Kuraishia MolischianaSp. Nov., the Teleomorph of Candida Molischiana

Abstract

Thirty-two strains, many of them isolated from wood-associated habitats, and designated as Kuraishia (Pichia) capsulata and Candida molischiana according to their phenotype, exhibited two types of HaeIII restriction fragment patterns of their small subunit rDNA with the neighboring ITS. One fragment pattern corresponded to that of the type strain of K. capsulata, whereas the other pattern was unique to the typestrain of C. molischiana. Sequencing of the D1/D2 domain of the large subunit rDNA confirmed that the different HaeIII restriction fragment patterns of small subunit rDNA with the neighboring ITS reliably distinguished K. capsulata from C. molischiana. Ascospore formation was observed in several C. molischiana strains and K. molischiana (type strain: NCAIM Y.01725, CBS 9993) is proposed as the teleomorphic state of Candida molischiana.

Full text

Kuraishia molischiana sp. nov., the teleomorph of Candida molischiana
Ga
´
bor Pe
´
ter
1,
*, De
´
nes Dlauchy
1
, Judit Tornai-Lehoczki
1
and Cletus P. Kurtzman
2
1
National Collection of Agricultural and Industrial Microorganisms, Corvinus University of Budapest, Faculty
of Food Sciences, Somlo
´
iu
´
t 14-16. H-1118 Budapest, Hungary;
2
Microbial Genomics and Bioprocessing
Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S.
Department of Agriculture, 1815 N. University Street, Peoria, Illinois 61604 USA; *Author for correspon-
dence (e-mail: gabor.peter@uni-corv inus.hu, phone: +36-1-482-6322)
Received 2 March 2005; accepted in revised form 12 May 2005
Key words: Kuraishia molischiana, New yeast species, Taxonomy
Abstract
Thirty-two strains, many of them isolated from wood-associated habitats, and designated as Kuraishia
(Pichia) capsulata and Candida molischiana according to their phenotype, exhibited two types of HaeIII
restriction fragment patterns of their small subunit rDNA with the neighboring ITS. One fragment pattern
corresponded to that of the type strain of K. capsulata, whereas the other pattern was unique to the
typestrain of C. molischiana. Sequencing of the D1/D2 domain of the large subunit rDNA confirmed that
the different HaeIII restriction fragment patterns of small subunit rDNA with the neighboring ITS
reliably distinguis hed K. capsulata from C. molischiana. Ascospore formation was observed in several
C. molischiana strains and K. molischiana (type strain: NCAIM Y.01725, CBS 9993) is proposed as the
teleomorphic state of Candida molischiana.
Introduction
Pichia capsulata (Wickerham) Kurtzman (1984a)
was describe d as Hansenula capsulata by Wicker-
ham (1951) based on strains isolated from frass in
the tunnels of larvae underneath the bark of cer-
tain conifers in North America. Together with the
other hat-spored Hansenula species, H. capsulata
was transferred to the genus Pichia by Kurtzman
(1984a). Based on the investigation of partial
sequences of 18S and 26S ribosomal RNAs of the
hat-spored, nitrate-assimilating Pichia species,
Yamada et al. (1994) proposed the genus Kurais-
hia to accommodate P. capsulata. Currently,
Kuraishia capsulata is the single species of the
genus.
Candida molischiana (Zikes) Torula S.A. Meyer
& Yarrow (Yarrow and Meyer 1978) was
described by Zikes (1911) as molischiana. The type
strain of the species was isolated from used
tanning bark. In the second edition of ‘The Yeasts,
A Taxonomic Study’, C. molischiana was consid-
ered to be the anamorphic state of P. capsulata
(Wickerham 1970). Since then, it has been con-
sidered so on the basis of the phenotypic similarity
of the two taxa (Kur tzman 1984b, 1998). How-
ever, Lee and Komagata (1983) noted that the
electophoretic patterns of the cellular enzymes of
the two species were different and raised the pos-
sibility that they may not represent an anamorph–
teleomorph pair. This assumption was confirmed
by Kurtzman and Robnett (1998), who found that
sequences of the D1/D2 domain of large subunit
(26S) rDNA of the type strains of the two species
were different, thus demonstrating that C. moli-
schiana is a distinct species.
Antonie van Leeuwenhoek (2005) 88: 241–247  Springer 2005
DOI 10.1007/s10482-005-7267-3

In the present study, ascospore formation was
observed in several C. molischiana strains. We
propose placement of this new ascosporic species in
the genus Kuraishia Yamada, Maeda & Mikata
(Yamada et al. 1994), as Kuraishia molischiana,the
teleomorph of C. molischiana.
Materials and methods
Organisms and physiological tests
The 32 strains investigated in this study are shown
in Table 1. Phenotypic characterization of the
strains was carried out according to the methods
described by Yarrow (1998).
DNA restriction patterns and sequence analysis
HaeIII restriction enzyme analysis of the small
subunit rDNA with the neighboring ITS region
was carried out according to Dlauchy et al. (1999).
The D1/D2 domain of the large subunit rDNA
from selected strains was sequenced as described
by Kurtzman and Robnett (1998), and those se-
quences were deposited with GenBank. A dataset
of D1/D2 sequences from specie s closely related to
C. molischiana was constructed by searching
GenBank using the BLAST 2.2.10 database search
program (Altschul et al. 1997) followed by se-
quence alignment using the ClustalX 1.81 program
(Thompson et al. 1997). A phylogenetic tree was
constructed by the neighbor-joining method (Sai-
tou and Nei 1987) with Schizosaccharomyces
pombe as the outgroup species. Bootstrap support
for the tree was determined from 1000 replications.
Results and discussion
All K. capsulata and C. molischiana strains inves-
tigated during this study (Table 1) showed a very
similar phenotype including colony appearance,
microscopic morphology and assimilation spec-
trum. However, the HaeIII restriction fragment
patterns of the DNA fragments amplified with
NS1 and ITS2 primers (from small subunit rDNA
with the neighboring ITS region) from the strains
under study formed two different groups (Fig-
ure 1). One restriction fragment pattern was
characterized by an extra band compared to the
other type. The group of strains exhibiting the first
pattern type included the type strain of K. capsu-
lata, while the other group comprising strains with
the extra band included the type strain of C. mo-
lischiana. To decide if the two types of rest riction
fragment patterns corresponded to K. capsulata
and C. molischiana, the D1/D2 domain of the large
subunit rDNA of 10 additional strains was se-
quenced (GenBank accession numbers are given in
Table 1). NRRL Y-1889 and NRRL YB-2441 (C.
molischiana) and NRRL YB-2520 (K. capsulata)
exhibited 100% sequence identity in the D1/D2
region with the corresponding type strains and, for
this reason , the seque nces were not deposited in
GenBank. The results of sequencing confirmed
that the HaeIII restriction fragment patterns of
small subunit rDNA with the neighboring ITS
region reliably distinguish K. capsulata from C.
molischiana. The HaeIII restriction fragmen t pat-
terns of small subunit rDNA without the neigh-
boring ITS region (amplified with NS1 and NS8
primers) made no distinction between the type
strains of the two species indicating that the dif-
ferentiating restriction site of the Hae III enzyme is
situated on the ITS region.
The phylogenetic tree derived from neighbor-
joining analysis of the D1/D2 domains of large
subunit rDNA of the type strains of K. capsulata,
C. molischiana, the teleomorph of C. molischiana
and some related species, is shown in Figure 2. Two
of the C. molischiana strains (NCAIM Y.01428 and
NCAIM Y.01585) and one K. capsulata strain
(NCAIM Y.01428) showed 1 or 2 substitutions,
respectively, in their D1/D2 sequences compared to
that of the type strains of C. molischiana and
K. capsulata. The strain CBS 4327 exhibited three
substitutions and one insertion compared to the
type strain of C. molischiana, which renders them
likely conspecific. However as Lee and Komagata
(1983) detected more than 4% difference between
the GC-content of the two strains, CBS 4327 is
designated here as Kuraishia cf. molischiana. Fur-
ther study is needed for clarifying the taxonomic
status of this strain.
The proposal of the genus Kuraishia (Yamada
et al. 1994), which was based on the comparison of
partial rRNA sequences, was not initially accepted
(Kurtzman, 1998) because the analysis included
relatively few species, leaving uncertain whether
this lineage was unique or part of a yet to be re-
solved larger clade. The study of Kurtzman and
242

Table 1. List of strains used in this study.
Species Strain accession no. GenBank
accession no.
D1/D2 large
subunit
CBS
a
NRRL
b
NCAIM
c
rDNA Source of isolation Ascospores
Kuraishia capsulata 1993
T
Y-1842 Y.01230 U75516
(d)
Insect frass, conifer, Canada +
4306 Soil, Finland +
YB-2236 Lichen, Wyoming, USA +
YB-2512 Insect frass, black spruce,
Ontario, Canada
+
YB-2520 Insect frass, yellow spruce,
Ontario, Canada
+
YB-2754 Insect frass, fir, Tokyo, Japan +
YB-2980 Insect frass, larch, Germany +
YB-2988 Insect frass, larch, Germany +
YB-3002 Insect frass, fir, Germany +
YB-4665 Resin, fir, Quebec, Canada +
9987 Y.01458 AY937231 Rotten wood, black pine,
Pilis Mountain, Hungary

9988 Y.01571 Rotten wood, Scotch pine,
Pilis Mountain, Hungary

9989 Y.01726 Rotten wood, Norway spruce,
Bu
¨
kk Mountain, Hungary
+
Kuraishia molischiana
(Including the anamorph:
Candida molischiana)
136
T(e)
Y-2237 U70178
(d)
Used tanning bark 
836 Y-2238 Non-slimy variant of CBS 136 
837 Y-2234 Water, at 40 C in wood-working
factory, Sweden

4683* Y.01723* AY937234 Soil +
5186 Non-mucoid variant of CBS 837 
7030 Soil, Japan 
Y-1889 Insect frass, red pine, Canada +
YB-2096 Insect frass, loblolly pine, Georgia, USA +
YB-2101 Insect frass, juniper, Montana, USA +
YB-2342 Insect frass, ponderosa pine,
Washington, USA
+
YB-2441 Insect frass, ponderosa pine,
Washington, USA
+
YB-2962 Insect frass, pine, Germany +
YB-3058 Insect frass, white pine, Wisconsin, USA +
9990 Y.01428* AY937232 Fruiting body, mushroom (Suillus sp.)
Pilis Mountain, Hungary
+
9991 Y.01585 AY937233 Rotten wood, black pine,
Pilis Mountain, Hungary
+
9992 Y.01724 Rotten wood, black locust,
Pilis Mountain, Hungary
+
9993 Y.01725
T(f)
AY937235 Rotten wood, European beech,
Pilis Mountain, Hungary
+
Y.01736 AY937236 Faeces, domesticated rabbit,
Budapest, Hungary

Kuraishia cf. molischiana 4327 Y.01719 DQ026030 Soil, Finland 
a
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
b
Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, Peoria, Illinois, USA.
c
National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary.
T
Type strain.
(d)
The sequence was deposited in GenBank by Kurtzman and Robnett (1998).
(e)
Candida molischiana.
(f)
Kuraishia molischiana.
*
Earlier maintained as Kuraishia (Pichia) capsulata.
243

Figure 1. HaeIII restriction analysis of small subunit rDNA with the neighboring ITS region. M: molecular size marker (Sigma P-
9577), lane 1–13: Kuraishia capsulata; lane 1: CBS 1993
T
, lane 2: CBS 4306, lane 3: NRRL YB-2236, lane 4: NRRL YB-2512, lane 5:
NRRL YB-2520, lane 6: NRRL YB-2754, lane 7: NRRL YB-2980, lane 8: NRRL YB-2988, lane 9: NRRL YB-3002, lane 10: NRRL
YB-4665, lane 11: NCAIM Y.01458, lane 12: NCAIM Y.01571, lane 13: NCAIM Y.01726; lane14–16: Kuraishia molischiana; lane 14:
CBS 136 (type strain of C. molischiana) lane 15: CBS 836, lane 16: CBS 837; lane 17: Kuraishia cf. molischiana, CBS 4327; lane 18–32:
Kuraishia molischiana; lane 18: CBS 4683, lane 19: CBS 5186, lane 20: CBS 7030, lane 21: NRRL YB-2096, lane 22: NRRL YB-2101,
lane 23: NRRL YB-2342, lane 24: NRRL YB-2441, lane 25: NRRL YB-2962, lane 26: NRRL YB-3058, lane 27: NCAIM Y.01428,
lane 28: NCAIM Y.01585, lane 29: NCAIM Y.01724, lane 30: NCAIM Y.01725 (type strain of K. molischiana), lane 31: NCAIM
Y.01736, lane 32: NRRL Y-1889 (the restriction pattern of this strain was obtained from a separate run). Those strains, whose D1/D2
domain sequences were determined are indicated with arrows.
Figure 2. Phylogenetic tree showing the placement of Kuraishia molischiana and some closely related species based on analysis of the
D1/D2 domain of large subunit rDNA. Sequences not generated during this study were obtained from GenBank. The tree was
constructed by neighbor-joining analysis of aligned sequences. The numbers at nodes indicate the bootstrap values from 1000 repli-
cations. The scale bar shows the proportional sequence divergence.
244

Robnett (1998), in which all known ascomycetous
yeasts were compared from divergence in the ca. 600
nucleotide sequence of domains 1 and 2 of large
subunit (26S) rDNA, showed K. capsulata and
C. molischiana to be members of a lineage that was
isolated from other known ascosporic genera, thus
lending support to the proposal that the genus Ku-
raishia is unique. Furthermore, the recently described
anamorphic species Candida hungarica and C. cidri
have expanded the Kuraishia clade (Figure 2).
Kuraishia (Pichia) capsulata and C. molischiana
were believed earlier to be conspecific because of
their similar appearance in culture and their
indistinguishable assimilation profiles (Kurtzman
1998). In this study, we were unable to find any
clear-cut differences between the two taxa on
standard physiological tests. Notably, most
C. molischiana strains grew well at 37 C, although
two of them (NRRL YB-2342 and NRRL YB-2441)
failed to do so. In contrast, most K. capsulata strains
failed to grow at 37 C, but one strain (NRRL YB-
3002) grew weakly at this temperature.
Not all strains identified earlier as K. capsulata
formed ascospores, but some strains initially clas-
sified as C. molischiana did form ascospores (Ta-
ble 1). For some C. molischiana strains, in addition
to the one or two-spored asci that are character-
istic for K. capsulata, four-spored asci were also
observed, although at rather low frequency.
Many of the strains examined share similar isola-
tion sources, i.e., they were isolated from wood-
associated habitats (Table 1). Wickerham (1951)
isolated numerous Hansenula capsulata (K. capsulata
and/or C. molischiana) strains from frass or tunnels
of larvae underneath the bark of certain conifers, and
it is likely that one of the primary habitats of the two
species is the larvae of wood-boring insects.
Kuraishia molischiana Dlauchy, Pe
´
ter,
Tornai-Lehoczki and Kurtzman sp. nov.
Status ascigerus Candida molischiana (Zikes) S.A.
Meyer and Yarrow. In agaro malti post dies tres in
25 C cultura est mucoidea vel butyrosa, alba,
glabra et nitida. Margine coloniae integro vel le-
viter undulato. Cellulae sunt spheroidae vel ellip-
soideae (1.2–5.0 · 1.9–6.0 lm), singulae, binae vel
raro racemis parvis conexae, undique gemm antes.
In extracto malti post dies tres in 25 C sedimen-
tum formatur, pellicula non formatur. In agaro
Zea maydis confecto post dies 10 in 25 C nec
pseudohyphae nec hyphae formantur. Asci con-
jugati vel inconjugati et deliquescentes, 1-4 as-
cosporas piliformes, liberi habent.
D-glucosum et trehalosum fermentantur, D-ga-
lactosum, maltosum, sucrosum, lactosum, et
raffinosum, non fermentantur.
D-glucosum, D-glu-
cosaminum, N-acetyl-
D-glucosaminum, D-ribo-
sum,
D-xylosum, L-arabinosum, D-arabinosum
(variabile),
L-rhamnosum (variabile), maltosum,
trehalosum, a-methyl-
D-glucosidum (lente, varia-
bile), cellobiosum, salicinum, arbutinum, lactosum
(tarde, exigue, variabile), melezitosum (variabile),
amylum solubile, glycerolum, meso-erytritolum,
ribitolum, xylitolum,
L-arabinitolum, D-glucito-
lum,
D-mannitolum, 2-ketogluconicum, D-glucon-
icum (variabile), succinatum, (variabilae)
methanolum (variabilae), ethanolum propane-1,
2-diolum (variabile) assi milantur, at non
D-galac-
tosum,
L-sorbosum, sucrosum, melibiosum, raffi-
nosum, inulinum, galactitolum, myo -inositolum,
D-glucuronicum, D-galacturonicum, DL-lactatum,
citratum, saccharatum, butane-2,3-diolum, hexa-
decanum. Kalium nitricum, natrium nitrosum,
ethylaminum hydrochloricum, lysinum, cadaveri-
num dihydrochloricum, glucosaminum assimilan-
tur at non creatinum, creatininum, imidazolum.
Materia amyloidea iodophila non formatur.
Vitamina externa crescentiae sun t necessaria.
Crescere potest in 30 C, in 37 C (variabile), at
non in 45 C. In agaro extracto fermenti confecto
50 partes glucosi per centum non crescit. Parte 0,1
cycloheximidi per mille crescit (variabile). Ureum
non finditur. Diazonium caeruleum B est negativum.
Typus strips NCAIM Y.01725 (CBS 9993).
Description of Kuraishia molischiana Dlauchy,
Pe
´
ter, Tornai-Lehoczki and Kurtzman sp. nov.
Growth on 5% malt extract agar.After3daysat
25 C, the streak culture is mucoid or butyrous,
tannish-white, smooth and glistening. The margin is
entire or slightly undulating. Cells are spherical to
ellipsoidal, 1.2–5.0 · 1.9–6.0 lm. Vegetative repro-
duction proceeds by multilateral budding. Cells are
single, in pairs and, rarely, in small clusters.
Growth in 5% malt extract. After 3 days at
25 C, coherent sediment and usually a thin ring
are present, the latter becomes more pronounced
after prolonged incubation. Pellicles are absent.
245

Dalmau plate on cornmeal agar. After 10 days at
25 C, neither pseudohyphae nor septate hyphae
are formed.
Formation of ascospores. In sporulating strains,
conjugation of independent cells or parent cell–
bud conjugation usually precedes spore formation,
but in some strains unconjugated asci also occa-
sionally occur. One to four easily liberating
hat-shaped ascospores are formed per ascus. Two-
spored asci are predominant (Figure 3), however,
four-spored asci were also observed, though very
rarely and only in three strains. The presence of
heterogamous conjugation is suggestive of homo-
thallism. Ascospores were observed after 5–10
days at 25 C, on at least one of the followin g
media: YM, 5% malt extract and cornmeal agars.
Physiological characteristics. The results of the
physiological characteristics tested are shown in
Table 2.
Type. The type strain was recovered from rotten
wood of Fagus sylvatica in Pilis Mountain (near
Budapest) in Hungary and is maintained as
NCAIM Y.01725 (CBS 9993) in the National
Collection of Agricultural and Industrial Micro-
organisms in Budapest (Hungary). The origin of
the other strains is shown in Table 1.
Etymology. The species epithet molischiana was
selected to emphasize that this species is the tele-
omorph pair of Candida molischiana.
Acknowledgements
We thank Vincent Robert (CBS) for providing the
CBS cultur es for this study. This research was
Figure 3. Kuraishia molischiana NCAIM Y.01725. Ascosporulating culture on cornmeal agar, 5 days, 25 C. Bar = 10 lm.
Table 2. Characteristics of Kuraishia molischiana.
Fermentation
D-Glucose + or s
D-Galactose 
Sucrose 
Maltose 
Lactose 
Raffinose 
a,a-Trehalose + or s
Utilization of carbon sources
D-Glucose +
D-Galactose 
L-Sorbose 
D-Glucosamine +
N-Acetyl-D-glucosamine + or s
D-Ribose +
D-Xylose +
L-Arabinose + or s or l
D-Arabinose v
L-Rhamnose v
Sucrose 
Maltose +
a,a-Trehalose +
Methyl-a-
D-Glucoside s or 
Cellobiose +
Salicin +
Arbutin +
Melibiose 
Lactose  or l,w
Raffinose 
Melezitose v
Inulin 
Starch +
Glycerol +
meso-Erythritol +
Ribitol +
Xylitol +
L-Arabinitol +
D-Glucitol +
246

partly supported by the Bolyai Ja
´
nos Research
Scholarship of the Hungarian Academy of
Sciences.
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Table 2. Continued.
Fermentation
D-Mannitol +
Galactitol 
myo-Inositol 
2-keto-
D-Gluconate +
D-Gluconate v
D-Glucuronate 
D-Galacturonate 
DL-lactate 
Succinate v
Citrate 
Saccharate 
Methanol v
Ethanol +
Propane 1,2 diol v
Butane 2,3 diol 
Hexadecane 
Utilization of nitrogen sources
Potassium Nitrate +
Sodium Nitrite +
Ethylamine hydrochloride +
L-Lysine +
Cadaverine dihydrochloride +
Creatine 
Creatinine 
Glucosamine +
Imidazole 
Growth in vitamin-free medium 
Growth at different temperatures:
30 C+
35 Cv
37 Cv
40 Cv
42 Cv
45 C 
Growth on 50% w/w glucose yeast
extract agar

Growth in 10% NaCl and 5% glucose
in yeast nitrogen base
v
Growth in 16% NaCl and 5% glucose
in yeast nitrogen base

Growth with 0.01% cycloheximide v
Growth with 1% acetic acid 
Formation of amyloid material 
Hydrolysis of urea 
Colour reaction with Diazonium Blue B 
s = slow; l = latent; w = weak, v = variable.
247