International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1887–1892
DOI : 10.1099/ijs.0.02209-0
Udeniomyces pannonicus sp. nov., a
ballistoconidium-forming yeast isolated from
leaves of plants in Hungary
1
2,3
Japan Collection of
Microorganisms, RIKEN
(The Institute of Physical
and Chemical Research),
Wako, Saitama 351-0198,
Japan
National Collection of
Agricultural and
Industrial Microorganisms2 and
Department of
Microbiology3 , Szent
Istva! n University, H-1118,
Budapest, Somlo! i u! t 1416, Hungary
Yasushi Niwata,1 Masako Takashima,1 Judit Tornai-Lehoczki,2
Tibor Deak3 and Takashi Nakase1†
Author for correspondence : Masako Takashima. Tel : j81 48 467 9560. Fax : j81 48 462 4617.
e-mail : masako!jcm.riken.go.jp
Fifteen ballistoconidium-forming yeasts, isolated from the leaves of plants in
Hungary, showed morphological, physiological and biochemical characteristics
similar to those of Udeniomyces pyricola. The identical sequences of internal
transcribed spacer regions for selected strains (HY-16T, HY-29, HY-111 and HY186) indicated that they should be classified as one species. Although a
representative strain, HY-16T, showed a closer relationship to Itersonilia
perplexans than to known Udeniomyces species in phylogenetic trees
constructed using 18S rDNA and the D1/D2 region of the 26S rDNA sequence,
this species was placed in the genus Udeniomyces on the basis of its
morphological and chemotaxonomic characteristics. Udeniomyces pannonicus
sp. nov. (type strain HY-16T l JCM 11145T l NCAIM Y 01556T l CBS 9123T) is
proposed.
Keywords : yeast, Udeniomyces pannonicus, Hungary
INTRODUCTION
The genus Udeniomyces Nakase & Takematsu was
established in 1992 for three species, Udeniomyces
pyricola (Stadelmann) Nakase & Takematsu, Udeniomyces puniceus (Komagata & Nakase) Nakase &
Takematsu and Udeniomyces megalosporus (Nakase
& M. Suzuki) Nakase & Takematsu (Nakase &
Takematsu, 1992). Prior to this paper, the genus
Bullera Derx had been known as a hymenomycetous
ballistoconidium-forming yeast, but it was reported to
be divided into two groups, the typical Bullera group
and the ‘ pyricola group ’, based on the morphology of
the ballistoconidia and the colour of colonies (Nakase,
1987, 1989).
Typical Bullera species produce symmetrical ballistoconidia and pale-coloured or orange colonies, while
species in the ‘ pyricola group ’ produce large, asymmetrical ballistoconidia and pinkish-white to palepink colonies. On the basis of the phylogenetic analyses
.................................................................................................................................................
† Present address : Yothi Research Unit, National Centre for Genetic
Engineering and Biotechnology (BIOTEC), National Science and Technology
Development Agency, 73/1 Rama VI Road, Bangkok 10400, Thailand.
Abbreviation : ITS, internal transcribed spacer.
The GenBank/EMBL /DDBJ accession numbers for the sequences determined
in this study are AB072225–AB072233 and AB077382.
of partial sequences of 18S rRNA, the ‘ pyricola group ’
was found to be phylogenetically far from Bulleromyces albus Boekhout & A. Fonseca [teleomorph of
Bullera alba (Hanna) Derx, type species of the genus]
and formed a single branch (Nakase et al., 1993). The
genus Udeniomyces was established on the basis of this
phylogenetic divergence (Nakase & Takematsu, 1992).
Suh & Nakase (1995) also drew a phylogenetic tree
based on almost complete 18S rDNA sequences, which
supported this separation of the genus Udeniomyces.
Fell et al. (2000) reported that this genus fits into the
Cystofilobasidiales Boekhout & Fell together with the
non-ballistoconidium-forming yeasts Cystofilobasidium Oberwinkler & Bandoni and Mrakia Y. Yamada
& Komagata in a phylogenetic tree based on the
sequences of the D1\D2 region of the 26S rDNA. In
the typical Bullera species, which produce symmetrical
ballistoconidia, phylogenetic heterogeneity has also
been reported (Suh & Nakase, 1995 ; Suh et al., 1996 ;
Takashima & Nakase, 1999 ; Fell et al., 2000), and
Takashima et al. (2001) recently restated the genus
Dioszegia Zsolt for one phylogenetically distinct
group.
In 1999, we isolated 204 ballistoconidium-forming
yeasts from the leaves of plants collected in various
places in Hungary. Fifteen strains were selected by
their morphological, physiological, biochemical and
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Y. Niwata and others
chemotaxonomic characteristics and used in this study.
They proliferate by means of budding cells and
ballistoconidia, possess Q-10 as a major ubiquinone
and contain xylose in the cells. Phylogenetically, they
showed an affinity with the Cystofilobasidiales on the
basis of sequences of the 18S rDNA and the D1\D2
region of the 26S rDNA, indicating that they belonged
to the genus Udeniomyces. A novel species, Udeniomyces pannonicus sp. nov. (type strain HY-16T l JCM
11145T l NCAIM Y 01557T l CBS 9123T), is proposed in this paper.
METHODS
Yeast strains. The strains used in this study, and their
sources, are shown in Table 1. They were isolated using the
ballistoconidium-fall method with YM agar (Difco) plates
(Nakase & Takashima, 1993).
Morphological, physiological and biochemical characteristics.
Most of the methods used to examine morphological,
physiological and biochemical characteristics were those
described by Yarrow (1998). Assimilation of nitrogen
compounds was investigated on solid media, using starved
inocula according to the method of Nakase & Suzuki (1986).
Vitamin requirements were investigated by following the
method of Komagata & Nakase (1976). The maximum
growth temperature was determined in YM broth (Difco)
using metal block heaters.
For analysis of the ubiquinone system, cells were grown in
500 ml Erlenmeyer flasks containing 250 ml YM broth on a
rotary shaker at 100 r.p.m. at 17 mC and were harvested in
the early stationary growth phase. The cells were washed
with distilled water. The extraction and purification of
ubiquinone was carried out according to the method of
Nakase & Suzuki (1986).
Xylose in the cells. Cells were grown in 500 ml Erlenmeyer
flasks containing 250 ml YM broth on a rotary shaker at
100 r.p.m. at 17 mC and were harvested in the early stationary
growth phase ; they were then washed with distilled water.
Acid hydrolysis of whole cells was performed as described by
Suzuki & Nakase (1988). The presence of xylose in the
whole-cell hydrolysate was analysed with an HPLC reducing-sugar analysis system (Shimadzu) according to the
instructions of the manufacturer.
DNA base composition. Cells were grown in 500 ml
Erlenmeyer flasks containing 250 ml YM broth on a rotary
shaker at 100 r.p.m. at 17 mC and were harvested in the
exponential growth phase and then washed with distilled
water. Isolation and purification of DNA were carried out
according to Takashima & Nakase (2000). The DNA base
composition was determined by HPLC after enzymic digestion of DNA to deoxyribonucleosides (Tamaoka &
Komagata, 1984). A DNA-GC kit (Yamasa Shoyu) was
used as a quantitative standard.
Nucleotide sequences and phylogenetic analysis. Nuclear
DNA was extracted by the method of Makimura et al.
(1994). The 18S rDNA and internal transcribed spacer (ITS)
Table 1. Strains used in this study
.................................................................................................................................................................................................................................................................................................................
Strains of Udeniomyces pannonicus sp. nov. were isolated from leaves collected in Hungary in June 1999. ATCC, American Type
Culture Collection, Manassas, VA, USA ; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands ; DSM,
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany ; IFO, Institute for Fermentation,
Osaka, Japan ; JCM, Japan Collection of Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Saitama,
Japan ; NCAIM, National Collection of Agricultural and Industrial Microorganisms, Szent Istva! n University, Budapest,
Hungary.
Strain
Source
Udeniomyces pannonicus sp. nov.
HY-16T (l JCM 11145T l NCAIM Y 01556T l CBS 9123T)
HY-17
HY-22
HY-29 (l JCM 11146 l NCAIM Y 01559)
HY-36
HY-39
HY-56
HY-87
HY-111 (l JCM 11148 l NCAIM Y 01558)
HY-117
HY-134
HY-142
HY-145
HY-186 (l JCM 11149 l NCAIM Y 01557)
HY-192
I. perplexans JCM 10245NT (l CBS 363.85NT)
U. megalosporus JCM 5269T (l CBS 7236T)
U. puniceus JCM 1535T (l ATCC 22020T l CBS 5689T l IFO 1337T)
U. pyricola JCM 2958T (l ATCC 34640T l CBS 6754T l DSM 70884T)
1888
1. Angelica sylvestris
1. Angelica sylvestris
1. Angelica sylvestris
3. Acer pseudoplatanus
4. Euonymus europaeus
4. Euonymus europaeus
7. Sambucus nigra
11. Elaeagnus angustifolia
14. Calistegia sepium
15. Corylus avellana
16. Chelidonium majus
17. Chelidonium majus
17. Chelidonium majus
22. Conium maculatum
22. Conium maculatum
Dacrymyces stillatus on twig of Fagus sp.
Dead leaf of Oryza sativa
Frozen salmon-stick
Leaf of Pyrus communis
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Udeniomyces pannonicus sp. nov.
regions including 5.8S rDNA were amplified by using a PCR
according to Sugita & Nakase (1999). The D1\D2 region of
the 26S rDNA was amplified according to the method of
Kurtzman & Robnett (1997). The PCR products were
sequenced directly using an ABI Prism BigDye Terminator
cycle sequencing ready reaction kit (Applied Biosystems)
and analysed by using an Applied Biosystems sequencer
(model 310) according to the instructions of the manufacturer. The sequences generated were aligned with sequences of other unicellular and filamentous basidiomycetes
(see Fig. 2) by using the program   (version 1.8)
(Thompson et al., 1997). Bulleromyces albus was used as an
outgroup. The phylogenetic trees were constructed by two
methods : the neighbour-joining method (Saitou & Nei,
1987) based on Kimura’s distance (Kimura, 1980) in
  and the maximum-parsimony method in the
program  version 4.0.43 (Xia & Xie, 2001). In making
phylogenetic trees, sites where gaps existed in any sequence
were excluded and bootstrap analyses (Felsenstein, 1985)
were performed from 100 random resamplings.
RESULTS AND DISCUSSION
Fifteen strains isolated from the leaves of plants in
Hungary in June 1999 showed similar morphological,
physiological and biochemical properties : vegetative
cells were spherical, ellipsoidal or cylindrical (Fig. 1) ;
ballistospores were apiculate, ellipsoidal or pyriform
(Fig. 1) ; mycelium and pseudomycelium were not
(a)
(b)
(c)
.................................................................................................................................................
Fig. 1. Udeniomyces pannonicus sp. nov. JCM 11145T. (a)
Vegetative cells grown in YM broth for 3 days at 17 mC. (b)
Vegetative cells with sterigma and ballistoconidia grown on YM
agar for 10 days at 17 mC and 3 days at room temperature. (c)
Ballistoconidia produced on cornmeal agar after 7 days at
17 mC. Bars, 10 µm.
formed ; fermentation was absent ; the diazonium blue
B colour test was positive ; the major ubiquinone was
Q-10 ; and xylose was present in the cells. The strains
were assumed to belong to one of the genera Udeniomyces or Bullera, and an especially close relationship
with U. pyricola was suspected on the basis of
physiological and biochemical properties.
Four strains (HY-16T, HY-29, HY-111 and HY-186)
were selected on the basis of their isolation sources and
the sequences of their ITS regions were determined.
The identity of the sequences (AB072229–AB072232)
indicated that the strains are conspecific (Sugita et al.,
1999). From the physiological and biological properties and the result of sequence analyses of ITS regions,
we concluded that these 15 strains should be included
in one species.
As shown in Fig. 2, strain HY-16T was positioned in
the Cystofilobasidiales on the basis of the 18S rDNA.
It is interesting that the novel strains showed a closer
relationship with Itersonilia perplexans Derx than with
Udeniomyces species, in spite of the physiological and
biochemical resemblance to U. pyricola. The same
topologies were obtained by the maximum-parsimony
method. In the D1\D2 region of the 26S rDNA, only
four base substitutions and one base deletion or
insertion were detected between strain HY-16T (accession no. AB077382) and I. perplexans (accession no.
AJ235274). Although the results showed that our
isolates and I. perplexans were very closely related, the
sequence similarities of ITS1 and ITS2 in our isolates
and I. perplexans were respectively 94n9 and 96n1 %,
indicating that they are two distinct species.
The genus Itersonilia was initially thought to contain
three species, I. perplexans Derx (Derx, 1948),
Itersonilia pyriformis Nyland (Nyland, 1949) and
Itersonilia pastinacae Channon (Channon, 1963). Because of mating compatibility, rather high DNA
similarities and an intergrading morphology, however,
the existence of separate species was later questioned
and the latter two are considered synonyms of I.
perplexans (Boekhout, 1991 ; Boekhout et al., 1991). I.
perplexans is characterized by the presence of hyphae
with clamp connections, inflated cells, ballistoconidia
and appressoria formed from germinating ballistoconidia. The yeast phase is formed under conditions of
high humidity or submerged growth and has never
been isolated from nature. Furthermore, the yeast
phase of I. perplexans produces monokaryotic hyphae
with pseudoclamps. We have observed no hyphae with
clamp connections, inflated cells or appressoria in our
isolates. The major ubiquinone of the genus Itersonilia
is Q-9 (Yamada & Konda, 1984) and that of our
isolates was Q-10, the same as that of the genus
Udeniomyces. From these morphological and chemotaxonomic characteristics, we decided to treat our
isolates as a member of the genus Udeniomyces and to
propose the name Udeniomyces pannonicus sp. nov. As
the physiological and biochemical properties of U.
pannonicus are very similar to those of U. pyricola as
described above, only a few characteristics can be used
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Y. Niwata and others
.....................................................................................................
Fig. 2. Phylogenetic trees of U. pannonicus
sp. nov. JCM 11145T and related species
based on 18S rDNA sequences. A total of
1760 bases were used for the analysis.
Numbers represent percentages from 100
replicate bootstrap samplings (frequencies
of less than 50 % are not shown).
Table 2. Differential characteristics among Udeniomyces pannonicus sp. nov., other Udeniomyces species and
Itersonilia perplexans
Characteristic
Assimilation of :*
N-Acetyl -glucosamine
Melibiose
Lactose
Inositol
Vitamin requirement
Major ubiquinone
GjC content (mol %)
Colour of colony
Sequence accession numbers :‡
D1\D2 region of 26S rDNA
18S rDNA
U. pannonicus
I. perplexans
U. megalosporus
U. puniceus
U. pyricola
k
j
k\
k
Biotin
10
58n9
Yellowish white
j
j
j
j\
None
9 a†
61n5b
Yellowish white
k
k
k
k
Biotin and thiamin
10
49n8c
Pinkish white
k
k
k
j
Biotin and thiamin
10
53n9d
Pinkish white
k
j
j
j
Biotin and thiamin
10
51n7c
Pinkish white
AB077382 (JCM 11145T)
AB072227 (JCM 11145T)
AJ235274 (CBS 363.85NT)
AB072228 (JCM 10245NT)
AF075510 (CBS 7236T)
D31657 (JCM 5269T)
AF075519 (CBS 5689T)
D31658 (JCM 1535T)
AF075507 (CBS 6754T)
D31659 (JCM 2958T)
* j, Positive ; , latent and weak ; , weak ; k, negative.
† Data taken from this study unless indicated by : a, Yamada & Konda (1984) ; b, Boekhout et al. (1991) ; c, Nakase & Suzuki (1986) ;
d, Nakase & Komagata (1971).
‡ Source strains in parentheses.
to distinguish them (Table 2). The GjC contents
(mol %) of nuclear DNA and the ITS sequence data,
however, show their separation clearly. U. pannonicus
is distinguishable from I. perplexans by a combination
of biological properties, in particular the assimilation
of N-acetyl -glucosamine and inositol, vitamin requirements and the major ubiquinone (Table 2).
U. pannonicus was isolated from 10 of 25 plants that we
sampled, indicating that this species is widely distributed in Hungary. We also isolated U. pyricola from
the same number of plants in the course of this study
(our unpublished data). Over 10 % of the total isolates
belonged to the genus Udeniomyces. This is rather
intriguing, as these yeasts were not isolated in Thailand
(Nakase et al., 2001), even though U. pyricola was
isolated from plants collected in Japan, New Zealand
and the islands of Tasmania, Australia, U. megalosporus from plants in Japan and U. puniceus from a
frozen fish in Japan (Nakase, 2000).
1890
Latin diagnosis of Udeniomyces pannonicus Niwata,
Takashima, Tornai-Lehoczki, Deak & Nakase sp. nov.
In liquido ‘ YM ’, post dies 5 ad 17 mC, cellulae vegetativae ovoideae, ellipsoideae vel cylindraceae, 4–10i6–
14 µm, singulae aut binae. Sedimentum formatur. Post
unum mensem ad 17 mC, annulus fragilis et imperfectus
vel comleta et sedimentum formantur. In agaro ‘ YM ’,
post unum mensem ad 17 mC, cultura griseo-flava,
seminitida, laevigata, mollis et margo glabra. In lamina
vitrea in ‘ CMA ’, post dies 14 ad 17 mC, mycelium et
pseudomycelium non formantur. Ballistosporae in
‘ CMA ’ abundanter formantae apiculatae, ellipsoideae
vel pyriformes, 2n6–6n5i4n3–10n4 µm. Fermentatio
nulla. Glucosum, galactosum, -sorbosum (vel lente, vel
lente et exiguum), saccharosum, maltosum, cellobiosum,
trehalosum, melibiosum, raffinosum, melezitosum, amylum solubile, -xylosum, -arabinosum, -arabinosum
(lente, lente et exiguum, vel nullum), -ribosum (exiguum, lente et exiguum, vel nullum), -rhamnosum (vel
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Udeniomyces pannonicus sp. nov.
lente), methanolum (exiguum vel lente et exiguum),
ethanolum (vel lente), glycerolum (vel lente, vel lente et
exiguum), -mannitolum (vel lente), -glucitolum (vel
lente, vel exiguum), methylum α--glucosidum, salicinum, glucono-δ-lactonum (vel lente, vel nullum), gluconatum (lente, lente et exiguum, vel nullum), acidum
2-ketogluconicum, acidum 5-ketogluconicum, acidum
succinicum, acidum citricum (vel exiguum, vel lente et
exiguum), saccharatum (lente vel lente et exiguum),
xylitolum, 1,2-propanediolum (exiguumn), acidum gluconicum et acidum -galacturonicum assimilantur,
autem lactosum (vel lente vel exiguum), inulinum, glucosaminum, N-acetylum -glucosaminum, erythritolum (vel lente et exiguum), ribitolum (vel lente, vel lente
et exiguum), galactitolum (vel lente et exiguum), lacticum (vel exiguum, vel lente et exiguum), inositolum,
hexadecanum, -arabitolum (vel exiguum, vel lente et
exiguum), et 2,3-butanediolum non assimilantur. Kalium
nitricum et natrium nitrosum assimilantur, autem ethylaminum, -lysinum (vel lente et exiguum) et cadaverinum non assimilantur. Ad crescentiam biotimum necessarium est. Maxima temperatura crescentiae : 24–25 mC.
Materia amyloidea iodophila formatur. Diazonium
caeruleum B positivum. Ureum hydrolysatur. Proportio
molaris guaninijcytosini in acido deoxyribonucleico :
58n9 mol % (per HPLC). Ubiquinonum majus : Q-10.
Xylosum in cellulis praesens. Holotypus : isolatus ex
folio Angelica sylvestri, JCM 11145T (l NCAIM Y
01556T l CBS 9123T ; originalitre ut HY-16T) conservatur in collectionibus culturarum quas Japan Collection of Microorganisms, Saitama, Japonia, National Collection of Agricultural and Industrial Microorganisms (NCAIM), Szent Istva! n University, Budapestinum, Pannonia, et Centraalbureau voor Schimmelcultures (CBS), Utrecht, Hollandia, sustentat.
Description of Udeniomyces pannonicus Niwata,
Takashima, Tornai-Lehoczki, Deak & Nakase sp. nov.
Udeniomyces pannonicus (pan.nonhi.cus. L. adj. pannonicus pertaining to Pannonia, the Roman name for
the area of modern Hungary, the country in which the
type strain was isolated).
In YM broth, after 5 days at 17 mC, the vegetative cells
are spherical, ellipsoidal or cylindrical, 4–10i6–
14 µm, single or in pairs. A sediment is formed. After
1 month at 17 mC, an incomplete or complete fragile
ring and sediment are present. On YM agar, after 1
month at 17 mC, the streak culture is greyish yellow,
semi-shining, smooth, soft and has an entire margin.
On slide culture on cornmeal agar, after 14 days at
17 mC, mycelia and pseudomycelia are not observed.
Ballistoconidia are produced abundantly on cornmeal
agar. They are apiculate, ellipsoidal or pyriform and
2n6–6n5i4n3–10n4 µm. Does not ferment glucose. Assimilates glucose, galactose, -sorbose (may be latent
or latent and weak), sucrose, maltose, cellobiose,
trehalose, melibiose, raffinose, melezitose, soluble
starch, -xylose, -arabinose, -arabinose (latent, latent and weak or negative), -ribose (weak, latent and
weak or negative), -rhamnose (may be latent), meth-
anol (weak or latent and weak), ethanol (may be
latent), glycerol (may be latent or latent and weak), mannitol (may be latent), -glucitol (may be latent or
weak), methyl α--glucoside, salicin, glucono-δ-lactone (may be latent or negative), -gluconate (latent,
latent and weak or negative), 2-ketogluconic acid, 5ketogluconic acid, succinic acid, citric acid (may be
weak or latent and weak), saccharate (latent or latent
and weak), xylitol, propane-1,2-diol (weak), -glucuronic acid and -galacturic acid. Does not assimilate
lactose (may be latent and weak), inulin, -glucosamine, N-acetyl -glucosamine, erythritol (may be
latent and weak), ribitol (may be weak or latent and
weak), galactitol (may be latent and weak), -lactate
(may be weak or latent and weak), inositol, hexadecane, -arabitol (may be weak or latent and weak) or
butane-2,3-diol. Assimilates potassium nitrate and
sodium nitrite. Does not assimilate ethylamine hydrochloride, -lysine (may be latent and weak) or
cadaverinehydrochloride.Biotinisrequiredforgrowth.
The maximum growth temperature is 24–25 mC.
Starch-like substances are produced. Growth does not
occur on 50 %(w\w) glucose\yeast extract agar.
Urease is positive. May or may not liquefy gelatin.
Does not hydrolyse fat. The diazonium blue B reaction
is positive. The GjC content of nuclear DNA is
58n9 mol % as determined by HPLC. The major
ubiquinone is Q-10. Xylose is present in the cell.
The type strain, HY-16T, was isolated from a leaf of
Angelica sylvestris collected in June 1999 in Hungary
by T. Nakase, J. Tornai-Lehoczki and M. Takashima.
This strain has been deposited in the Japan Collection
of Microorganisms (JCM), Wako, Saitama, as JCM
11145T, in the National Collection of Agricultural and
Industrial Microorganisms (NCAIM), Szent Istva! n
University, Budapest, Hungary, as NCAIM Y 01556T
and in the Centraalbureau voor Schimmelcultures
(CBS), Utrecht, The Netherlands, as CBS 9123T.
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