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Leucosporidium drummii sp. nov., a member of the
Microbotryomycetes isolated from soil
A. M. Yurkov, A. M. Scha¨fer and D. Begerow
Correspondence
Andrey Yurkov
andrey.yurkov@rub.de
Geobotanik, Fakulta¨t fu
¨r Biologie und Biotechnologie, Ruhr-Universita¨ t Bochum, Universita¨tsstraße
150, 44801 Bochum, Germany
Two strains of a novel teleomorphic basidiomycete were isolated from grassland soil. Standard
phenotypic tests and phylogenetic analyses of 26S rRNA gene (D1/D2 domains) and ITS region
sequences showed that the species belongs to the core group of the genus Leucosporidium.A
novel species, Leucosporidium drummii sp. nov., is proposed to accommodate the two strains,
with SEG-3-2-AY220
T
(5CBS 11562
T
5MUCL 52878
T
) as the type strain. In addition,
phylogenetic analysis revealed great genetic variability in the Leucosporidium scottii complex.
INTRODUCTION
One of the aims of the German Biodiversity Exploratories
initiative (http://www.biodiversity-exploratories.de/) is to
better understand biodiversity in relation to land use and
management. In this context, we have been studying soil
yeast communities (Yurkov et al., 2012). During this
survey, several novel species have been discovered, three
of which, Clavispora reshetovae,Barnettozyma vustinii
and Holtermanniella takashimae, were described recently
(Yurkov et al., 2009a, b; Wuczkowski et al., 2011). Mul-
tiple cultures of non-pigmented yeasts with phenotypic
characters resembling those of the basidiomycetous genus
Rhodotorula (Pucciniomycotina, Microbotryomycetes) were
used for experiments aiming to induce the teleomor-
phic stage. Of these, two cultures produced hyphae with
teliospores. The absence of pigmentation, the lack of
production of starch-like compounds, the inability to grow
on inositol and the formation of hyphae suggested that
they were related to members of the order Leucospori-
diales (Microbotryomycetes, Pucciniomycotina). Phylo-
genetic analysis based on the D1/D2 domains of the 26S
rRNA gene and the complete 5.8S–internal transcribed
spacer (ITS) gene region confirmed the placement of the
strains in the genus Leucosporidium and indicated that they
represent a novel species for which the name Leuco-
sporidium drummii sp. nov. is proposed.
METHODS
The two novel strains were isolated from grassland soil collected in
the UNESCO Biosphere Reserve Schorfheide-Chorin (plot ID S526,
SEG 3; approx. coordinates: 53.1028uN 13.9857uE), Germany,
following a previously described procedure (Yurkov et al., 2012).
Strain CBS 11561 was isolated as a separate colony and strain CBS
11562
T
grew in a mixed culture with Trichosporon porosum. The latter
culture was purified on modified Browns’ nitrogen deficient media
(Brown et al., 1962) supplemented with imidazole (according to LaRue
& Spencer, 1967) and cycloheximide (according to Danielson &
Jurgensen, 1973). Cultures were maintained on MYP medium
(Sampaio et al., 2003). Phenotypic characterization of purified isolates
was carried out according to Yarrow (1998) using both solid and liquid
media. Plates were incubated at room temperature and checked after 7,
14 and 21 days. Growth tests in liquid media were performed in test
tubes with 2.5 ml media, which were shaken on an orbital shaker at
200 r.p.m. All assimilation profiles of members of the genera
Leucosporidium and Leucosporidiella were obtained from the CBS
database (www.cbs.knaw.nl) except for that of Leucosporidium
golubevii, which was described by Sampaio et al. (2003).
Both strains had a similar white and mucilaginous look, although CBS
11562
T
displayed some hyphal growth. Formation of hyphae was
induced in cultures after prolonged incubation (3 weeks) on MYP
medium at 4 and 16 uC. Agar blocks containing hyphae were cut out,
transferred to new plates and incubated at 16 uC in order to facilitate
development of the hyphal stage. The last step was repeated four times.
Teliospore germination was investigated after 3 to 6 months of
incubation. Alternatively, agar blocks containing teliospores were cut
out of a 1-year-old culture grown on MYP at 16 uC and transferred to a
new plate. Germination of teliospores normally occurred on the day
after the transfer. For observation of nuclei, a modified Giemsa staining
protocol described by Sampaio et al. (2001) was used. For staining, agar
blocks of a 1-year-old culture incubated on MYP agar at 16 uC for
7 days were used.
The DNA extraction protocol, PCR amplification, purification and
sequencing were performed as described previously (Glushakova et al.,
2010; Yurkov et al., 2012). The assembly and editing of sequence data were
performed using Sequencher 4.8 (Gene Codes Corporation). Alignments
were made using the MAFFT algorithm (Katoh et al.,2002).Themodelof
DNA substitution (GTR+G+I) and parameters for maximum-like-
lihood analysis were derived by using MODELTEST version 3.7 (Posada &
Crandall, 1998). Maximum-likelihood analysis was performed using
RAxML (-m GTRGAMMA option) version 7.0.3, with 1000 rounds of
bootstrap replicates (Felsenstein, 1985; Stamatakis et al., 2008). Additional
sequences were retrieved from GenBank (www.ncbi.nlm.nih.gov) and CBS
(www.cbs.knaw.nl) databases. All accession numbers and strain numbers
areindicatedonthephylogenetictree(Fig.1).
Abbreviations: ITS, internal transcribed spacer; LSU, large subunit.
The GenBank/EMBL/DDBJ accession numbers for the LSU rRNA
gene (D1/D2 domains) and ITS sequences of Leucosporidium drummii
CBS 11562
T
are FN428965 and FN908919, respectively.
International Journal of Systematic and Evolutionary Microbiology (2012), 62, 728–734 DOI 10.1099/ijs.0.027102-0
728 027102 G2012 IUMS Printed in Great Britain
RESULTS
Ecology
Although the novel species was isolated from soil, its low
incidence and abundance in this substrate suggest that soil
is probably not the primary habitat of Leucosporidium
drummii. Both strains of the novel species were isolated
from the same mixed soil sample collected at a grassland
plot in northern Germany (Brandenburg). We did not
observe this species in central or southern Germany, i.e. in
Thuringia or the Swabian Alps, respectively.
Members of the Leucosporidiales are psychrotolerant
dimorphic fungi that occur in water, decaying plant
material and soil (Fell et al., 1969; Sampaio et al., 2003).
They are considered to be saprothrophs, although the
possession of colacosomes suggests a parasitic nature
(Sampaio et al., 2003; Bauer et al., 2006). Strain CBS
11562
T
, recovered from the mixed culture with Tricho-
sporon porosum, displayed vigorous hyphal growth. Inter-
estingly, Trichosporon porosum was recently reported to
produce cellobiose lipids, which are fungicidal agents with
a very broad inhibition spectrum (Kulakovskaya et al.,
2010). However, these glycolipids did not inhibit growth of
Fig. 1. Phylogenetic placement of Leucosporidium drummii obtained by maximum-likelihood analysis of the LSU rRNA gene
(D1/D2 domains) and ITS region. The numbers given on branches are frequencies (.75 %) with which a given branch
appeared in 1000 bootstrap replications. Bar, number of expected substitutions accumulated per site. The tree is rooted with
Rhodosporidium toruloides CBS 349
T
(AF444489/AF070426) and Sporidiobolus salmonicolor CBS 490
T
(AY015434/
AF070439). Where no sequence accession numbers are given, data were retrieved from the CBS database; sequences
determined in this study are given in bold. Mating types (MT) for Leucosporidium scottii and closely related species are given
according to Fell & Statzell-Tallman (1982), Suh et al. (1993) and the CBS database; SF, self-fertile; ANA, anamorphic; ND, no
data.
Leucosporidium drummii sp. nov.
http://ijs.sgmjournals.org 729
any members of the Leucosporidiales (Kulakovskaya
et al., 2010). This finding is in agreement with our observa-
tions, i.e. the growth and development of Leucospori-
dium drummii was not suppressed during co-culture with
Trichosporon porosum. Therefore, dimorphic fungi of the
genus Leucosporidium seem to possess adaptations such as
tolerance to some antifungal agents that probably facilitate
their success as fungal parasites inhabiting litter or soil.
Phylogenetic placement
The genus Leucosporidium (Leucosporidiales, Microbotryo-
mycetes, Pucciniomycotina) in its current state is polyphy-
letic and comprises five species (Sampaio et al., 2003).
Phylogenetic analysis based on partial 26S rRNA gene
sequences demonstrated that the two species Leucospo-
ridium antarcticum and Leucosporidium fasciculatum are not
related to Leucosporidium scottii,Leucosporidium golubevii
and Leucosporidium fellii, comprising the core group. The
Leucosporidium core group also includes the teleomorphic
species Mastigobasidium intermedium and several ana-
morphic species recently transferred to the genus Leucospo-
ridiella, namely Leucosporidiella creatinivora,Leucosporidiella
fragaria,Leucosporidiella muscorum and Leucosporidiella
yakutica (Sampaio et al., 2003). Phylogenetic analysis of the
partial large subunit (LSU) rRNA gene (D1/D2 domains) and
the ITS region sequences performed in the current study
using the maximum-likelihood algorithm clearly suggests
relatedness of Leucosporidium drummii with a species group
that includes Leucosporidium scottii,Leucosporidiella creatini-
vora,Leucosporidiella yakutica,andLeucosporidiella mus-
corum (Fig. 1). The nearest sequence match among currently
recognized species was obtained with the type strain of
Leucosporidiella muscorum, which showed 10 nt substitutions
and one indel in the D1/D2 domain of the LSU rRNA gene.
Additionally, our phylogenetic analysis revealed intraspe-
cific variability of the Leucosporidium scottii strains
deposited in the CBS culture collection. They showed 6–
7 nt substitutions in the D1/D2 domains and 6–10
nt substitutions in the 5.8S–ITS region, respectively.
Interestingly, sequences of two Leucosporidiella yakutica
strains, CBS 8248 and CBS 8621
T
, differed from those of
Leucosporidium scottii strains CBS 2300 and CBS 9467 in
1 nt substitution each in the ITS region and showed
identical D1/D2 domain sequences. In the CBS database,
Leucosporidium scottii CBS 2300 is designated mating type
A1B2; mating information for the other strain of this
species is unavailable (Fig. 1). Similarly, Leucosporidiella
creatinivora CBS 8620
T
differed in 1 nt substitution in the
D1/D2 domain from Leucosporidium scottii strains CBS
5930
T
, 5932, 614, 8162 and 9490. Out of six variable
positions in the ITS region among these cultures, Leuco-
sporidiella creatinivora CBS 8620
T
had only one unique
substitution compared with these Leucosporidium scottii
strains. This group of Leucosporidium scottii strains
comprises both haploid mating-compatible strains and
self-fertile cultures (Fell & Statzell-Tallman, 1982; CBS
database). According to the original description, both
Leucosporidiella creatinivora and Leucosporidiella yakutica
were tested in mating experiments with the mating types
reported by Fell et al. (1969), namely strains CBS 5930
T
and CBS 5931, corresponding to MT A2B2 and MT A1B1,
respectively (Golubev, 1998). However, multiple allelic
incompatibility of Leucosporidium scottii, containing at
least 5 A and 3 B specificities, was demonstrated by Fell
& Statzell-Tallman (1982). In summary, our observa-
tions imply that Leucosporidiella creatinivora as well as
Leucosporidiella yakutica might represent anamorphs of
Leucosporidium scottii or that D1/D2 and ITS sequence
data are not sufficient to resolve these species. Therefore,
we suggest using multigene phylogenies coupled with
mating experiments to reassess anamorph/teleomorph
relationships within the Leucosporidium core group.
Latin diagnosis of Leucosporidium drummii
Yurkov, Scha
¨fer et Begerow sp. nov.
Cultura in striis in agaro cum extracto malti,extracto levidinis
et peptono (MYP)post unum mensem ad 20–22 uC cremea,
nitens,mucosa,laevis.In agaro MYP post 5dies ad 16 uC,
cellulae ovoideae vel ellipsoidae (3–4615–35 mm), interdum
elongatae simile hyphae,binae aut catenae breves (Fig. 2).
Flosculi sunt polares.Ballistosporae absunt.Status teleo-
morphicus post hebdomades tres ad 16 uCin agaro MYP
observatus,homothallicus.Mycelium verum (2–3mmin
diametro)et teliosporae sunt.Teliosporae intercalares aut
terminals,sphericae (8–11 mmin diametro), granulatae (Fig.
2). Teliosporae basidia aut hyphae septata germinant.Basidia
transversaliter septata,3–5635–45 mm. Basidiosporae ovoi-
deae,3–4625–35 mm. Teliosporae interdum hyphis aseptatis
in apicem cum metabasidia curvata et septata germinant.
Fermentatio (glucosum)nulla.D-Glucosum,D-galactosum
(lente), D-glucosaminum,D-xylosum,L-rhamnosum,sucrosum,
maltosum,trehalosum,cellobiosum,raffinosum,melezitosum,
glycerolum,erythritolum,sorbitolum,D-mannitolum,acidum
D-glucuronicum et acidum succinicum (lente)assimilantur at
non L-sorbosum,L-arabinosum,D-arabinosum,lactosum,
inulinum,amylum solubile,ribitolum,acidum DL-lacticum,
acidum citricum nec inositolum.Assimilatio kalii nitrati,natrii
nitrosi,L-lysini et ethylamini.Materia amyloidea iodophila
non formantur.Ureum finditur.Diazonium caeruleum B est
positivum.Temperatura maxima crescentiae: 25 uC.Ad cre-
scentiam vitaminae externae non necessariae sunt.
Cultura typica SEG-3-2-AY220
T
isolata ex solo prato in
Brandenburg,Germania,viva et exsiccata numero CBS
11562
T
(5MUCL 52878
T
)in collectione zymotica Centraal-
bureau voor Schimmelcultures, Trajectum ad Rhenum,
Hollandia,sustentat.
Description of Leucosporidium drummii Yurkov,
Scha
¨fer et Begerow sp. nov.
Leucosporidium drummii (drum9mi.i. N.L. gen. masc. n.
drummii of Drumm, named after the late German botanist
A. M. Yurkov, A. M. Scha¨ fer and D. Begerow
730 International Journal of Systematic and Evolutionary Microbiology 62
Dr Klaus Drumm for being an inspiring teacher and
mentor in all fields of botany and mycology).
Streak culture after 1 month at 20–22 uCiscreamcoloured,
shiny and mucilaginous with a smooth surface. Yeast cells
after4daysonMYPagar(Sampaioet al., 2003) are ovoid to
elongate, 3–4615–35 mm (Fig. 2). Ballistospores are absent.
Some hyphal strands are evident. The teleomorphic stage
(homothallic, self-fertile cultures) was obtained independently
for both strains after cultivation on MYP for 3 weeks at 16 uC.
Fig. 2. Light microscopic images illustrating the different stages of Leucosporidium drummii sp. nov. (a) budding cells; (b) hyphal
structures; (c) teliospores in chains; (d–f, i–k) various modes of teliospore germination; (g) teliospore germination showing
monokaryotic cells (black arrows); (h) dikaryotic hyphal structures (white arrow); (l) fungal structures with mono- (black arrows) or
dikaryotic (white arrows) cells. (a–c, e) Differential interference contrast images; (g–i) Giemsa-stained images. Bars, 10 mm.
Leucosporidium drummii sp. nov.
http://ijs.sgmjournals.org 731
Hyphae are 2–3 mm in diameter and devoid of clamp
connections (Fig. 2). Teliospores are spherical, 8–11 mmin
diameter, terminal or intercalary, single and in short chains of
2–3 spores (Fig. 2). Teliospore germination mode is variable.
Teliospore germinates with basidium or hyphal segments.
Basidia are transversally septate, measuring 3–5635–45 mm.
Basidiospores are ovoid, measuring 3–4625–35 mm(Fig.2).
In some cases, teliospores germinate to produce several
aseptate hyphae resulting in the formation of curved
phragmometabasidia.
Sugars are not fermented. Assimilates the following carbon
compounds: D-glucose, D-galactose (weak, delayed), D-
glucosamine, D-xylose, L-rhamnose, sucrose, maltose, tre-
halose, cellobiose, raffinose, melezitose, glycerol, D-glucitol,
D-mannitol, ethanol, D-glucuronic acid and succinic acid
(delayed). No growth occurs on L-sorbose, L-arabinose, D-
arabinose, lactose, inulin, starch, ribitol, myo-inositol, DL-
lactic acid or citric acid. Assimilates the following nitrogen
compounds: potassium nitrate, sodium nitrite, L-lysine and
ethylamine. Grows in the presence of 0.01% cycloheximide.
Maximal growth temperature: 25 uC. Positive for urease
activity. Diazonium blue B reaction is positive. Growth on
vitamin-free medium is positive. Starch-like compounds are
not produced.
The type strain is SEG-3-2-AY220
T
(5CBS 11562
T
5
MUCL 52878
T
), isolated from grassland soil in
Brandenburg, Germany.
Additional remarks
In addition to the salient characters distinguishing currently
recognized members of the Leucosporidiales (Sampaio et al.,
2003), the novel species differs from the other species in
sorbose-utilizing ability.
Although this description is made based on homothallic self-
fertile cultures, we believe that the respective mating types
exist in nature. This assumption is based on the two different
life cycles observed for Leucosporidium scottii, the type
species of genus Leucosporidium, which has heterothallic and
self-sporulating cultures (Fell et al., 1969). The observed
teleomorphic stage of the novel species clearly resembles the
following morphological characters of Leucosporidium scottii
self-fertile cultures: mycelium devoid of clamp connections
and teliospores with bud-like projections developing into
the new teliospores (Fell et al., 1969). Unlike Leucosporidium
scottii, the novel species displayed irregular and variable
modes of teliospore germination. Teliospores of a 3- to 6-
month-old culture germinated in agar with hyphal segments
in addition to the typical basidia-like structures (Fig. 2e).
Giemsa staining showed that yeast cells of Leucosporidium
drummii contained one nucleus, whereas the hyphal stage
was dikaryotic (Fig. 2h, l). Teliospore germination resulted
in monokaryotic cells (Fig. 2g). An interesting observation
was made after 1-year-old teliospores grown on MYP at
16 uC were harvested and transferred onto a new MYP plate.
In addition to hyphal segments and basidia-like structures
(Fig. 2f, i–k), we observed teliospores producing several
aseptate hyphae, which gave rise to curved phragmobasidia
(Fig. 2d). This type of teliospore germination resembles
the characteristic features of the genus Mastigobasidium
(Golubev, 1999; Sampaio et al., 2003).
DISCUSSION
In this study, we propose a novel species in the sexual
genus Leucosporidium. This species produces hyphae
without clamp connections and intercalary teliospores,
which germinate with either typical basidia or hyphal
segments. In the last revision of the genus Leucosporidium,
Sampaio (2011) pointed out that the irregular mode of
teliospore germination exemplified by Leucosporidium
antarcticum should be considered as asexual, i.e. when the
number of supposedly ‘basidial’ compartments varies within
a culture. In our opinion, this irregular mode of germination
does not contradict the presence of the teleomorphic stage.
It was demonstrated that members of the Microbotryales,
the sister lineage to Leucosporidiales, sometimes display
variable modes of germination, e.g. the number of basidial
compartments in Microbotryum violaceum varied between
different samples (Hood et al., 2001). In Microbotryum
lychnidis-dioicae, the germination mode depended on
environmental conditions, such as nutrient availability and
temperature (Hood & Antonovics, 1998; Scha
¨fer et al.,
2010). Similarly, depending on the conditions, teliospores of
Leucosporidium drummii produced hyphae, which origi-
nated in curved metabasidia similar to that reported for
Mastigobasidium intermedium (Golubev, 1999; Sampaio
et al., 2003). Ingold (1989) reported germination with
multiple basidia-like structures for Microbotryum succisae,
so-called metabasidium branching. Interestingly, the same
type of germination was observed in Sporidiobolus para-
roseus (Sporidiobolales, Microbotryomycetes) but, due to
the absence of typical basidia, this species was considered to
be asexual (Vale
´rio et al., 2008). These examples imply that
different modes of teliospore germination might be a
common attribute of the Microbotryomycetes.
The examples mentioned above also reflect striking
differences between criteria used to classify saprobic and
parasitic taxa in the Microbotryomycetes. The parasitic
fungi are commonly described on the basis of observed
infection symptoms on their hosts and the morphology of
the probasidia (e.g. Va
´nky, 1994) even though there is
variance in teliospore germination patterns. Furthermore,
many species of the genus Microbotryum (e.g. all parasites on
the Polygonaceae) do not germinate on artificial media
(Fischer & Holton, 1957). Because a parasitic origin of
Leucosporidiales and Sporidiobolales has been suggested
(Sampaio et al., 2003), there is no serious reason to keep
distinct approaches for classification of yeast-like and
smut fungi. Here, we argue for a common solution for
classification of dimorphic basidiomycetes and taxonomic
simplicity. Basidiomycetous yeasts are spread around three
phyla and thus should be classified together with allied taxa.
A. M. Yurkov, A. M. Scha¨ fer and D. Begerow
732 International Journal of Systematic and Evolutionary Microbiology 62
The placement of species producing teliospores in ana-
morphic genera would make the current state of systematics
neither simple nor stable.
The use of a single name for the same fungus has been
repeatedly necessitated in the last few years. As a result of past
discussions during IMC9 nomenclature sessions, the use of
one name for one fungus was accepted by the majority of
delegates (Norvell et al., 2010). In light of all these facts,
further use of double nomenclature in a well-supported
phylogenetic clade, like the Leucosporidium core group, is
debatable and the use of anamorphic names should be
limited to strictly asexual fungi. Thus, the placement of
Leucosporidium drummii, which produces mycelium with
teliospores, in the asexual genus Leucosporidiella due to the
variance in germination mode is impractical and rather
confusing for many mycologists. Additionally, the teleo-
morphic species Mastigobasidium intermedium, which is a
member of the Leucosporidium core group (Fig. 1; Sampaio
et al., 2003), might be transferred to the genus Leucospori-
dium. Ballistoconidia production and teliospore germination
mode morphologically distinguish Mastigobasidium from
members of the genus Leucosporidium (Sampaio et al., 2003).
However, our observations suggest that teliospores of
Leucosporidium drummii sometimes originate multiple
aseptate hyphae and curved basidia. Additionally, formation
of ballistoconidia is apparently a variable feature among the
Microbotryomycetes and is exemplified with several closely
related species assigned to the genera Bensingtonia,
Sporobolomyces and Rhodotorula (e.g. Sporobolomyces singu-
laris and Rhodotorula lignophila;Bensingtonia yamatoana and
Rhodotorula arctica) based on this morphological attribute
(Sampaio et al., 2003; Vishniac & Takashima, 2010). Because
the mode of teliospore germination, as well as other
morphological attributes, might vary considerably within
the Microbotryomycetes, the application of molecular
markers provides a solid background for the classification
of these dimorphic fungi. Notably, molecular markers are also
consistent with ultrastructural markers, septal pore morpho-
logy and the presence of colacosomes (Sampaio et al., 2003).
ACKNOWLEDGEMENTS
Authors are grateful to participants and collaborators of the DFG
Biodiversity Exploratories for exchanging and sharing soil samples,
and the CBS for the uncountable amount of data about yeasts
available through their databases. Niko Bias and Ilse Weßel are
acknowledged for assistance in the laboratory. This study was
supported by DFG (BE 2201/9-1 and YU 152/1-1).
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