-arabinosum (infirme), D-ribosum
(infirme), D-glucosaminum (lente), ethanolum (lente), gly-
cerolum,ribitolum,D-mannitolum,D-glucitolum,methyl-a-
D-glucosidum (lente), salicinum (lente), acidum succinicum,
acidum citricum (infirme)et hexadecanum (infirme)assim-
ilantur at non lactosum,melibiosum,raffinosum,inulinum,
amylum solubile,L-arabinosum,L-rhamnosum,methanolum,
erythritolum,galactitolum,acidum DL-lacticum nec
inositolum.Ammonium sulfatum,ethylaminum,L-lysinum
et cadaverinum assimilantur at non kalium nitricum nec
natrum nitrosum.Ad crescentiam vitaminae externae
necessariae sunt.Maxima temperatura crescentiae:33
uC.
Materia amyloidea iodophila non formantur.Diazonium
caeruleum Bnon respondens.Ureum non hydrolysatur.Typus:
isolatus ex fructu Asparagus filicinus, SN 15-1
T
,depositus in
collectione China General Microbiological Culture
Collection Center, Academia Sinica (AS 2.2526
T
).
Description of Candida asparagi Bai et Lu
sp. nov.
Candida asparagi (as.pa9ra.gi. N.L. gen. n. asparagi of
Asparagus, referring to the genus name of Asparagus
filicinus, the source of the type strain of the species).
Growth in YM medium: after 3 days at 25 uC, cells are
ellipsoidal to elongate (1?2–4?561?8–5?5mm) and occur
singly, in pairs or in groups (Fig. 2c). Budding is multi-
lateral. After 1 month at 25 uC, sediment is present. Growth
on YM agar medium: after 1 month at 25 uC, the streak
culture is butyrous, cream and semi-glossy, with an entire to
slightly undulating margin. Dalmau plate culture on corn-
meal agar: after 7 days at 25 uC, pseudohyphae are not
formed. Ascospores are not formed. Glucose and galactose
are fermented; sucrose, maltose, lactose and raffinose are not
fermented. Glucose, galactose, L-sorbose, sucrose, maltose,
cellobiose, trehalose, melezitose, D-xylose, D-arabinose
(weak), D-ribose (weak), D-glucosamine (delayed), ethanol
(delayed), glycerol, ribitol, D-mannitol, D-glucitol, methyl
a-D-glucoside (delayed), salicin (delayed), succinic acid,
citric acid (weak) and hexadecane (weak) are assimilated;
lactose, melibiose, raffinose, inulin, soluble starch,
L-arabinose, L-rhamnose, methanol, erythritol, galactitol,
DL-lactic acid and inositol are not. Ammonium sulfate,
ethylamine hydrochloride, L-lysine and cadaverine dihy-
drochloride are assimilated; potassium nitrate and sodium
nitrite are not. Growth in vitamin-free medium is negative.
Maximum growth temperature is 33 uC. Starch-like com-
pounds are not produced. Diazonium blue B reaction is
negative. Urease activity is negative.
The type strain, SN 15-1
T
, was isolated from Asparagus
filicinus fruit collected in Shennongjia, Hubei Province,
China, in October 2002. This strain has been deposited
in the CGMCC, Academia Sinica, Beijing, China, as AS
2.2526
T
(=CBS 9770
T
).
Acknowledgements
This study was supported by grants no. KSCX2-SW-101C from the
Chinese Academy of Sciences and no. 2001AA227131 of the ‘863
program’ from the Ministry of Science and Technology, China.
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