Download Kloeckera taiwanica sp. nov., an ascomycetous apiculate yeast

International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1434–1437
DOI 10.1099/ijs.0.034231-0
Kloeckera taiwanica sp. nov., an ascomycetous
apiculate yeast species isolated from mushroom
fruiting bodies
Chin-Feng Chang,1 Li-Yin Huang,1 Shan-Fu Chen2 and Ching-Fu Lee1
Correspondence
1
Ching-Fu Lee
[email protected]
2
Department of Applied Science, National Hsinchu University of Education, 521 Nanda Road,
Hsinchu 30014, Taiwan, ROC
Department of Hospitality Management, Chu Hwa University of Medical Technology,
91 Wenhua 1st Street, Zender, Tainan 717, Taiwan, ROC
Three apiculate yeast strains, EJ7M09T, GJ5M15 and GJ15M04, isolated from mushrooms in
Taiwan were found to represent a novel species of the genus Kloeckera. The phylogenetically
closest relative of this novel species is Hanseniaspora occidentalis, but the type strain of
H. occidentalis differed by 4.6 % divergence (25 substitutions; 5 gaps) in the sequence of the
D1/D2 domain of the large subunit rRNA gene. This difference clearly suggests that the three
strains represent a distinct species. As none of the strains that were examined in this study
produced ascospores or exhibited conjugation on common sporulation medium either alone or in
a pairwise mixture, this species could be considered as an anamorphic member of the genus
Hanseniaspora, and a novel species, Kloeckera taiwanica sp. nov., is proposed, with EJ7M09T
(5BCRC 23182T5CBS 11434T) as the type strain.
The ascosporogenous genus Hanseniaspora and its anamorphic genus Kloeckera are characterized by bipolar
budding as apiculate yeasts (Smith, 1998a, b). Several
novel apiculate yeasts have been described since Reess
(1870) proposed a novel species Saccharomyces apiculatus
(5Kloeckera apiculata, an anamorph of Hanseniaspora
uvarum) to accommodate yeast strains that reproduce by
bipolar budding. Meyer et al. (1978) carried out detailed
taxonomic studies based on DNA–DNA reassociation and
classified these apiculate yeasts into six species in the genus
Hanseniaspora, proposed by Zikes (1911), and one species
in the anamorphic genus Kloeckera, proposed by Janke
(1923). The classification was confirmed by other molecular studies based on sequence analysis of ribosomal genes
and two protein-coding genes (Boekhout et al., 1994;
Esteve-Zarzoso et al., 2001; Kurtzman & Robnett, 2003;
Yamada et al., 1992), electrophoretic karyotyping and
RAPD-PCR analysis (Cadez et al., 2002). Since then, seven
novel Hanseniaspora species, Hanseniaspora clermontiae, H.
lachancei, H. meyeri, H. opuntiae, H. pseudoguilliermondii,
H. singularis and H. thailandica, and one nonascosporous
Kloeckera species, Kloeckera hatyaiensis, have been reported
(Cadez et al., 2003, 2006; Jindamorakot et al., 2009).
Abbreviation: LSU, large subunit.
The GenBank/EMBL/DDBJ accession numbers for sequences of the
D1/D2 domain of the large subunit rRNA gene of strains EJ7M09T,
GJ5M15 and GJ15M04 are EF653942, FJ873496 and FJ873507,
respectively; accession numbers for the internal transcribed spacer
regions are FJ873604, FJ873578 and FJ873584, respectively.
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Therefore, 13 Hanseniaspora species and two Kloeckera
species are currently recognized.
Members of the genera Hanseniaspora and Kloeckera have
been reported in various habitats such as fruit, flowers, soil
and fermenting juice, as well as insect-associated samples
(Jindamorakot et al., 2009; Cadez et al., 2003, 2006).
During an investigation of yeast diversity in mushrooms in
Taiwan, many Hanseniaspora and Kloeckera strains were
also isolated from the fruiting bodies of mushrooms. Of
these isolates, three strains (EJ7M09T, GJ5M15 and
GJ15M04) were distinctly different from members of
currently recognized species. These strains were highly
similar to each other in their morphological and molecular
characteristics, indicating that they are conspecific. Strains
EJ7M09T, GJ5M15 and GJ15M04 were isolated from the
fruiting bodies of the mushrooms Russula sp., Mycena sp.,
and Bisporella sp., respectively; EJ7M09T was found in
Hsinchu county, whereas GJ5M15 and GJ15M04 were
from Pingtung county, Taiwan. Based on our data, we
propose that these strains represent a novel species.
The yeast strains were isolated from mushroom fruiting
bodies as described by Hsieh et al. (2010). Approximately
1.0 g fruiting body from each sample was placed into a
tube containing 9 ml yeast extract-malt extract (YM) broth
(1 % glucose, 0.5 % peptone, 0.3 % yeast extract, 0.3 % malt
extract, pH 5.4) supplemented with 50 mg chloramphenicol ml21 and then vortex-mixed. One-tenth of a millilitre of successive decimal dilutions was spread on acidified
YM agar (pH 3.5) or DRBC (dichloran rose Bengal
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Kloeckera taiwanica sp. nov.
Fig. 1. Phylogenetic tree drawn from neighbour-joining analysis of the LSU rRNA gene sequence depicting the relationships of
Kloeckera taiwanica sp. nov. with closely related taxa. Bootstrap values over 50 % (1000 bootstrap replicates) are shown.
Reference sequences were from the type strains of species retrieved from GenBank under the accession numbers indicated.
Pichia anomala NRRL Y-366T (U74592) was used as an outgroup. Bar, 0.01 substitutions per position.
chloramphenicol agar; Merck) and the plates were incubated at 24 uC for 3 days. Representative colonies were
selected and the yeasts were purified by streaking onto YM
agar followed by cultivation for 3 days at 24 uC. All of the
isolated strains were subsequently preserved in a freezer at
270 uC and/or on YM agar at 4 uC.
Morphological, physiological and biochemical characteristics were examined by standard methods used for yeast
taxonomy (Yarrow, 1998). The strains used for the
examination of ascospores were incubated alone or in
pairs on potato dextrose agar, McClary’s acetate agar,
cornmeal agar and malt extract agar (Yarrow, 1998) for
1 month at 18 uC and 25 uC.
Nuclear DNA was extracted using a Biokit Genome DNA
Extraction kit. The D1/D2 domain of the large subunit
(LSU) of the rRNA gene was amplified by PCR and
sequenced using the primer pair NL1 and NL4 (Kurtzman &
Robnett, 1998). Sequences were aligned using the program
CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were
constructed by the neighbour-joining method using the
MEGA version 4.0 software package (Tamura et al., 2007)
based on evolutionary distances calculated using the Kimura
2-parameter model. Bootstrap analyses (Felsenstein, 1985)
were performed from 1000 random resamplings.
Sequence comparison and species delineation
Phylogenetic analysis based on sequences of the D1/D2
domain of the LSU rRNA gene showed that there was no
difference between strains GJ5M15 and GJ15M04 and only
one substitution difference between strain EJ7M09T and
the other two strains. Phylogenetic analysis of the D1/D2
domain of the LSU rRNA gene sequence placed the three
http://ijs.sgmjournals.org
strains in the Saccharomyces clade (Kurtzman & Robnett,
1998); this differed by a 4.6 % divergence (25 substitutions,
5 gaps) from the closest relative, strain H. occidentalis
NRRL Y-7946T (Fig. 1). This difference clearly suggests that
the three strains represent a novel yeast species.
The results showed that the phenotypic characteristics of
the novel species are similar to those of H. occidentalis but
the isolates can be distinguished by the assimilation of
maltose and the inability to assimilate glycerol.
Molecular and physiological analysis demonstrated that
these strains were related to members of the genus Hanseniaspora but they did not produce ascospores or exhibit
conjugation in common sporulation medium, either alone
or in pairwise mixtures. As ascosporulation could not be
induced, these yeast isolates are assigned to the genus
Kloeckera, the anamorph state of the genus Hanseniaspora,
as Kloeckera taiwanica sp. nov.; the type strain is EJ7M09T.
Ecology
During a study of yeast diversity in Taiwan between 2006
and 2008, 39 Hanseniaspora- and Kloeckera-related strains
were isolated from various natural samples: 25 strains were
isolated from soil samples; seven strains were isolated from
the fruiting bodies of mushrooms; and seven strains
were isolated from phylloplanes. Based on the molecular,
morphological, and physiological evidence, these 39 isolates were identified as members of seven yeast species of
the genus Hanseniaspora and three yeast species of the
genus Kloeckera (including the novel species, K. taiwanica,
proposed in this study). In this study, the 39 isolates, like
all currently recognized Hanseniaspora and Kloeckera
species, were isolated from various natural substrates. A
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C.-F. Chang and others
relationship was observed between the yeast communities
found and the type of habitat. The genus Hanseniaspora
and its imperfect counterpart Kloeckera are the most
frequently encountered genera of apiculate yeasts in
various natural products (Nakase & Komagata, 1970).
Latin diagnosis of Kloeckera taiwanica Lee
sp. nov.
In medio agaro YM post dies 3 ad 25 uC, cellulae apiculatae,
ovoideae aut ellipsoideae (2–464–12 mm), singulae aut
binae, gemmatione bipolari repruducentes. Post unum mensem ad 25 uC, annulus, pellicilum et sedimentum formatur.
Cultura in agaro YM post dies 3 (25 uC) parva, glabra, nitida,
cremea et margine glabra. Pseudohyphae non formantur. Asci
non formantur. D-Glucosum et sucrosum fermentantur, at
non galactosum, maltosum, methyl a-D-glucosidum, trehalosum, melibiosum, lactatum, cellobiosum, melezitosum, raffinosum, inulinum, amylum solubile et xylosum. Assimilantur
D-glucosum, sucrosum, maltosum (exigue), cellobiosum, salicinum, arbutinum et D-glucono-1,5-lactonum (exigue). Non
assimilantur D-galactosum, L-sorbosum, D-glucosaminum,
D-ribosum, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, trehalosum, methyl a-D-glucosidum, melibiosum,
lactosum, raffinosum, melezitosum, inulinum, amylum solubile,
glycerolum, erythritolum, ribitolum, xylitolum, L-arabitolum,
D-glucitolum, D-mannitolum, galactitolum, myo-inositolum,
2-ketogluconatum, 5-ketogluconatum, acidum D-gluconicum,
D-glucuronatum, acidum galacturonicum, DL-lactatum, succinatum, citratum, methanolum, ethanolum, propane-1,2diolum, butane-2,3-diolum et N-acetyl-D-glucosaminum. Non
assimilantur kalii nitratum, natrium nitrosum, ethylaminum,
L-lysinum, cadaverinum et creatinum. In 25 uC crescit, at non
in 30 uC. Non crescere potest in medio cum 0.01 % cycloheximide, 1 % acido acetico, 10 % NaCl et 50 % glucosum.
Vitaminae externae ad crescentiam necessariae sunt. Amylum
non formatur. Acidum non formatur. Ureum haud hydrolysatum, reactio Diazonium blue B infitialis.
Typus stirps EJ7M09T (5BCRC 23182T5CBS 11434T)
isolatus ex terea, in Hsinchu, Taiwan. Conservatur in
Bioresource Collection and Research Center, Food Industry Research and Development Institute (BCRC), Hsinchu,
Taiwan, et Centraalbureau voor Schimmelcultures (CBS),
Utrecht, The Netherlands.
Description of Kloeckera taiwanica Lee sp. nov.
Kloeckera taiwanica (tai.wa.ni9ca. N.L. fem. adj. taiwanica
of or belonging to Taiwan, where the yeast was originally
isolated).
Grows in YM broth and, after 3 days at 25 uC, cells are
apiculate, ovoid to elongate, 2–464–12 mm, and occur
singly or in pairs (Fig. 2). Asexual reproduction is by
bipolar budding. Sediment is present and a very thin ring
is formed after 1 month. Streak cultures are smooth,
glistening and cream in colour, with entire margins after
1 month at 25 uC. Pseudohyphae are not produced on
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Fig. 2. Morphology of Kloeckera taiwanica EJ7M09T as determined by light microscopy. Vegetative cells grown on YM agar for
3 days at 25 6C. Bar, 10 mm.
cornmeal agar in Dalmau plate cultures after 2 weeks of
incubation. Ascospores are not produced on potato
dextrose agar, McClary’s acetate agar, cornmeal agar or
malt extract agar after incubation at 18 uC and 25 uC.
Glucose and sucrose are fermented, but galactose, maltose,
methyl a-D-glucoside, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and xylose are
not fermented. D-Glucose, sucrose, maltose (weak),
cellobiose, salicin, arbutin, D-glucono-1,5-lactone (weak)
are assimilated, but D-galactose, L-sorbose, D-glucosamine,
D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose,
trehalose, methyl a-D-glucoside, melibiose, lactose, raffinose, melezitose, inulin, soluble starch, glycerol, erythritol,
ribitol, xylitol, L-arabinitol, D-glucitol, D-mannitol, galactitol, myo-inositol, 2-keto-D-gluconate, 5-keto-D-gluconate, D-gluconate, D-glucuronate, D-galacturonic acid,
DL-lactate, succinate, citrate, methanol, ethanol, propane-1,
2-diol, butane-2,3-diol and N-acetyl-D-glucosamine are not
assimilated. Potassium nitrate, sodium nitrite, ethylamine,
L-lysine, cadaverine, and creatine are not utilized. Growth
occurs on YM agar at 25 uC, but not at 30 uC. Growth in
the presence of 0.01 % cycloheximide is negative and no
growth is observed on 1 % acetic acid, 50 % glucose and
10 % NaCl plus 5 % glucose agar. Growth in vitamin-free
medium is negative. Production of starch-like compounds
is absent. Acetic acid production on chalk agar is negative. Diazonium blue B reaction and urease activity are
negative.
The type strain is EJ7M09T (5BCRC 23182T5CBS
11434T), isolated from fruiting body of Russula sp. in
Hsinchu, Taiwan.
Acknowledgements
This work was supported by a Grant-in-Aid from the National
Science Council, Executive Yuan, Taiwan (NSC 97-2621-B-134-001MY3).
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Kloeckera taiwanica sp. nov.
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