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ar1
i1
1 Department of Biology, Biotechnical Faculty, University of Ljubljana,
Vena pot 111, SI-1000 Ljubljana, Slovenia
2 CBS Fungal Biodiversity Centre, P.O. Box 85167, NL-3508 AD Utrecht, The
Netherlands
Correspondence: Polona Zalar,
polona.zalar{at}bf.uni-lj.s
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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) and elongase
(ELO). Two globally ubiquitous varieties were distinguished: var.
pullulans, occurring particularly in slightly osmotic substrates and
in the phyllosphere; and var. melanogenum, mainly isolated from
watery habitats. Both varieties were commonly isolated from the sampled Arctic
habitats. However, some aureobasidium-like strains from subglacial ice from
three different glaciers in Kongsfjorden (Svalbard, Spitsbergen), appeared to
represent a new variety of A. pullulans. A strain from dolomitic
marble in Namibia was found to belong to yet another variety. No molecular
support has as yet been found for the previously described var.
aubasidani. A partial elongase-encoding gene was successfully used as
a phylogenetic marker at the (infra-)specific level. Taxonomic novelties: Aureobasidium pullulans var. subglaciale Zalar, de Hoog & Gunde-Cimerman, var. nov.; Aureobasidium pullulans var. namibiae Zalar, de Hoog & Gunde-Cimerman, var. nov.
Keywords Arctic / Aureobasidium / black yeasts / elongase / glacier / ITS / LSU / phylogeny / polar environment / rDNA / sea ice / seawater / taxonomy / translation elongation factor / β-tubulin
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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-1,6-maltotriose). This component is a promising biomaterial
(Rekha & Sharma 2007), and
is currently used among others for the packaging of food and drugs
(Singh et al. 2008).
Its biotechnological potential is also seen in the production of a variety of
hydrolytic enzymes (Federici
1982, Chi et al.
2006, Wang et al.
2007, Li et al.
2007, Ma et al.
2007, Zhiqiang et al.
2008).
Aureobasidum pullulans was taxonomically characterised by de Hoog
& Yurlova (1994) on the
basis of its morphology and nutritional physiology. These authors noted some
differences in growth with galactitol, glucono-
-lactone, creatine and
creatinine, and in gelatin liquefaction. Since the species shows considerable
variability in its morphological and physiological properties, three varieties
have been described during the last decades, viz. Aureobasidium
pullulans var. pullulans
(Viala & Boyer 1891),
A. pullulans var. melanogenum Hermanides-Nijhof
(1977), and A.
pullulans var. aubasidani Yurlova
(Yurlova & de Hoog 1997).
The first two of these were distinguishable by culture discolouration, while
the latter is unique in its production of aubasidan-like EPS (glucans with
-1,4-D-, ß-1,6-D- and ß-1,3-D-glycosidic bonds).
Diagnostically, var. aubasidani is unique due to the absence of
assimilation of methyl--D-glucoside and lactose and by N-source
assimilation for the production of EPS. In a further study using PCR
ribotyping (rDNA RFLP and UP-PCR/hybridisation), Yurlova et al.
(1996) divided the
Aureobasidium strains into four groups, which, however, do not
correlate with morphological differences. Yurlova et al.
(1999) also revealed close
relationships between Kabatiella lini (Laff.) Karak., the teleomorph
species Discosphaerina (Columnosphaeria) fagi (H.J. Huds.) M.E. Barr
and Aureobasidium pullulans.
Aureobasidum pullulans is a ubiquitous and widespread oligotrophe
that can be found in environments with fluctuating water activities, such as
the phyllosphere (Andrews et al.
1994), bathrooms, food and feeds
(Samson et al. 2004).
It can also be found in osmotically very stressed environments, such as
hypersaline waters in salterns
(Gunde-Cimerman et al.
2000), and rocks and monuments
(Urzí et al.
1999). Due to the production of large quantities of yeast-like
propagules, this fungus disperses globally, although thus far it has only
rarely been reported in cold environments. This may be because most
investigations on the occurrence and diversity of fungi in the cold have been
limited to frozen Antarctic soils and Siberian permafrost, where
basidiomycetous yeasts prevail (Abyzov
1993, Babjeva & Reshetova
1998, Deegenaars & Watson
1998, Golubev
1998, Ma et al.
1999,
2000,
2005,
Margesin et al. 2002,
Onofri et al. 2004, Price
2000, Vishniac
2006, Vishniac & Onofri
2003). Thus far, no investigations of mycobiota in ice had been
carried out. We recently investigated ice originating from glacial and
subglacial environments of three different polythermal Arctic glaciers in
Svalbard (Spitsbergen, Norway) (Butinar et al.
2007,
2008,
Sonjak et al. 2006).
During these studies, aureobasidium-like fungi were found among the dominant
ascomycetous mycota. Given the known adaptive ability of A. pullulans
to low water activity (aw) and oligotrophic conditions, it appeared
likely that ice from cryocarstic formations and subglacial ice in polythermal
glaciers constitute a potential natural habitat. Since some of the Arctic
aureobasidium-like isolates deviated phenetically from the pan-global
population, a taxonomic study into the genus Aureobasidium was
performed. Isolates obtained from different niches in Arctic, temperate and
tropical climates were compared by multilocus analyses of rDNA internal
transcribed spacers (ITS), partial large subunit of rDNA (LSU), and partial
introns and exons of genes coding β-tubulin (TUB),
translation elongation factor (EF1) and elongase
(ELO). The main aims of the study were to describe the total
diversity of A. pullulans, to redefine its entities, to describe
potentially new varieties, and to correlate these with their ecology, focusing
on the Arctic sampling area investigated.
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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Physico-chemical parameters (pH, Na+, Mg2+ and K+ concentrations, and total phosphorus content) were determined for five basal ice samples (originating from Kongsvegen), a sample of subglacial meltwater, and three samples of seawater, as described by Gunde-Cimerman et al. (2003).
Isolation and preservation
Ice samples were transported to the laboratory, where they were processed.
The surface layer of ice was aseptically melted at room temperature and
discarded. The remaining ice was transferred to another sterile container and
melted. The resulting water, as well as directly sampled glacier meltwater and
seawater, were filtered immediately (Millipore membrane filters; 0.22-µm
and 0.45-µm pore sizes) in aliquots of up to 100 mL. The membrane filters
were placed on general-purpose isolation media [DRBC: Dichloran
(2,6-dichloro-4-nitroanilin) Rose Bengal Agar (Oxoid CM729) and Malt Extract
Agar (MEA)], as well as on a medium for the detection of moderate xerophiles
[18 % dichloran glycerol agar (DG18;
Hocking & Pitt 1980)], and
on selective media with high concentrations of salt (MEA with addition of 5 %
to 15 % NaCl) or sugar (malt extract yeast extract with 20 %, 35 % and 50 %
glucose). For prevention of bacterial growth, chloramphenicol (50
mg/L-1) was added to all of the media. One drop of the original
water sample was applied onto a membrane and was dispersed with a Drigalski
spatula. For each sample and medium, at least four and up to 10 aliquots were
filtered in parallel, and average numbers of colony forming units (CFUs) were
calculated (Gunde-Cimerman et al.
2000). The plates were incubated for up to 14 wk at 4, 10 and 24
°C.
Subcultures were maintained at the Culture Collection of Extremophilic Fungi (EXF, Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia), while a selection have been deposited at the Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands). Reference strains were obtained from the CBS, and were selected either on the basis of strain history, name, or on the basis of their ITS rDNA sequence. The strains were maintained on MEA and preserved for long periods in liquid nitrogen or by lyophilisation. The strains studied are listed in Table 1. A detailed map of the sampling area, with the sites of retrieved isolates marked, is shown In Fig. 1.
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Cultivation and microscopy
For growth rate determination and the phenetic description of colonies, the
strains were point-inoculated onto potato-dextrose agar (PDA; Oxoid CM139),
and Blakeslee's MEA (Samson et
al. 2004), and then incubated at 25 °C for 7–14 d
in darkness. Surface colours were rated using the colour charts of Kornerup
& Wanscher (1978). For
microscopic morphology, MEA blocks of about 1 x 1 cm2 were
cut out aseptically, placed on sterile microscope slides, and inoculated at
the upper four edges by means of a conidial suspension
(Pitt 1979). Inoculated agar
blocks were covered with sterile cover slips and incubated in moist chambers
for 2, 4, and 7 d at 25 °C in the dark. The structure and branching
pattern of the immersed hyphae were examined under magnifications of
100x and 400x in intact slide cultures under the microscope
without removing the cover slips from the agar blocks. For higher
magnifications (400x, 1000x) the cover slips were carefully
removed and mounted in 60 % lactic acid.
DNA extraction, sequencing and analysis
For DNA isolation, the strains were grown on MEA for 7 d. Their DNA was
extracted according to Gerrits van den Ende & de Hoog
(1999), by mechanical lysis of
approx. 1 cm2 of mycelium. A fragment of rDNA including ITS region
1, 5.8S rDNA and ITS 2 (ITS) was amplified using the ITS1 and ITS4 primers
(White et al. 1990).
LSU (partial 28 S rDNA) was amplified and sequenced with the NL1 and NL4
primers (Boekhout et al.
1995). For amplification and sequencing of the β-tubulin
(TUB) gene, primers Bt2a and Bt2b were used
(Glass & Donaldson 1995).
Translation elongation factor EF-1 (EF1) was amplified
and sequenced with the primers EF1-728F and EF1-986R
(Carbone & Kohn 1999). For
amplification and sequencing of the partial elongase gene (ELO), the
ELO2-F (5'-CAC TCT TGA CCG TCC CTT CGG-3') and ELO2-R (5'-GCG GTG ATG TAC TTC
TTC CAC CAG-3') primers were used, designed for Aureobasidium
pullulans. Reactions were run in a PCR Mastercycler Ep Gradient
(Eppendorf) with a profile of initial denaturation of 2 min at 94 °C,
followed by 6 cycles of 15 s at 94 °C, 15 s at 58 °C and of 45 s at 72
°C, and 30 cycles of 15 s at 94 °C, 15 s at 56 °C and of 45 s at
72 °C, with a final elongation of 7 min at 72 °C. BigDye terminator
cycle sequencing kits were used in sequence reactions (Applied Biosystems,
Foster City, CA, U.S.A.). Sequences were obtained with an ABI Prism 3700
(Applied Biosystems). They were assembled and edited using SeqMan 3.61
(DNAStar, Inc., Madison, U.S.A.). Sequences downloaded from GenBank are
indicated in the gene trees by their GenBank accession numbers; newly
generated sequences are indicated by their strain numbers (see also
Table 1).
Phylogenetic analyses
Sequences were automatically aligned using ClustalX 1.81
(Jeanmougin et al.
1998). Alignments were adjusted manually using MEGA4
(Tamura et al. 2007).
Gene trees were generated with MrBayes software, applying Bayesian inference
(Huelsenbeck & Ronquist
2001, Ronquist &
Huelsenbeck 2003). Three parallel runs were performed for three
million generations with mixed amino-acid models, the default temperature and
five chains. The gene trees were sampled every 100 generations. Gene trees
sampled before the analysis that reached stationarity of likelihood values,
and those sampled before the mean standard deviation of the split frequencies
decreased to under 0.5 % were excluded from the final analysis. The
stationarity of likelihood values was checked using the Tracer software
(Rambaut & Drummond: MCMC Trace Analysis Tool, version 1.4,
2003–2007). In phylogenetic analysis of LSU rDNA the LSU sequence of
Elsinoe veneta (DQ678060
[GenBank]
) was selected as an outgroup, according to
Schoch et al. (2006).
Isolates were grouped on the basis of multilocus analyses and representative
strains were selected for morphological analyses.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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Phylogenetic analyses
Alignments for the phylogenetic analyses included 599 base pairs for LSU,
488 for ITS, 704 for ELO, 323 for EF1, and 425 for
TUB. Internodes were considered strongly supported if they received
posterior probabilities 
95 % (Lutzoni
et al. 2004). Good convergence of the runs was reached
when constructing all of the gene trees with MrBayes. The likelihood values
reached plateaus after approximately 24,000 (LSU), 4,000 (ITS), 6,000
(TUB), 7,000 (EF1) and 15,000 (ELO)
generations, while the mean standard deviations of the split frequencies
dropped below 1 % after 600,000 (LSU), 300,000 (ITS), 800,000 (TUB),
300,000 (EF1) and 200,000 (ELO) generations. The
first 6,000 (LSU), 3,000 (ITS), 8,000 (TUB), 3,000
(EF1) and 2,000 (ELO) trees were discarded as
burn-in.
According to the LSU rDNA analysis (Fig.
2), a high level of support was evident for the clade containing
A. pullulans (groups 1–4) together with Selenophoma
mahoniae A.W. Ramaley (CBS
388.92), Kabatiella caulivora (Kirchn.) Karak
(CBS 242.64) and
Kabatiella microsticta Bubák
(CBS 114.64). Group
7, consisting of Sydowia polyspora (Bref. & Tavel) E. Müll.,
Pringsheimia smilacis E. Müll., Delphinella
strobiligena (Desm.) Sacc. ex E. Müll. & Arx and Dothichiza
pithyophila (Corda) Petr., formed a well supported, but separate, clade.
Separate well-supported clades (groups 5 and 6) joined arctic strains of no
affinity to any of the known taxa. Clade Aureobasidium pullulans was
badly supported (85 posterior probability). Groups 1 and 2 within this clade
were statistically supported, while groups 3 and 4 reached a poor posterior
probability value. Group 1 contained the ex-neotype strain of A.
pullulans var. pullulans
(CBS 584.75), its
supposed teleomorph Discosphaerina (Columnosphaeria) fagi, the
ex-type strain of Kabatiella lini
(CBS 125.21), the
ex-type strain of Dematoidium nigrescens Stautz
(CBS 146.30), the
ex-type strain of A. pullulans var. aubasidani
(CBS 100524), and a
strain of Kabatiella microsticta
(CBS 342.66).
Another strain of K. microsticta was placed on the basal branch as
the sister taxon of K. caulivora
(CBS 242.64) and
Selenophoma mahoniae
(CBS 388.92). Group
2 contained the ex-type strain of A. pullulans var.
melanogenum. Group 3 contained exclusively Arctic strains, while
group 4 consisted of one strain only
(CBS 147.97).
Analyses of the more variable ITS spacers
(Fig. 3A), and ELO
(Fig. 3B), EF1
(Fig. 3C), and TUB
(Fig. 3D) introns and exons
almost consistently supported the first three groups, with only a few
exceptions. For example, several strains of group 2 were dispersed outside the
clade of group 2 in ITS analysis, while in other analyses they formed a
monophyletic group. In analyses of ITS and ELO, group 4 was
supported, whereas based on TUB it was grouped together with group 2,
but on a separate and long branch. The amplification of the
EF1 gene failed in the only strain of group 4; therefore, its
phylogenetic position concerning this gene is unknown.
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TAXONOMY |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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Aureobasidium pullulans (de Bary) Arn. var. aubasidani Yurlova in Yurlova & de Hoog 1997 (MB 442903; T = CBS 100524)
Candida malicola D.S. Clark & R.H. Wallace 1955 (MB 294033; T = CBS 701.76)
Dematoidium nigrescens Stautz 1931 (MB 272259; T = CBS 146.30)
Cultural characteristics: Colonies on MEA/PDA at 25 °C attaining about 40/30 mm diam after 7 d, appearing smooth and slimy due to abundant sporulation, pinkish (pinkish white, 7A2) to yellowish (light yellow, 3A4), reverse yellowish (pale yellow (4A3) to light yellow (4A4)). Black sectors composed of dark pigmented hyphae or conidia develop in some isolates after 14 d. Margin composed of arachnoid mycelium, sometimes in sectors. No aerial mycelium. Deviations: White aerial mycelium at the edge of cultures present in some strains (CBS 109800, EXF-915), some strains entirely filamentous (dH 12637), some develop white, setae-like mycelial formations in colony centre and marginal leathery mycelium (CBS 701.76). Strain CBS 146.30 was black and filamentous already after 1 wk of incubation.
Microscopy: Vegetative hyphae hyaline, smooth, thin-walled, 4–12 µm wide, transversely septate, in older cultures sometimes locally converted to dark-brown hyphae. Conidiogenous cells undifferentiated, intercalary or terminal on hyaline hyphae. Conidia produced synchronously in dense groups from small denticles, and also formed percurrently on short lateral denticles. Conidia hyaline to dark brown. Hyaline conidia one-celled, smooth, ellipsoidal, very variable in shape and size, 7.5–16 x 3.5–7 µm, often with an indistinct hilum. Dark brown conidia (measured in strain CBS 100524, developed after 2 wk) 1–2 celled, one celled 10–17 x 5–7 µm, two celled slightly constricted at septum, 14–25 x 5–11 µm. Budding of hyaline and dark brown conidia frequently seen, with the secondary conidia being smaller than the primary ones. Conidia in old cultures transfer to globose, brownish structures of 10–15 µm diam. Endoconidia, about 6 x 3 µm occasionally seen in intercalary cells.
Maximum tolerated salt concentration: 15 % NaCl.
Cardinal temperatures: Minimum at 4 °C, optimum at 25 °C, maximum at 30 °C.
Specimens examined: France, fruit of Vitis vinifera, 1974, coll. and isol. E.J. Hermanides-Nijhof, ex-neotype culture CBS 584.75; for additional specimens, see Table 1.
Aureobasidium pullulans (de Bary) G. Arnaud var. melanogenum Hermanides-Nijhof – Stud. Mycol. 15: 161, 1977. MycoBank MB352628. Fig. 6.
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Pullularia fermentans Wynne & Gott var. schoenii (Roukhelman) Wynne & Gott 1956 (MB 352450)
Aureobasidium pullulans (de Bary) G. Arnaud var. melanogenum Hermanides-Nijhof 1977 (MB 352628; T = CBS 105.22)
Cultural characteristics: Colonies on MEA/PDA at 25 °C attaining 25 mm diam after 7 d, appearing smooth and slimy due to abundant sporulation and EPS formation, olive brown (4F3-4F8) to black in centre, towards margin mustard yellow (3B6), margin yellowish white (3A2); reverse olive-grey (3E2) at the centre, towards margin dull yellow (3B4), at the margin yellowish white (3A2). Margin composed of arachnoid to thick undulating hyphae growing into the agar, sometimes sectorial. After 14 d the entire colonies are green to black. Aerial mycelium develops in some parts of the colonies. Deviations: White aerial mycelium present in strain CBS 621.80.
Microscopy: Vegetative hyphae in the central part of colonies, dark brown, smooth to slightly roughened, thick walled, 6–12 µm wide, transversely septate, constricted at septa, embedded in EPS, disarticulating to 1–2-celled, dark brown chlamydospores, one celled 13–16 x 8–12 µm, two celled 17–24 x 10–12 µm. Vegetative hyphae at colony edge hyaline, smooth, thin-walled, 2–10 µm wide, transversely septate, getting thicker and darker with age. Immersed hyphae with multiple lateral pegs. Conidiogenous cells undifferentiated, intercalary or terminal on hyaline hyphae, sometimes grown in the form of an outgrowth with three denticles. Conidia produced synchronously in dense groups from small denticles (1.0–2.5 µm long), and also formed percurrently alongside hyphae and on short lateral branches. Conidia hyaline and dark brown. Hyaline conidia one-celled, smooth, ellipsoidal, very variable in shape and size, 8–30 x 3.5–5 µm, often with an indistinct hilum. Dark brown conidia 1–2-celled, smooth, ellipsoidal when one celled, 7 x 6 µm, slightly constricted at septa when two celled, 12–20 x 4–12 µm. Unilateral and bilateral budding of hyaline conidia frequently seen, with the secondary conidia being smaller than the primary ones. Endoconidia not seen.
Maximum tolerated salt concentration: 10 % NaCl.
Cardinal temperatures: Minimum at 10 °C, optimum at 30 °C, maximum at 35 °C.
Specimens examined: Unknown, culture ex-type CBS 105.22 = ATCC 12536 = CECT 2658 = IMI 062460 = NRRL Y-7469, isolated by M. Church; additional specimens see Table 1.
Aureobasidium pullulans (de Bary) G. Arnaud var. subglaciale Zalar, de Hoog & Gunde-Cimerman, var. nov. MycoBank MB512380. Fig. 7.
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Holotype: CBS H-20186
Cultural characteristics: Colonies on MEA/PDA at 25 °C attaining 20 mm (10–35 mm) diam after 7 d, appearing smooth and matt due to abundant sporulation, pinkish (pinkish white, 7A2), reverse pale orange (5A3). After 14 d central areas of colonies remain pinkish, towards the margin becoming dark-brown (greyish brown, 5F3). Margin composed of thick undulating superficial and immersed branched hyphae, sometimes with sectors. Aerial mycelium absent.
Deviations: Culture EXF-2479 develops more intensively pigmented colonies than others, pink in centre and yellowish orange towards the colony margin on MEA, and golden-yellow on PDA.
Microscopy: Vegetative hyphae hyaline, smooth, thin-walled, 2–10 µm wide, transversely septate, in older cultures locally converted to dark brown, thick-walled hyphae of 5–9 µm diam. Conidiogenous cells mostly undifferentiated, intercalary or terminal on hyaline hyphae, sometimes developed in clusters as conidiophore-like structure. Conidia produced synchronously in dense groups from small denticles, and also percurrently on short lateral branches. Conidia hyaline to dark brown. Hyaline conidia one-celled, smooth, ellipsoidal, very variable in shape and size, 5.5–28 x 2–6.5 µm, often with an indistinct hilum. Dark conidia 1–2-celled, one celled 8–16 x5–9 µm, two celled 9–25 x 5.5–7.5 µm. Budding frequently seen, with secondary conidia being smaller than the primary ones. Endoconidia, about 8 x 3 µm, sometimes present in intercalary cells.
Maximum tolerated salt concentration: 10 % NaCl.
Cardinal temperatures: Minimum at 4 °C, optimum and maximum at 25 °C.
Specimen examined: Norway, Spitsbergen, subglacial ice from sea water, 2003, coll. and isol. N. Gunde-Cimerman, Holotype CBS H-20186, culture ex-neotype EXF-2481 = CBS 123387; additional specimens see Table 1.
Aureobasidium pullulans (de Bary) Arnaud var. namibiae Zalar, de Hoog & Gunde-Cimerman, var. nov. MycoBank MB512381. Fig. 8.
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Holotype: CBS H-20184
Cultural characteristics: Colonies on MEA at 25 °C attaining 25 mm diam after 7 d, appearing smooth and shiny due to the leathery structure of colonies, pinkish (pinkish white, 7A2) with brownish (greyish brown, 5E3) central part, margin white (5A1), reverse yellowish (greyish yellow, 4B4); margin composed of superficial aerial mycelium. Colonies on PDA at 25 °C attaining 20 mm diam after 7 d, appearing smooth and shiny due to abundant sporulation, orange-white (5A2) with olive-brown (4F3) centre, sometimes hyphal and with setae, reverse apricot (orange white, 5A2). No aerial mycelium.
Microscopy: Vegetative hyphae hyaline, smooth, thin-walled, 2–13 µm wide, transversely septate, locally converted to dark brown, thick-walled hyphae. Conidiogenous cells undifferentiated, intercalary or terminal on hyaline hyphae and on larger transformed conidia. Conidia produced synchronously in dense groups from small denticles, later formed percurrently on short lateral branches. Conidia hyaline and dark brown. Hyaline conidia one celled, smooth, ellipsoidal, very variable in shape and size, 7–17 x 3.5–7.0 µm, often with an indistinct hilum. Dark brown conidia 1-2-celled, one celled 8–13 x 5–9 µm, two celled 8–24 x 2–10 µm, surrounded by granular EP; if two-celled, constricted at the septum. Budding frequently seen, with secondary conidia smaller than the primary ones. Endoconidia not seen.
Maximum tolerated salt concentration: 10 % NaCl.
Cardinal temperatures: Minimum at 10 °C, optimum at 25 °C and maximum at 30 °C.
Specimen examined: Namibia, dolomitic marble in Namib Desert, 1997, coll. and isol. U. Wollenzien, holotype CBS H-20184, culture ex-type CBS 147.97.
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONReferences |
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ar et al.
2008); this would have diminished its value for routine studies.
The gene provided excellent resolution of the Aureobasidium complex
and thus could reliably be used for tree reconstruction. The anamorph genus Aureobasidium phylogenetically belongs to Ascomycota, order Dothideales, family Dothideaceae (Schoch et al. 2006). The fungi have been known since the late 19th century, when Viala & Boyer (1891) described A. vitis as a common coloniser of the sugary surface of grapes (Vitis vinifera). Type material is not known to be preserved. In her revision of the genus, Hermanides-Nijhof (1977) neotypified Dematium pullulans De Bary (1884) with CBS 584.75, thus establishing A. pullulans as the oldest name for the type species of Aureobasidium. The genus was circumscribed using criteria of conidiogenesis, i.e., synchronous holoblastic conidium production. This feature is also known in sporodochial Kabatiella species forming defined leaf spots on specific host plants. When these fungi are cultured, the sporodochia fall apart, and the micromorphology becomes very similar to that of Aureobasidium pullulans. For this reason, Hermanides-Nijhof (1977) classified all Kabatiella species in Aureobasidium, even though most Kabatiella species have not been cultured and are only known from the sporodochial anamorph on the host plant. LSU sequences of the few species thus far available for study indeed show affinity to A. pullulans.
Kabatiella zeae Narita & Y. Hirats. (Hermanides-Nijhof 1977) is found in isolated positions away from other aureobasidia (de Hoog et al. 1999, Yurlova et al. 1999). Synchronous conidiation is thus polyphyletic. However, in addition to molecular differences for some species, morphological distinctions may also be possible, since most Kabatiella species have sickle-shaped conidia, such as K. caulivora, K. harpospora (Bres. & Sacc.) Arx, K. phoradendri (Darling) Harvey f. umbellulariae Harvey, and K. zeae. In Kabatiella lini, a species clustering within A. pullulans (Fig. 2), the conidia have similar shape, but are slightly larger than in the var. pullulans. Kabatiella microsticta, K. caulivora and another, related pycnidial fungus, Selenophoma mahoniae deviate in all of the genes studied at the variety level. Thus, they might be regarded as separate varieties of A. pullulans, but the possibility cannot be excluded that unintentially the ubiquitous phyllosphere fungus A. pullulans was isolated instead of the pathogen. It appears likely that the plant-invading, host-specific pathogens are consistently different from A. pullulans, which on host plants colonises surfaces only, but unambiguously identified strains are needed to prove this.
Our multilocus analysis shows that Aureobasidium pullulans
consists of three robust main groups, two of which have high statistic support
in LSU and show the same topology with all of the genes sequenced. The
ex-neotype of the species, CBS
584.75, is in group 1, A. pullulans var.
pullulans. This group also contains
CBS 146.30, the
ex-type strain of Dematoideum nigrescens Stautz,
CBS 701.76, the
ex-type strain of Candida malicola D.S. Clark & R.H. Wallace, and
CBS 100524, the
ex-type strain of A. pullulans var. aubasidani Yurlova,
which should thus be regarded as synonyms. The production of aubasidan rather
than pullulan as the main extracellular exopolysaccharide
(Yurlova & de Hoog 1997)
is apparently strain dependent. Although the production of EPS and other
previously described diagnostic characters for this variety were not evaluated
in this study, we believe that used multilocus approach as molecular
diagnostic tool would show the difference of var. aubasidani to other
varieties. The ex-type strain of Dematoidium nigrescens
(CBS 146.30) was
the only initially darkly pigmented strain in group 1, which is probably due
to its degeneration. The var. pullulans, which is newly defined, is
phenetically characterised by rapidly expanding, pinkish cultures that can
develop radial dark brown sectors due to the local presence of thick-walled,
melanised hyphae. Most isolates attributed to this variety originate from
sugary or osmotically fluctuating habitats, such as saline water in the
salterns, tree slime flux, fruit surfaces and phyllosphere
(Table 1). This well supported
variety was obtained pan-globally from temperate to tropical habitats, and was
also found trapped in Spitsbergen glaciers and in ice released from these
glaciers into the sea water. Its distribution is wide, ranging from the Arctic
to the Mediterranean coast. Given the small degree of diversity with TUB,
ELO and EF1, the taxon can be regarded as being
relatively recent.
Group 2, A. pullulans var. melanogenum contains
CBS 105.22, the
ex-type strain of this variety, and an authentic strain,
CBS 123.37 with the
invalid description of Torula schoenii Roukhelman. Earlier data
(Yurlova et al. 1995)
suggested that this taxon cannot be distinguished from var.
pullulans, but current sequence data show that the groups are
strictly concordant. Cultures are characteristically black from the beginning.
They produce an abundance of dark, ellipsoidal conidia, which can either
originate from disarticulating hyphae (arthroconidia) or transfer from hyaline
conidia. The hyaline conidia are ellipsoidal and emerge from inconspicuous
scars alongside undifferentiated hyphae; the process of conidiogenesis is
synchronous in addition to percurrent, the latter being identical to that in
the anamorph genus Hormonema. The sources of isolation of the strains
of this variety, as far as is known, are low-nutrient, mostly low-strength
environments, such as moist metal and glass surfaces, showers, fountains, as
well as ocean water. Only one strain of this variety was retrieved from a
human patient, but it is also possible that this was a culture contaminant,
since it is often reported in air, especially in warmer climates
(Punnapayak et al.
2003). Strains of this variety have a world-wide distribution,
from the Arctic to the tropics. Given its marked diversity with TUB,
ELO and EF1, this may be an ancestral taxon, the introns
having accumulated more mutations than var. pullulans.
Group 3, A. pullulans var. subglaciale Zalar et al. is exclusively known from Kongfjorden glacial and subglacial ice and sea water. Its psychrotolerant nature is in line with its active metabolism under conditions of permanently cold in Arctic glaciers.
Group 4 consists of a single isolate, CBS 147.97, the ex-type of the monotypic variety A. pullulans var. namibiae, isolated from marble in Namibia, Africa. The strain takes an isolated position with all sequenced genes, but has not drifted far away from the ancestral variety.
Other related groups are 5 and 6, which are aureobasidium-like but consistently different. Strains of these groups thus far have only been recovered from glacial ice in Spitsbergen. The species occurred with very high densities in subglacial ice in microchannels, and in gypsum-rich ice at high pH. During their travel through the glacier, these cells have been subjected to extreme variations in aw due to ice freezing and thawing. These conditions are highly selective, for which reason this entity is likely to be restricted to small endemic areas, such as Kongsfjorden (Skidmore et al. 2005). The description as novel species will be the subject of a later paper.
The overall phylogenetic structure of A. pullulans suggests that the species is strictly clonal. A possible teleomorph, Discosphaerina fagi, has been suggested on the basis of ITS sequence similarity (de Hoog et al. 2000), but this finding awaits confirmation with multilocus analysis and re-isolation from single ascospores.
The varieties of Aureobasidium pullulans are markedly different for melanin production. This can be of biotechnological interest, since the organism is highly significant for its pullulan and aubasidan production (Yurlova & de Hoog 1997). Melanin contamination leads to low pullulan quality. Attempts have been made to grow non-pigmented yeast cells, e.g. by culturing A. pullulans in a two-stage fermentation process in media with a special nutrient combination (Shabtai & Mukmenev 1995), or with melanin-deficient mutants (Gniewosz & Duszkiewicz-Reinhard 2008). From the present study, it is apparent that the use of strains of the variety pullulans is recommended.
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- plant associated;
- originating from
Arctic ice;
- originating from
hyperosmotic environment;
- clinical strain.
- water.
