Bullera penniseticola sp. nov. and Kockovaella sacchari sp. nov., two new yeast species isolated from plants in Thailand.

Abstract

Two strains of ballistocondium-forming yeasts, isolated from plants collected in the south-east seacoast of Bangkok, Thailand, were described. The strains (K-272(T) and K-337(T)) were assigned to the genera Bullera and Kockovaella, respectively, based on morphological and chemotaxonomical characteristics. Phylogenetically, strain K-272(T) is close to Bullera hannae, and strain K-337(T) is close to Kockovaella thailandica and Kockovaella imperatae. These two strains represent new species based on DNA-DNA reassociation experiments. Bullera penniseticola Takashima et Nakase sp. nov. and Kockovaella sacchari Takashima et Nakase sp. nov. are proposed for K-272(T) (=JCM 9857(T)) and K-337(T) (=JCM 9858(T)), respectively.

Full text

Page 1

international Journal of Systematic Bacteriology (1 998), 48, 1025-1 030
Printed in Great Britain
Bullera penniseticola sp. nov, and Kockovaella
sacchari sp. nov., two new yeast species
isolated from plants in Thailand
Masako Takashima and Takashi Nakase
Author for correspondence : Masako Takashima. Tel : + 8 1 48 467 9560. Fax : + 8 1 48 462 461 7.
e-mail : masako@ulmus.riken.go.jp
Japan Collection of Micro-
organisms, The Institute of
Physical and Chemical
Research (RIKEN), Wako,
Saitama 351-0198, Japan
Two strains of ballistoconidium-forming yeasts, isolated from plants collected
in the south-east seacoast of Bangkok, Thailand, were described. The strains
(K-272T and K-3373 were assigned to the genera Bullera and Kockowaella,
respectively, based on morphological and chemotaxonom ica I characteristics.
Phylogenetically, strain K-272l is close to Bullera hannae, and strain K-337T is
close to Kockowaella thailandica and Kockovaella imperatae. These two strains
represent new species based on DNA-DNA reassociation experiments. Bullera
penniseticola Takashima et Nakase sp. nov. and Kockowaella sacchari
Takashima et Nakase sp. nov. are proposed for K-272T (= JCM 98573 and K-337T
(= JCM 98583, respectively.
Keywords: yeast, Bullera penniseticola sp. nov., Kockovaella sacchari sp. nov.,
Thailand
INTRODUCTION
In 1990, two ballistoconidium-forming yeasts, K-272T
and K-337T, were isolated from plant leaves collected
in Thailand. Strain K-272T, assigned to the genus
Bullera, was characterized by coenzyme Q- 10, the
presence of xylose in the cells, and proliferation by
ballistoconidia and budding cells. Strain K-337T,
assigned to the genus Kockovaella, was characterized
by Q- 10 as major ubiquinone, the presence of xylose in
the cells, and proliferation by stalked conidia, ballisto-
conidia and budding cells. Bullera sp. K-272T showed
physiological and biochemical characteristics similar
to Bullera hannae, while Kockovaella sp. K-337T was
found to be similar to Kockovaella thailandica and
Kockovaella imperatae. DNA-DNA reassociation ex-
periments, however, revealed that these yeasts repre-
sented new species of the genera Bullera and
Kockovaella. They are described as Bullera penni-
seticola Takashima et Nakase sp. nov. and Kockovaella
sacchari Takashima et Nakase sp. nov.
METHODS
Yeast strains. The strains K-272T and K-337T reported in this
paper were isolated from leaves of Pennisetum sp. and
. . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . , . . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . , . . . , , . , . , . , , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviation: SSU, small subunit.
The GenBanWEMBUDDBJ accession number for the sequences reported in
this paper are A6005452 (Bullera penniseticola J C M 98577, A8005453
(Kockovaella sacchari J C M 98583 and AB005561 (K. imperatae J C M 7826').
Saccharum oficinarum, respectively, which were collected
from the south-east seacoast of Bangkok, Thailand, using
the ballistoconidium-fall method with YM agar (Difco)
plates (Nakase & Takashima, 1993).
Examination of morphological, physiological and biochemi-
cal characteristics. Most of the morphological, physiological
and biochemical characteristics were examined according to
the methods of van der Walt & Yarrow (1984). Assimilation
of nitrogen compounds was investigated on solid media
using starved inocula according to the method of Nakase &
Suzuki (1986). Vitamin requirements were investigated
according to the method of Komagata & Nakase (1967). The
maximum growth temperature was determined in YM broth
(Difco) using metal block baths.
Ubiquinone system. Cells were grown in 500 ml Erlenmeyer
flasks containing 250 ml YM broth on a rotary shaker at 150
r.p.m. at 25 "C and were harvested in the early stationary
growth phase. The cells were washed with distilled water.
Extraction, purification and identification of ubiquinones
were 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 150
r.p.m. at 25 "C and were harvested in the early stationary
growth phase. The cells were washed with distilled water and
dried with acetone. Xylose in the cells was analysed as
described by Suzuki i5r Nakase (1988).
DNA base composition. Cells were grown in 500 ml Erlen-
meyer flasks containing 250 ml YM broth on a rotary shaker
at 150 r.p.m. at 25 "C and were harvested in the exponential
growth phase. The cells were washed with distilled water and
0.067 M phosphate buffer containing 1 mM EDTA (pH
00678 0 1998 IUMS
1025

Page 2

M. Takashima and T. Nakase
7.4). Isolation and purification of DNA were carried out
according to the methods of Nakase & Suzuki (1986). The
DNA base composition was determined by HPLC after
enzymic digestion of DNA to deoxyribonucleosides. The
DNA-GC Kit (Yamasa Shoyu) was used as a quantitative
standard.
DNA-DNA relatedness. DNA-DNA reassociation experi-
ments were carried out using a membrane-filter method
according to the method of Hamamoto & Nakase (1995).
Sequencing and phylogenetic analysis. The small subunit
(SSU) rDNA sequence was determined after amplifying the
gene using PCR. Both strands were sequenced by the dideoxy
method as described by Takashima & Nakase (1996). The
SSU rDNA sequences of Bullera penniseticola JCM 9857T
(K-272T), Kockovaella sacchari JCM 9858T (K-337T) and K.
imperatae JCM 7826T were determined in this study. Gen-
erated sequences were aligned with other unicellular and
filamentous basidiomycetes using the CLUSTAL w computer
program (Thompson et al., 1994). Sporidiobolus johnsonii
and Sporobolomyces roseus were used as outgroups. The
fixed gap and floating gap penalty values used in CLUSTAL
were 10. The alignment is available upon request. The
phylogenetic tree was constructed from the evolutionary
distance data according to Kimura (1980) using neighbour-
joining method (Saitou & Nei, 1987) in the CLUSTAL w
computer program. Sites where gaps existed in any sequences
were excluded. Bootstrap analyses (Felsenstein, 1985) were
performed from 1000 random resamplings.
RESULTS AND DISCUSSION
DNA base composition and DNA-DNA relatedness
Fifteen species are now recognized in the genus Bullera.
Suh & Nakase (1995) and Suh et al. (1996) described
the phylogenetic heterogeneity of the genus Bullera
based on SSU rDNA sequences. The wide range of the
DNA base composition, namely 39-62 mol% G + C,
also predicts the heterogeneity of the genus.
The DNA base composition of Bullera sp. K-272T was
44.2 mol% G + C, and similar to that of B. hannae and
B. huiaensis (Hamamoto & Nakase, 1996). In the
neighbour-joining tree based on SSU rDNA se-
quences, however, Bullera sp. K-272T showed close
relationship (99.6 YO sequence similarity) to B. hannae
JCM 8937 and was located in a cluster different from
that of B. huiaensis JCM 8933 (Fig. 1). DNA-DNA
reassociation experiments, carried out between Bullera
sp. K-272T and B. hannae, revealed DNA relatedness
values of 23 and 26 Y,
This suggests that Bullera sp. K-
272T is a species different from B. hannae.
Two species, K. thailandica and K. imperatae, have
been described in the genus Kockovaella (Nakase et al.
1991). The DNA base composition of Kockovaella sp.
K-337T was found to be 48-0 mol% G+ C, and similar
to those of the described species in the genus. As shown
in Fig. 1, Kockovaella sy. K-337T constituted a cluster
with K. thailandica JCM 7824T and K. imperatae JCM
7826T in the neighbour-joining tree based on SSU
rDNA sequences. DNA-DNA reassociation experi-
ments carried out between Kockovaella sp. K-337T, K.
thailandica and K. imperatae showed low DNA related-
ness values (5-15 %) (Table 1).
Latin diagnosis of Bullera penniseticola Takashima et
Nakase sp. nov.
In YM', post dies 5 ad 25 "C, cellulae vegetativae
ovoideae, ellipsoidales aut elongatae, (2.040) x (3.3-
10- 1) pm, singulae aut binae. Sedimenturn formatur.
Post unum mensem ad 17 "C, annulus imperfectus,
insulae et sedimenturn formantur. In agaro ' YM ', post
unum mensem ad 17 OC, cultura subJava, glabra, nitida,
mollis et rnargine glabra. Mycelium et pseudomycelium
non formantur. Ballistosporae globosae vel napiformes,
(24-3.5) x (2.6-4.1) pm. Fermentatio nulla. Glucosurn,
galactosum, saccharosum, maltosum, cellobiosum, tre-
halosum, lactosum, melibiosurn, rafinosum, melezi-
tosum, amyhm solubile, D-XylOSUm, L-arabinosum, D-
arabinosum, o-ribosurn, L-rhamnosum (lente), glycero-
lum (lente), ribitolum (lente et exiguum), galactitolum,
D-mann it ohm, D-glucit ohm, methyl a-D-ghcosidum,
salicinum (lente et exiguum), glucono-d-lactonurn, acid-
urn 2-ke togluconicum, acidum 5 - ketogluconicum, aci-
durn succinicum (exiguum), acidum citricum (exiguum)
et inositolum assirnilantur at non L-sorbosum, inulinum,
ethanolum, erythritolum nec acidum DL-lacticum.
Kalium nitricum, natrium nitrosum, ethylaminurn, L-
lysinum et cadaverinum non assimilantur. Maxima
temperatura crescentiae 1 30-3 1 "C. Ad crescentiam
thiaminum necessarium est. Diazonium caeruleum B
positivum. Proportio molaris guanini + cytosini in acido
deoxyribonucleico : 44.2 molyo (per HPLC). Ubiqui-
nonum majus : Q-10. Xylosurn in cellulis praesens.
Holotypus: Isolatus ex folio Pennisetum species, JCM
9857T (originaliter ut K-272T) conservatur in collection-
ibus culturarum quas ' Japan Collection of Micro-
organisms ', Wako, Saitama sustentat.
Description of Bullera penniseticola Takashima et
Nakase sp. nov.
In YM broth, after 5 d at 25 "C, the vegetative cells are
ovoidal, ellipsoidal or elongate, (2.0-4.0) x (3.3-10.1)
pm, single or in pairs. A sediment is formed. After 1
month at 17 "C, an incomplete ring, islets and a
sediment are present. On YM agar, after 1 month at
17 "C, the streak culture is pale yellow, shining, smooth
and delicately wrinkled near the bottom, and has an
entire margin. On Dalmau plate culture on corn meal
agar, mycelia and pseudomycelia are not observed.
Ballistoconidia are abundantly produced on corn
meal agar. They are globose to napiform, (24-3-5) x
(2-6-4-1) pm (Fig. 2). Does not ferment glucose.
Assimilates glucose, galactose, sucrose, maltose, cello-
biose, trehalose, lactose, melibiose, raffinose, melezi-
tose, soluble starch, D-xylose, L-arabinose, D-arabi-
nose, D-ribose, L-rhamnose (slow), glycerol (latent),
ribitol (latent and weak), galactitol, D-mannitol, D-
glucitol, methyl a-D-glucoside, salicin (latent and
weak), glucono-6-lactone, 2-ketogluconic acid, 5-keto-
gluconic acid, succinic acid (weak), citric acid (weak)
and inositol. Does not assimilate L-sorbose, inulin,
ethanol, erythritol or DL-lactic acid. Does not as-
similate potassium nitrate, sodium nitrite, ethylamine
hydrochloride, L-lysine hydrochloride, or cadaverine
-
International Journal of Systematic Bacteriology 48
1026

Page 3

Bullera penniseticola and Kockovaella sacchari spp. nov.
L
Bullera globispora (JCM 2957 I 031 650)
Temella globospora (U00976)
99.8
Tsuchiyaea wingfie/dii (JCM 7368 I 064121)
LFilobssidie/la neofonnans (IAM 1421 1 I D12804)
Bullem dendrophila (JCM 5357 I 031 649)
Fibulobasidium inmnspicuum (JCM 6898 I D64123)
,TBlomyces polyborus (JCM 6908 I D64117)
Kockovaella fhailandica (JCM 7824 I D64133)
sacchar/ (K-337) (JCM 9858 / AB005453)
imperatae (JCM 7826 I AB005561)
Tremella mrifomjs (U00977)
Bullera pseudoalba (JCM 5290 I D31660)
100 BU//SM p d ~ ~ t / W h
(K-272) (JCM 9857 / AB005452)
L6ullera hannae (JCM 8937 1078327)
Bullera unica (JCM 8932 I D78330)
9~Bullemmyces albus (MUCL 30301 I X60179)
Fig. 7. Phylogenetic tree of Bullera, Kockovaella, Udeniomyces and related taxa based on SSU rDNA sequences. The tree
was constructed from the evolutionary distance data according to Kimura (1980) using the neighbour-joining method
(Saitou & Neil 1987) with bootstrapping (Felsenstein, 1985). The numerals represent the percentages from 1000 replicate
bootstrap samplings. Sequences were retrieved from the DDBJ/EMBUGenBank nucleotide sequence databases under the
accession numbers indicated.
0.01 Knuc
H
59.7 -
Table 1. DNA-DNA relatedness of Kockovaella sp. K-337T and other Kockovaella species
h
i
i
-
Species
59.9
Strain
G + C mol% Percentage relative binding of DNA
from :
-r
-
75.6
58.1
Bullera variabilis (JCM 5275 I D31654)
K-337T JCM 7824T JCM 7826r
-
76.5
-
Xerocomus chrysentemn (TDB 635 I M94340)
Thanarephorus praticola (M92990)
Coprinus cinereus (M929 1 1 )
Amelia bombacina (ATCC 20629 I M55638)
Pseudohydnum gelatinosum (UC 14751 14 I L22260)
Auricularia polytricha (KW 21 5.1 1 I L22255)
loo
Kockovaella sp.
K. thailandica
K. imperatae
K-337T
JCM 7824T
JCM 7826T
48.0
47.5
47.7
100
15
12
5
100
8
100
hternational Journal of Systematic Bacteriology 48
1027

Page 4

M. Takashima and T. Nakase
Fig. 2 . Bullera penniseticola. (a) Vegetative cells grown in YM
broth for 5 d at 25 "C. (b) Ballistospores produced on corn meal
agar after 5 d at 25 "C. Bars, 10 pm.
dihydrochloride. Maximum growth temperature is
30-3 1 "C. Thiamine is required for growth. No starch-
like substances are produced. Growth does not occur
on 50 YO (w/w) glucose-yeast extract agar. Urease-
positive. Does not liquefy gelatin. Does not hydrolyse
fat. The diazonium blue B reaction is positive. The
G+C content of nuclear DNA is 44.2 mol% as
determined by HPLC. The major ubiquinone is Q- 10.
Xylose is present in the cells. The type strain of B.
penniseticola, K-272T, was isolated by T. Nakase and
B. Fungsin in December 1990 from a leaf of Penni-
setum sp. collected along the south-east seacoast of
Bangkok, Thailand. This strain has been deposited in
Japan Collection of Microorganisms (JCM), Wako,
Saitama, as JCM 9857T. The physiological and bio-
chemical characteristics of B. penniseticola are similar
to those of B. hannae, but the maximum growth
temperature, 30-31 "C for B. penniseticola and 22-
23 "C for B. hannae, can clearly distinguish between
these species.
Latin diagnosis of Kockowaella sacchari Takashima et
Nakase sp. nov.
In liquid0 ' YM', post dies 5 ad 25 "C, cellulae vege-
tativae sphaerical vel ovoideae, (2-0-6-3) x (3.3-7-3) pm,
singulae, binae, in catenis aut in fasciculis, propagantes
formatione conidiorurn stipitatorum et gernmarum blas-
ticarum. Conidia stipitata ex cellula conidiogena multi-
lateraliter. Sedimentum formatur. Post unurn mensem
ad 17 "C, anulus, insulae et sedirnentum formantur. In
agaro ' YM ', post unum mensem ad 17 "C, cultura
griseo-Java, glabra, nitida, butyracea et margine glabra.
Mycelium et pseudomycelium non formantur. Ballisto-
sporae globosae vel napqormes, (2.2-4.0) x (3.1-M)
pm. Fermen tat io nulla. Glucosum, galactosum, sacchar-
osurn, rnaltosum, cellobiosum, trehalosum, lactosum,
melibiosum, rafinosum, melezitosum, amylurn solubile,
D-xylosurn, L-arabinosum, D-arabinosum (lente), D-ribo-
. . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . , . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fig. 3 . Kockovaella sacchari. (a) Vegetative cells grown in YM
broth for 5 d at 25 "C. (b) Ballistospores produced on corn meal
agar after 5 d at 25 "C. Bars, 10 pm.
sum, L-rhamnosum (lente), ribitolum (lente), galacti-
tolum (len te), D-mann itolurn (len te), D-glucit olum
(lente), methyl a-D-glucosidum, sakinum, glucono-6-
lactonurn (exiguum), acidium succinicum (lente), acidum
citricum (exiguum) et inositolum (lente et exiguum)
assimilantur at non L-sorbosurn, inulinum, ethanolum,
glycerolurn, erythritolum, acidum 2-ketogluconicum,
acidum 5-ketogluconicum nec acidurn DL-lacticum. L-
Lysinum assimilatur at non kalium nitricum, natrium
nitrosum, ethylaminum nec cadaverinum. Maxima tem-
peratura crescentiae : 3 1-32 "C. Ad crescentiam thia-
rninurn necessarium est. Diazonium caeruleum B posi-
tivurn. Proportio molaris guanini + cytosini in acido
deoxyribonucleico : 48-0 mol% (per HPLC). Ubiqui-
nonum majus Q- 10. Xylosum in cellulis praesens.
Holotypus : Isolatus ex folio Saccharum oficinarurn,
JCM 985gT (originaliter ut K-337T) conservatur in
collectionibus culturarum quas ' Japan Collection of
Microorganisms', Wako, Saitama sustentat.
Description of Kockowaella sacchari Takashima et
Nakase sp. nov.
In YM broth, after 5 d at 25 "C, the vegetative cells are
spherical to ovoidal (2.0-6-3) x (3.3-7.3) pm, single, in
pairs or in clusters, reproduced by budding and stalked
conidia. Stalks develop multilaterally from a conidio-
genous cell. A sediment is formed. After 1 month at
17 "C, a complete ring and islets, and a sediment are
present. On YM agar, after 1 month at 17 "C, the
streak culture is greyish-yellow, shining, smooth,
butyrous and has an entire margin. On Dalmau plate
culture on corn meal agar, mycelia and pseudomycelia
are not formed. Ballistoconidia are abundantly pro-
duced on corn meal agar. They are globose to
napiform (2-2-4.0) x (3.1-4.6) pm (Fig. 3). Does not
ferment glucose. Assimilates glucose, galactose, su-
crose, maltose, cellobiose, trehalose, lactose, melibiose,
raffinose, melezitose, soluble starch, D-xylose, L-ar-
1028
International Journal of Systematic Bacteriology 48

Page 5

Bullera penniseticola and Kockovaella sacchari spp. nov.
Table 2 . Salient characteristics of Kockovaella sacchari and other Kockovaella species
+, Positive; L, latent; W, weak; LW, latent and weak; -, negative.
. . . . . . . . . . . . .
I.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Species Strain Assimilation of:
Galactitol Methyl
a-D-glucoside
+
L/ -
-
Glucono-&-lactone 2-Ketogluconic 5-Ketogluconic
acid acid
K. sacchari
K. thailandica
K. imperatae
JCM 985gT
JCM 7824T, JCM 7825
JCM 7826T
L
-
L
W
-
L
-
+
LW
-
+
LW
abinose, D-arabinose (latent), D-ribose, L-rhamnose
(latent), ribitol (latent), galactitol (latent), D-mannitol
(latent), D-glucitol (latent), methyl a-D-glucoside, sali-
cin, glucono-d-lactone (weak), succinic acid (latent),
citric acid (weak), and inositol (latent and weak). Does
not assimilate L-sorbose, inulin, ethanol, glycerol,
erythritol, 2-ketogluconic acid, 5-ketogluconic acid, or
DL-lactic acid. Assimilates L-lysine hydrochloride.
Does not assimilate potassium nitrate, sodium nitrite,
ethylamine hydrochloride, or cadaverine dihydro-
chloride. Maximum growth temperature is 3 1-32 "C.
Thiamine is required for growth. No starch-like
substances are produced. Growth does not occur on
50 % (w/w) glucose-yeast extract agar. Urease posi-
tive. Does not liquefy gelatin. Does not hydrolyse fat.
The diazonium blue B reaction is positive. The G + C
content of nuclear DNA is 48-0 mol% as determined
by HPLC. The major ubiquinone is Q-10. Xylose is
present in the cells. The type strain of K. sacchari, K-
337T, was isolated by T. Nakase and B. Fungsin in
December 1990 from a leaf of Saccharurn oficinarurn
collected along the south-east seacoast of Bangkok,
Thailand. This strain has been deposited in Japan
Collection of Microorganisms, Wako, Saitama, as
JCM 985gT. Kockovaella sacchari is distinguished from
K. thailandica by the assimilation of galactitol, 2-
ketogluconic acid, 5-ketogluconic acid, and from K.
irnperatae by the assimilation of methyl a-D-glucoside
(Table 2). This is the third species of the genus
Kocko vaella.
ACKNOWLEDGEMENTS
We thank Miss P. Atthasampunna and Mr B. Fungsin,
Thailand Institute of Scientific and Technological Research,
Bangkok, Thailand, for their help in collecting plant
materials and the isolation of the strain reported in this
paper. This study was supported in part by special co-
ordination funds for promoting science and technology of
the Science and Technology Agency of the Japanese Govern-
ment.
REFERENCES
Felsenstein, J. (1985). Confidence limits on phylogenies : an
approach using the bootstrap. Evolution 39, 783-791.
Hamamoto, M. & Nakase, T. (1995). Ballistosporous yeasts found
on the surface of plant materials collected in New Zealand. 1.
Six new species in the genus Sporobolomyces. Antonie Leeuwen-
hoek 67, 151-171.
Hamamoto, M. & Nakase, T. (1996). Ballistosporous yeasts found
on the surface of plant materials collected in New Zealand. The
genera Bensingtonia and Bullera with descriptions of five new
species. Antonie Leeuwenhoek 69,279-29 1.
Kimura, M. (1980). A simple method for estimating evolutionary
rate of base substitutions through comparative studies of
nucleotide sequences. J Mol Evoll6, 11 1-120.
Komagata, K. & Nakase, T. (1967). Microbiological studies on
frozen foods. V. General properties of yeasts isolated from
frozen foods. J Food Hyg Soc Jpn 8, 53-57 (in Japanese).
Nakase, T. & Suzuki, M. (1986). Bullera megalospora, a new
species of yeast forming large ballistospores isolated from dead
leaves of Oryza sativa, Miscanthus sinensis, and Sasa sp. in
Japan. J Gen Appl Microbiol32, 225-240.
Nakase, T. & Takashima, M. (1993). A simple procedure for the
high frequency isolation of new taxa of ballistosporous yeasts
living on the surface of plants. RIKEN Rev 3, 33-34.
Nakase, T., Itoh, M., Takematsu, A., Mikata, K., Banno, 1. &
Yamada, Y. (1991). Kockovaella, a new ballistospore-forming
anamorphic yeast genus. J Gen Appl Microbiol37, 175-1 97.
Saitou, N. 8 1 Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol4,
406-425.
Suh, S.4. 81 Nakase, T. (1995). Phylogenetic analysis of the
ballistosporous anamorphic genera Udeniomyces and Bullera,
and related basidiomycetous yeasts, based on 18s rDNA
sequence. Microbiology 141, 901-906.
Suh, S.4.. Takashima, M., Hamamoto, M. & Nakase, T. (1996).
Molecular phylogeny of the ballistoconidium-forming ana-
morphic yeast genus Bullera and related taxa based on small
subunit ribosomal DNA sequences. J Gen Appl Microbiol42,
50 1-509.
Suzuki, M. & Nakase, T. (1988). The distribution of xylose in the
cells of ballistosporous yeasts - application of high perform-
ance liquid chromatography without derivatization to the
analysis of xylose in whole cell hydrolysates. J Gen Appl
Microbiol34, 95-103.
Takashima, M. & Nakase, T. (1996). A phylogenetic study of the
genus Tilletiopsis, Tilletiaria anomala and related taxa based on
the small subunit ribosomal DNA sequences. J Gen Appl
Microbiol42, 421429.
International Journal of Systematic Bacteriology 48
1029