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1 Biotechnical Faculty, Department of Biology, Ve
na pot 111, SI-1000
Ljubljana, Slovenia
2 CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The
Netherlands
3 Institute for Biodiversity and Ecosystem Dynamics, University of
Amsterdam, Kruislaan 315, 1098 SM Amsterdam, The Netherlands
4 Agricultural Institute of Slovenia, Hacquetova 17, p.p. 2553, 1001
Ljubljana, Slovenia
*
Correspondence: Polona Zalar,
polona.zalar{at}bf.uni-lj.si
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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Taxonomic novelties: Cladosporium dominicanum Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. fusiforme Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. halotolerans Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. psychrotolerans Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. salinae Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. spinulosum Zalar, de Hoog & Gunde-Cimerman, sp. nov., C. velox Zalar, de Hoog & Gunde-Cimerman, sp. nov.
Keywords Actin / β-tubulin / halotolerance / ITS rDNA / phylogeny / SSU rDNA / taxonomy
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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We therefore hypothesised that C. sphaerospermum represents a complex of species having either narrow or wide ecological amplitudes. The molecular diversity of strains identified as C. sphaerospermum has not yet been determined and isolates from humans have not yet been critically compared with those from environmental samples. Therefore, a taxonomic study was initiated with the aim to define phylogenetically and morphologically distinct entities and to describe their in vitro osmotolerance and their natural ecological preferences.
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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ovlje salterns (Slovenia) were collected
once per month in 1999. Samples from the Santa Pola salterns and Ebre delta
river saltern (Spain) were taken twice (July and November) in 2000. A saltern
in Namibia and one in the Dominican Republic were sampled twice (August and
October) in 2002. Various salinities, ranging from 15 to 32 % NaCl were
encountered in these ponds.
Isolation and maintenance of fungi
Strains were isolated from salterns using filtration of hypersaline water
through membrane filters (pore diam 0.45 µm), followed by incubation of the
membrane filters on different culture media with lowered water activity
(Gunde-Cimerman et al.
2000). Only colonies of different morphology on one particular
selective medium per sample were analysed further. Strains were carefully
selected from different evaporation ponds, collected at different times, in
order to avoid sampling of identical clones. Subcultures were maintained at
the Culture Collection of Extremophilic Fungi (EXF, Biotechnical Faculty,
Ljubljana, Slovenia), while a selection was deposited at the Centraalbureau
voor Schimmelcultures (CBS, Utrecht, The Netherlands) and the Culture
Collection of the National Institute of Chemistry (MZKI, Ljubljana, Slovenia).
Reference strains were obtained from CBS, and were selected either on the
basis of the strain history, name, or on the basis of their ITS rDNA sequence.
Strains were maintained on oatmeal agar (OA; diluted OA, Difco: 15 g of Difco
255210 OA medium, 12 g of agar, dissolved in 1 L of distilled water) with or
without 5 % additional NaCl. They were preserved in liquid nitrogen or by
lyophilisation. Strains studied are listed in
Table 1.
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Cultivation and microscopy
For growth rate determination and phenetic description of colonies, strains
were point inoculated on potato-dextrose agar (PDA, Difco), OA and Blakeslee
malt extract agar (MEA, Samson et
al. 2002) and incubated at 25 °C for 14 d in darkness.
Surface colours were rated using the colour charts of Kornerup & Wanscher
(1967). For studies of
microscopic morphology, strains were grown on synthetic nutrient agar (SNA,
Gams et al. 2007) in
slide cultures. SNA blocks of approximately 1 x 1 cm were cut out
aseptically, placed upon 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 7 d at 25 °C in darkness. The structure and branching pattern of
conidiophores were observed at magnifications x 100, x 200 and
x 400 in intact slide cultures under the microscope without removing the
cover slips from the agar blocks. For higher magnifications (x 400,
x 1 000) cover slips were carefully removed and mounted in lactic acid
with aniline blue.
Morphological parameters
Morphological terms follow David
(1997), Kirk et al.
(2001) and Schubert et
al. (2007 - this volume).
Conidiophores in Cladosporium are usually ascending and sometimes
poorly differentiated. Though the initiation point of conidiophore stipes
could sometimes be determined only approximately, their lengths were in some
cases useful for distinguishing morphologically similar species when observed
in slide cultures. The branching patterns can be rotationally symmetric or
unilateral. Characters of conidial scars were studied by light and scanning
electron microscopy (SEM). Conidial chains show different branching patterns,
determined by the numbers of conidia in unbranched parts, the nature of
ramoconidia as well as their distribution in conidial chains. Measurements are
given as (i) n1-n2 or (ii)
(n1-)n3-n4(-n2), with
n1 = minimum value observed; n2 = maximum value
observed; n3/n4 = first/third quartile. For conidia and
ramoconidia also average values and standard deviations are listed. The values
provided are based on at least 25 measurements for the conidiophores of each
strain, and at least 50 measurements for conidia.
Ecophysiology
To determine the degree of halotolerance, strains were point-inoculated on
MEA without and with additional NaCl at concentrations of 5, 10, 17 and 20 %
NaCl (w/v) and incubated at 25 °C for 14 d. To determine cardinal
temperature requirements for growth, plates were incubated at 4, 10, 25, 30
and 37 °C, and colony diameters measured after 14 d of incubation.
DNA extraction, sequencing and analysis
For DNA isolation strains were grown on MEA for 7 d. DNA was extracted
according to Gerrits van den Ende & de Hoog
(1999) by mechanical lysis of
approx. 1 cm2 of mycelium. A fragment of the rDNA including the
Internal Transcribed Spacer region 1, 5.8S rDNA and the ITS 2 (ITS) was
amplified using the primers V9G (de Hoog
& Gerrits van den Ende 1998) and LS266
(Masclaux et al.
1995). Sequence reactions were done using primers ITS1 and ITS4
(White et al. 1990).
For amplification and sequencing of the partial actin gene, primers ACT-512F
and ACT-783R were applied according to Carbone & Kohn
(1999). For amplification and
sequencing of the β-tubulin gene primers T1 and T22 were used according
to O'Donnell & Cigelnik
(1997). A BigDye terminator
cycle sequencing kit (Applied Biosystems, Foster City, CA, U.S.A.) was used in
sequence reactions. Sequences were obtained with an ABI Prism 3700 DNA
Analyzer (Applied Biosystems). They were assembled and edited using SeqMan v.
3.61 (DNAStar, Inc., Madison, U.S.A.). Sequences downloaded from GenBank are
indicated in the trees by their GenBank accession numbers; newly generated
sequences are indicated by strain numbers (see also
Table 1). Sequences were
automatically aligned using ClustalX v. 1.81 (Jeanmougin et al.
1998). The alignments were adjusted manually using MEGA3
(Kumar et al. 2004).
Phylogenetic relationships of the taxa were estimated from aligned sequences
by the maximum parsimony criterion as implemented in PAUP v. 4.0b10
(Swofford 2003). Data sets of
the SSU rDNA, ITS rDNA and the β-tubulin and actin genes are analysed
separately. Species of Cladosporium s. str. were compared with
various taxa of the Mycosphaerellaceae using SSU rDNA sequences and
Fusicladium effusum G. Winter (Venturiaceae) as outgroup.
The other data sets focus on Cladosporium s. str., using
Cladosporium salinae Zalar, de Hoog & Gunde-Cimerman as an
outgroup, because this species was most deviant within Cladosporium
in the SSU rDNA analysis (see below). Heuristic searches were performed on all
characters, which were unordered and equally weighted. Gaps were treated as
missing characters. Starting tree(s) were obtained via stepwise, random, 100
times repeated sequence addition. Other parameters included a
"MaxTrees" setting to 9 000, the tree-bisection-reconnection as
branch-swapping algorithm, and the "MulTrees" option set to
active. Branch robustness was tested in the parsimony analysis by 10 000
search replications, each on bootstrapped data sets using a fast step-wise
addition bootstrap analysis. Bootstrap values larger than 60 are noted near
their respective branches. Newly generated sequences were deposited in GenBank
(www.ncbi.nlm.nih.gov);
their accession numbers are listed in Table
1. Alignments and trees were deposited in TreeBASE
(www.treebase.org).
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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The analyses of sequences of the ITS and the β-tubulin and actin gene introns and exons (Figs 2, 3 and 4) do not allow the full elucidation of phylogenetic relationships among these Cladosporium species. Statistical support of the interior tree branches resulting from analyses of the β-tubulin and actin genes is low (bootstrap values mostly < 50 %). While the sister group relationship of C. sphaerospermum and C. fusiforme is highly supported in the analysis based on the β-tubulin gene, analysis of the ITS rDNA indicate that these two species are unrelated, and that C. sphaerospermum is closely related to C. dominicanum. It is clear from the data that the species morphologically resembling C. sphaerospermum are not phylogenetically closely related and that the data we present here do not allow their classification in natural subgroups of the genus Cladosporium. Only C. spinulosum was placed in all analyses among species of the C. herbarum complex and all analyses supported close relatedness of C. langeronii and C. psychrotolerans.
The majority of species described here have slightly ornamented conidia ranging from minutely verruculose (C. fusiforme, C. langeronii, C. psychrotolerans, C. sphaerospermum, C. velox) to verrucose (C. halotolerans) (Fig. 5). The verrucose conidia of C. halotolerans can be recognised also under the light microscope and used as a distinguishing character. Almost smooth to minutely verruculose conidia are encountered in C. dominicanum and C. salinae (Fig. 5). Cladosporium spinulosum, a member of the C. herbarum species complex, has conidia with a digitate ornamentation that can appear spinulose under the light microscope; however, when using the SEM it became clear that its projections have parallel sides and a blunt end (Fig. 5).
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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The concept of distinguishing ramoconidia from secondary ramoconidia has been adopted from Schubert et al. (2007). In the species described here, ramoconidia have been observed often in C. sphaerospermum, sometimes in C. psychrotolerans, C. langeronii and C. spinulosum, and only sporadically in all other species. Therefore, ramoconidia can be seen as important for distinguishing species although sometimes, they can be observed only with difficulty. When using ramoconidia as a diagnostic criterion, colonies only from SNA and not older than 7 d should be taken into account.
Cladosporium sphaerospermum was described by Penzig (1882) from decaying Citrus leaves and branches in Italy. He described C. sphaerospermum as a species with (i) branched, septate and dark conidiophores having a length of 150-300 µm and a width of the main conidiophore stipe of 3.5-4 µm, (ii) spherical to ellipsoid, acrogenously formed conidia of 3.4-4 µm diam, and (iii) ramoconidia of 6-14 x 3.5-4 µm. Penzig's original material is not known to be preserved. Later, a culture derived from CBS 193.54, originating from a human nail, was accepted as typical of C. sphaerospermum. However, de Vries (1952), incorrectly cited it as "lectotype", and thus the same specimen is designated as neotype in this study (see below), with the derived culture (CBS 193.54) used as ex-neotype strain. Numerous strains with identical or very similar ITS rDNA sequences as CBS 193.54 were isolated from hypersaline water or organic substrata including plants or walls of bathrooms. It is not clear yet whether surfaces in bathrooms and of plants, colonised by C. sphaerospermum, can have a similar low water activity as salterns. In our experiments, the strains of this species, however, grew under in vitro conditions at a water activity of up to 0.860, while Hocking et al. (1994) and Aihara et al. (2002) reported that it can grow even at 0.815. Therefore, we consider C. sphaerospermum as halo- or osmotolerant. Hardly any reports are available unambiguously proving that C. sphaerospermum is a human pathogen. It is therefore possible that CBS 193.54 was not involved in any disease process but rather occurred as a contaminant on dry nail material. Cladosporium sphaerospermum is a phylogenetically well-delineated species (Figs 2, 3 and 4).
Strains of C. halotolerans were isolated sporadically from substrata such as peanut cell suspension, tissue culture, bathroom walls and as culture contaminants. This surprising heterogeneity of substrata suggests that C. halotolerans is distributed by air and that it can colonise whatever substrata available, although it may have its natural niche elsewhere. We have recurrently isolated it from hypersaline water of salterns and other saline environments and it was also detected with molecular methods (but not isolated) from skin of a salt water dolphin. There are only few reports of this species from plants (Table 1). It is therefore possible that C. halotolerans is a species closely linked to salty or hypersaline environments although additional sampling is necessary to prove that. Cladosporium halotolerans is morphologically recognisable by relatively oblong to spherical, coarsely rough-walled conidia. The ITS rDNA sequence of a fungus in the skin of a bottlenose dolphin, suffering from lobomycosis, is identical to the sequences of C. halotolerans. This sequence was deposited as Lacazia loboi Taborda, V.A. Taborda & McGinnis (GenBank AF035674 [GenBank] ) by Haubold et al. (1998), who apparently concluded wrongly that a fungus with a cladosporium-like ITS rDNA sequence similar to that of C. halotolerans can be the agent of lobomycosis. Later, Herr et al. (2001) showed that Lacazia loboi phylogenetically belongs to the Onygenales on the basis of amplified SSU rDNA and chitin synthase-2 gene sequences generated from tissue lesions. By this, they confirmed an earlier supposition by Lacaz (1996) who reclassified the organism as Paracoccidioides loboi O.M. Fonseca & Silva Lacaz (Onygenales). It is therefore possible that C. halotolerans was not the main etiologic agent for the lobomycosis and it was colonising the affected dolphin skin secondarily while inhabiting other seawater habitats.
Cladosporium langeronii and C. psychrotolerans are closely related but C. langeronii is particularly well distinguishable from all other Cladosporium species by its slow growing colonies (1-7 mm diam/14 d) and relatively large conidia (4-5.5 x 3-4 µm). Cladosporium psychrotolerans has smaller conidia (3-4 x 2.5-3 µm) but a similar length : width ratio and faster expanding colonies (8-18 mm diam/14 d). Cladosporium langeronii is most likely a complex of at least two species. Strains isolated from the Arctic and the Antarctic may need to be distinguished from C. langeronii s. str. on species level. This inference is particularly supported by analyses of the β-tubulin and actin genes (Figs 3-4). Cladosporium langeronii s. str., represented by an authentic strain of Hormodendrum langeronii Fonseca, Leão & Nogueira, CBS 189.54 (Trejos 1954), has been isolated from a variety of substrata but is tolerating only up to 10 % NaCl. It was originally described by da Fonseca et al. (1927a, b) and subsequently reclassified as Cladosporium langeronii by Vuillemin (1931). The authentic strain derived from an ulcerating nodular lesion on the arm of a human patient. Because other strains of this species are ubiquitous saprobes originating from various substrata, we suspect that C. langeronii is not an important human pathogen. Cladosporium psychrotolerans has been isolated from hypersaline environments only, and tolerates up to 20 % NaCl in culture media.
In general, the human- or animal-pathogenic role of the C. sphaerospermum-like species described here seems to be limited. It is possible that pathogenic species of Cladophialophora Sacc. have been misidentified as C. sphaerospermum or as other species of Cladosporium (de Hoog et al. 2000). Alternatively, true Cladosporium species isolated as clinical strains could have been secondary colonisers since they are able to dwell on surfaces poor in nutrients, possibly in an inconspicuous dormant phase and may then be practically invisible. More likely, they could be air-borne contaminations of lesions, affected nails etc. (Summerbell et al. 2005) or are perhaps disseminated by insufficiently sterilised medical devices, as melanised fungi can be quite resistant to disinfectants (Phillips et al. 1992). They can easily be isolated and rapidly become preponderant at isolation and thus difficult to exclude as etiologic agents of a disease. For example, in 2002, a case report on an intrabronchial lesion by C. sphaerospermum in a healthy, non-asthmatic woman was described (Yano et al. 2002), but we judge the identification of the causal agent to remain uncertain, as it was based on morphology alone and no culture is available. The present authors have the opinion that all clinical cases ascribed to Cladosporium species need careful re-examination.
General characteristics and description of Cladosporium sphaerospermum-like species
The present paper focuses on Cladosporium strains isolated from
hypersaline environments. Comparison of data from deliberate sampling and
analysis of reference strains from culture collections inevitably leads to
statistical bias, and therefore a balanced interpretation of ecological
preferences of the species presented is impossible. Nevertheless, some species
appeared to be consistent in their choice of habitat, and for this reason we
summarise isolation data for all species described. Strains belonging to a
single molecular clade proved to have similar cultural characteristics and
microscopic morphology. Although within most of the species there was some
molecular variation noted (particularly when intron-rich genes were analysed),
some consistent phenetic trends could be observed.
Conidiophores of all C. sphaerospermum-like species lack nodose inflations (McKemy & Morgan-Jones 1991). They are usually ascending and can sometimes be poorly differentiated from their supporting hyphae. Though the initiation point of conidiophore stipes could sometimes be determined only approximately, their lengths were in some cases useful for distinguishing morphologically similar species when observed in slide cultures. Generally, the branched part of a conidiophore forms a complex tree-like structure. The number and orientation of early formed secondary ramoconidia, however, determines whether it is rotationally symmetric or unilateral.
The variability in ITS rDNA sequences observed in all C. sphaerospermum-like species (about 10 %) spans the variation observed in all members of the genus Cladosporium sequenced to date. Thus, the C. sphaerospermum-like species described here may not present a single monophyletic group but may belong to various species complexes within Cladosporium. Verifying existing literature with sequence data of these species (Wirsel et al. 2002, Park et al. 2004), we noticed that names of the common saprobes seem to be distributed nearly at random over phylogenetic trees. For most commonly used names, no type material is available for sequencing. Also verification of published reports is difficult without available voucher strains.
Cladosporium cladosporioides was incorrectly lectotypified based on CBS 170.54 (de Vries 1952), which Bisby considered a standard culture of C. herbarum. The C. cladosporioides species complex requires revision, and will form the basis of a future study. Cladosporium herbarum is maintained as a dried specimen in the Leiden herbarium; Prasil & de Hoog (1988) selected CBS 177.71 as a representative living strain. Strains, earlier accepted as living representatives of C. herbarum, CBS 177.71 and CBS 812.71 (Prasil & de Hoog 1988, Wirsel et al. 2002) and ATCC 66670 (Braun et al. 2003, as Davidiella tassiana) have been re-identified as C. bruhnei Linder by Schubert et al. (2007 - this volume). Ho et al. (1999) used strain ATCC 38027 as a representative of C. tenuissimum Cooke and this strain has identical ITS sequences as the non-deposited C. tenuissimum material used by Moricca et al. (1999). We tentatively accept this concept although we could not include ATCC 38027 in our analyses. The ITS sequence of strain CBS 125.80, identified by Wirsel et al. (2002) as C. oxysporum, is identical to the sequence of ATCC 38027. Strain ATCC 76499, published by Ho et al. (1999) as C. oxysporum, appears to be identical to a number of currently unidentified Cladosporium strains from Slovenian salterns that compose a cluster separate from all remaining species. Strains of this cluster, represented in Fig. 2 by strain ATCC 76499, morphologically resemble C. oxysporum.
Strain CBS 300.96 has not been identified to species level in the present study. It clusters outside the species clade of C. sphaerospermum, with the latter being its nearest relative. CBS 300.96 differs from C. sphaerospermum by having smaller structures: conidiophore stipes [(5-)20-80(-150) x (2-)2.5-3(-4) µm], 0-1 septate ramoconidia [(13-)19-27(-32) x 2-2.5 µm], conidia [(2.5-)3-3.5(-4) x (2-)2-2.5(-3) µm] and secondary ramoconidia [(5-)9-18(-30) x (2-)2.5-2.5(-3) µm]. However, based on a single isolate, we currently refrain from describing it as a new species.
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Key to species treated in this study |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONKey to species treated...References |
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Description of Cladosporium species
Cladosporium dominicanum Zalar, de Hoog &
Gunde-Cimerman, sp. nov. MycoBank
MB510995.
Fig. 6.
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Conidiophora lateralia vel terminalia ex hyphis rectis oriunda; stipes longitudine variabili, (5-)10-100(-200) x (1.5-)2-2.5(-3.5) µm, olivaceo-brunneus, levis vel leniter verruculosus, tenuitunicatus, plerumque unicellularis, simplex vel ramosus. Conidiorum catenae undique divergentes, ad 8 conidia in parte continua continentes. Cellulae conidiogenae indistinctae. Conidia levia vel leniter verruculosa, dilute brunnea, unicellularia, plerumque breviter ovoidea, utrinque angustata, (2.5-)3-3.5(-5.5) x (2-)2-2.5(-2.5) µm, long.: lat. 1.4-1.6; ramoconidia secundaria cylindrica vel quasi globosa, 0-1-septata, (4-)6.5-13(-24.5) x (2-)2.5-3(-4.5) µm, ad 4 cicatrices terminales ferentia; cicatrices inspissatae, protuberantes, 0.5-1.2 µm diam. Hyphae vagina polysaccharidica carentes.
Mycelium without extracellular polysaccharide-like material. Conidiophores arising laterally and terminally on erect hyphae, micronematous and semimacronematous, stipes of variable length, (5-)10-100(-200) x (1.5-)2-2.5(-3.5) µm, olivaceous-brown, smooth to minutely verruculose, thin-walled, almost non-septate, unbranched or branched. Conidial chains branching in all directions, up to eight conidia in the unbranched parts. Conidiogenous cells undifferentiated. Ramoconidia rarely formed. Conidia smooth to minutely verruculose, subhyaline to light brown, non-septate, usually short-ovoid, narrower at both ends, length : width ratio = 1.4-1.6; (2.5-)3-3.5(-5.5) x (2-)2-2.5(-2.5) µm [av. (± SD) 3.4 (± 0.6) x 2.2 (± 0.2)]; secondary ramoconidia cylindrical to almost spherical, 0-1-septate, (4-)6.5-13(-24.5) x (2-)2.5-3(-4.5) µm [av. (± SD) 10.3 (± 5.2) x 2.7 (± 0.6)], with up to four distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.5-1.2 µm diam.
Cultural characteristics: Colonies on PDA reaching 18-36 mm diam, olive-yellow (2D6), hairy granular, flat or slightly furrowed, with flat margin. Droplets of light reseda-green (2E6) exudate sometimes present. Reverse dark green to black. Colonies on OA reaching 19-34 mm diam, olive (2F5), loosely powdery with raised central part due to fasciculate bundles of conidiophores. Reverse dark green. Colonies on MEA reaching 30-32 mm diam, reseda green (2E6), velvety, furrowed, with undulate margin. Reverse dark green-brown. Colonies on MEA + 5 % NaCl reaching 37-41 mm diam, reseda-green (2E6), radially furrowed, velvety, sporulating in the central part or all over the colony, margin white and regular. Reverse brownish green.
Maximum tolerated salt concentration: 75 % of tested strains develop colonies at 20 % NaCl after 7 d, while after 14 d all strains grow and sporulate.
Cardinal temperatures: No growth at 4 and 10 °C, optimum 25 °C (30-32 mm diam), maximum 30 °C (2-15 mm diam), no growth at 37 °C.
Specimen examined: Dominican Republic, from hypersaline water of salt lake Enriquillo, coll. Nina Gunde-Cimerman, Jan. 2001, isol. P. Zalar 25 Feb. 2001, CBS H-19733, holotype, culture ex-type EXF-732 = CBS 119415.
Habitats and distribution: Fruit surfaces; hypersaline waters in (sub)tropical climates.
Differential parameters: No growth at 10 °C, ovoid conidia, large amounts of sterile mycelium.
Strains examined: CPC 11683, EXF-696, EXF-718, EXF-720, EXF-727, EXF-732 (= CBS 119415; ex-type strain).
Note: Cultures of C. dominicanum sporulate less abundantly than C. sphaerospermum and C. halotolerans and tend to lose their ability to sporulate with subculturing.
Cladosporium fusiforme Zalar, de Hoog & Gunde-Cimerman, sp. nov. MycoBank MB510997. Fig. 7.
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Conidiophora erecta, lateralia vel terminalia ex hyphis rectis oriunda; stipes longitudine variabili, (10-)25-50(-100) x (2-)2-3.5(-4) µm, olivaceo-brunneus, levis, crassitunicatus, compluries septatus (cellulis 9-23 µm longis), plerumque simplex. Conidiorum catenae undique divergentes, in parte continua ad 5 conidia continentes. Cellulae conidiogenae indistinctae. Conidia leniter verruculosus, dilute brunnea, unicellularia, plerumque fusiformia, utrinque angustata, (2.5-)3.5-5(-6.5) x (2-)2-2.5(-3) µm, long.: lat. 1.8-2.0; ramoconidia secundaria cylindrica, 0(-1)-septata, (5-)6-11(-22) x (2.5-)2.5-3(-3) µm, ad 4 cicatrices terminales ferentia; cicatrices inspissatae, conspicuae, 0.7-1.0 µm diam. Hyphae vagina polysaccharidica carentes.
Mycelium without extracellular polysaccharide-like material. Conidiophores erect, arising laterally and terminally from straight hyphae, stipes of variable length, (10-)25-50(-100) x (2-)2-3.5(-4) µm, olivaceous-brown, smooth- and thick-walled, regularly-septate (cell length 9-23 µm), mostly unbranched. Conidial chains branching in all directions, up to 5 conidia in the unbranched parts. Conidiogenous cells undifferentiated. Ramoconidia rarely formed. Conidia minutely verruculose, light brown, aseptate, usually fusiform and narrower at both ends, length : width ratio = 1.8-2.0; (2.5-)3.5-5(-6.5) x (2-)2-2.5(-3) µm [av. (± SD) 4.4 (± 0.8) x 2.2 (± 0.2)]; secondary ramoconidia cylindrical, 0(-1)-septate, (5-)6-11(-22) x (2.5-)2.5-3(-3) µm [av. (± SD) 9.0 (± 4.7) x 2.6 (± 0.3)], with up to 4 distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.7-1.0 µm diam.
Cultural characteristics: Colonies on PDA reaching 20-26 mm diam, dull green (30E3), granular due to profuse sporulation, flat, with flat margin. Sterile mycelium absent. Reverse blackish green. Colonies on OA reaching 24-28 mm diam, olive (3F3), granular in concentric circles, consisting of two kinds of conidiophores (low and high), flat, with flat margin. Reverse black. Colonies on MEA reaching 23-28 mm diam, olive (3E5), deeply furrowed, velvety (sporulating all over) with undulate, white margin. Reverse brownish green. Colonies on MEA + 5 % NaCl reaching 28-43 mm diam, olive (3E6), granular due to profuse sporulation, slightly furrowed with flat, olive-grey (3F2) margin. Reverse dark green.
Maximum tolerated salt concentration: Only one of three strains tested (CBS 452.71) developed colonies at 17 % NaCl after 14 d, the other two strains grew until 10 % NaCl.
Cardinal temperatures: For one of three strains (CBS 452.71) the minimum temperature of growth was 4 °C (6 mm diam), for the other two 10 °C (8-9 mm diam); optimum 25 °C (23-28 mm diam), maximum 30 °C (only strain CBS 452.71 grew 5 mm diam), no growth at 37 °C.
Specimen examined: Slovenia, from hypersaline water of
Seovlje salterns, coll. and isol. L. Butinar, Dec. 1999,
CBS H-19732,
holotype, culture ex-type EXF-449 =
CBS 119414.
Habitats and distribution: Osmotic environments worldwide.
Differential parameters: Oblong conidia, relatively low degree of halotolerance.
Strains examined: CBS 452.71, EXF-397, EXF-449 (= CBS 119414; ex-type strain).
Cladosporium halotolerans Zalar, de Hoog & Gunde-Cimerman sp. nov. MycoBank MB492439. Fig. 8.
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Etymology: Refers to its halotolerant habit.
Conidiophora erecta, lateralia vel terminalia ex hyphis rectis oriunda; stipes longitudine variabili, (5-)10-50(-300) x (2-)2.5-3(-5.5) µm, pallide olivaceo-brunneus, levis vel leniter verruculosus, tenuitunicatus, 0-3-septatus, interdum pluriseptatus, simplex, denticulatus. Conidiorum catenae undique divergentes, terminales ad 9 conidia continentes. Cellulae conidiogenae indistinctae. Conidia verrucosa, brunnea vel fusca, unicellularia, plerumque subglobosa vel globosa, raro breviter ovoidea, utrinque angustata, (2-)3-4(-6) x (2-)2.5-3(-5) µm, long.: lat. 1.2-1.5; ramoconidia secundaria cylindrica vel quasi globosa, 0(-1)-septata, (5-)7-12(-37.5) x (2-)2.5-3(-6.5) µm, ad 4 cicatrices terminales ferentia; cicatrices inspissatae, conspicuae, protuberantes, 0.7-1.0(-1.5) µm diam. Hyphae vagina polysaccharidica carentes. Mycelium partly submerged, partly superficial; hyphae without extracellular polysaccharide-like material. Conidiophores erect, arising laterally and terminally from straight hyphae, stipes of variable length, (5-)10-50(-300) x (2-)2.5-3(-5.5) µm, pale olivaceous-brown, smooth to minutely verruculose, thin-walled, 0-3-septate, unbranched, with pronounced denticles. Conidial chains branching in all directions, terminal chains with up to 9 conidia. Conidiogenous cells undifferentiated. Ramoconidia rarely formed. Conidia verrucose, brown to dark brown, non-septate, usually subglobose to globose, less often short-ovoid, narrower at both ends, length : width ratio = 1.2-1.5; (2-)3-4(-6) x (2-)2.5-3(-5) µm [av. (± SD) 3.5 (± 0.7) x 2.7 (± 0.5)]; secondary ramoconidia cylindrical to almost spherical, 0-1-septate, (5-)7-12(-37.5) x (2-)2.5-3(-6.5) µm [av. (± SD) 10.3 (± 4.8) x 2.9 (± 0.6)], with up to 4 distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.7-1.0(-1.5) µm diam.
Cultural characteristics: Colonies on PDA reaching 27-43 mm diam, olive (2F5), slightly furrowed, often covered with grey secondary mycelium, except at the marginal area where only sporulating structures can be observed. Margin white and regular, with submerged hyphae. Reverse pale green to black. Colonies on OA reaching 29-40 mm diam, olive (2F6), flat, uniform, granular due to profuse sporulation and fasciculate bundles of conidiophores, without sterile mycelium. Reverse dark green to black. Colonies on MEA reaching 18-44 mm diam, highly variable in colour, but mainly olive (2E5), and from flat with regular margin to deeply furrowed with undulate margin. Colony centre wrinkled with crater-shaped appearance. Reverse pale to dark green. Colonies on MEA + 5 % NaCl reaching 24-48 mm diam, olive (3E8), furrowed, velvety, with more pale, undulate margins. Reverse dark green to black.
Maximum tolerated salt concentration: Only 15 % of tested strains develop colonies at 20 % NaCl after 7 d, whereas after 14 d all cultures grow and sporulate.
Cardinal temperatures: No growth at 4 °C, optimum 25 °C (18-44 mm diam), maximum 30 °C (6-23 mm diam). No growth at 37 °C.
Specimen examined: Namibia, from hypersaline water of salterns, coll. Nina Gunde-Cimerman, 1 Sep. 2000, isol. P. Zalar, 1 Oct. 2000, CBS H-19734, holotype, culture ex-type EXF-572 = CBS 119416.
Habitats and distribution: Hypersaline water in subtropical climates; indoor environments; Arctic ice; contaminant in lesions of humans and animals; plant phyllosphere; rock.
Literature: Haubold et al. (1998), Meklin et al. (2004).
Differential parameters: Verrucose conidia, short unbranched and non-septate conidiophores which arise laterally alongside erect hyphae.
Strains examined: CBS 191.54, CBS 573.78, CBS 626.82, dH 12862, dH 12991, dH 13911, EXF-228, EXF-380, EXF-565, EXF-567, EXF-571, EXF-572 (= CBS 119416; ex-type strain), EXF-646, EXF-698, EXF-703, EXF-944, EXF-972, EXF-977, EXF-1072, EXF-2372.
Notes: Cladosporium halotolerans strongly resembles C. sphaerospermum. Several strains of this species such as dH 12862, dH 12941, CBS 191.54 and UAMH 7686 have been isolated sporadically from various indoor habitats in Europe, Brazil and the U.S.A. and repeatedly from bathrooms in Slovenia (Table 1). Probably sometimes as uncertain culture contaminations, it has been isolated from plants (GenBank accession no. L25433 [GenBank] ), inner organs of a diseased frog (AY361982 [GenBank] ) and human brain (Kantarcioglu et al. 2002). The presence of C. halotolerans species in gypsum sediments entrapped in Arctic ice, the fact that it was repeatedly isolated from hypersaline water and possibly its presence in dolphin skin (see Discussion) suggest that it has a clear preference for (hyper)osmotic habitats. This is supported by its ability to grow at 20 % NaCl.
The teleomorph of C. halotolerans is predicted to be a Davidiella species. Strain CBS 280.49 was isolated by J.A. von Arx from teleomorphic material of a fungus labelled as Mycosphaerella hyperici (Auersw.) Starbäck on Hypericum perforatum in Switzerland. According to Aptroot (2006) this species may belong in Davidiella and produces a Septoria anamorph. In the original herbarium specimen, CBS H-4867, a Mycosphaerella teleomorph was present, but no sign of a Cladosporium anamorph. We assume that CBS 280.49 was a culture contaminant.
Cladosporium langeronii (Fonseca, Leão & Nogueira) Vuill., Champ. Paras.: 78. 1931. Fig. 9.
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Cladosporium langeronii (Fonseca, Leão &
Nogueira) Cif., Manuale di Micologia Medica, ed. 2: 488 (1960), comb.
superfl. Mycelium partly submerged, partly superficial; hyphae sometimes enveloped in polysaccharide-like material. Conidiophores erect or ascending, micronematous and macronematous, stipes of variable length, (20-)50-130(-200) x (3-)3.5-4.5(-6.5) µm, dark brown, rough- and thick-walled, regularly septate (cell length 9-22 µm), arising laterally and terminally from submerged or aerial hyphae, branched. Conidial chains dichotomously branched, up to 6 conidia in the unbranched parts. Conidiogenous cells undifferentiated, sometimes seceding and forming ramoconidia. Ramoconidia cylindrical, 0-1 septate, (10-)11-22(-42) x (3-)3.5-4.5(-5) µm, base broadly truncate, 2-3.5 µm wide, slightly thickened and somewhat darkened. Conidia irregularly verruculose to sometimes loosely verrucose, dark brown, non-septate, usually ovoid, length : width ratio = 1.3-1.5; conidial size (3-)4-5.5(-8) x (2-)3-4(-5) µm [av. (± SD) 4.8 (± 1.0) x 3.5 (± 0.6)]; secondary ramoconidia cylindrical to almost spherical, mostly 0-1(-2)-septate, (5.5-)7.5-12.5(-35.5) x (2.5-)3-4.5(-5.5) µm [av. (± SD) 10.7 (± 4.7) x 3.6 (± 0.8)], with 2, rarely 3 distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.9-1.5(-2.3) µm diam.
Cultural characteristics: Colonies on PDA, OA and MEA with restricted growth, attaining 2.5-4.5, 1.5-7.0 and 1.0-5.5 mm diam, respectively. Colonies flat or heaped (up to 3 mm), dark green (30F4), with black reverse and slightly undulate margin with immersed mycelium. Sporulating on all media. On MEA + 5 % NaCl growth is faster, colonies attaining 8.5-12.0 mm diam, sporulating and growing deeply into the agar.
Maximum tolerated salt concentration: All strains develop colonies at 17 % NaCl after 14 d.
Cardinal temperatures: No growth at 4 °C, optimum/maximum 25 °C (1.0-5.5 mm diam), no growth at 30 °C.
Specimen examined: Brazil, from man ulcero-nodular mycosis of hand and arm, 1927, coll. and isol. da Fonseca, CBS H-19737, holotype, culture ex-type CBS 189.54.
Habitats and distribution: Polar ice and biomats; conifer wood and window frame in Europe; humans; strains originating from nasal mucus (Buzina et al. 2003) have 100 % sequence homology with the strains studied, as well as with a clone from mycorrhizal roots (Menkis et al. 2005). The species is distributed worldwide, without any apparent predilection for a particular habitat. The strains from clinical cases probably were culture contaminants.
Literature: da Fonseca et al. (1927a, b).
Differential parameters: Restricted growth; lowest salt halotolerance taxon of all C. sphaerospermum-like species.
Strains examined: CBS 189.54 (ex-type strain), CBS 601.84, CBS 101880, CBS 109868, dH 11736, dH 12459 = EXF-999, dH 13833 = EXF-1933.
Notes: De Vries (1952) synonymised the isolate identified as Hormodendrum langeronii with C. sphaerospermum. Strains of this species have often been identified as C. cladosporioides (Buzina et al. 2003, Menkis et al. 2005) although it has slightly longer conidia.
Cladosporium psychrotolerans Zalar, de Hoog & Gunde-Cimerman, sp. nov. MycoBank MB492428. Fig. 10.
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Mycelium partim submersum; hyphae vagina polysaccharidica carentes. Conidiophora erecta vel adscendentia; stipes (10-)50-100(-150) x (3-)3.5-4(-7.5) µm, olivaceo-brunneus, levis, crassitunicatus, compluries regulariter septatus (cellulis 10-40 µm longis), identidem dichotome ramosus. Conidiorum catenae undique divergentes, terminales partes simplices ad 4 conidia continentes. Cellulae conidiogenae indistinctae. Ramoconidia primaria cylindrica, (18-)19-22(-43) x (2.5)3-3.5(-4.5) µm, 0(-1)-septata. Conidia leves vel leniter verruculosa, dilute brunnea, unicellularia, globosa vel ovoidea, (2.5-)3-4(-4.5) x (2-)2.5-3(-3) µm, long.: lat. 1.3-1.4; ramoconidia secundaria cylindrica, 0-1(-2)-septata, (5-)8-16(-36) x (2-)2.5-3(-5) µm, ad 4 cicatrices terminales ferentia; cicatrices inspissatae, conspicuae, 0.5-2 µm diam.
Mycelium partly superficial partly submerged; hyphae without extracellular polysaccharide-like material. Conidiophores erect or ascending, macronematous, stipes (10-)50-100(-150) x (3-)3.5-4(-7.5) µm, olivaceous-brown, smooth or almost so, thick-walled, regularly septate (cell length 10-40 µm), arising laterally from aerial hyphae, repeatedly dichotomously branched. Conidial chains branching in all directions, up to 4 conidia in the unbranched parts. Ramoconidia sometimes formed, cylindrical, (18-)19-22(-43) x (2.5)3-3.5(-4.5) µm, aseptate, rarely 1-septate, with a broadly truncate base, up to 2 µm wide, unthickened or slightly thickened, somewhat darkened-refractive. Conidia smooth to minutely verruculose, light brown, non-septate, spherical to ovoid, length : width ratio = 1.3-1.4; conidial size (2.5-)3-4(-4.5) x (2-)2.5-3(-3) µm [av. (± SD) 3.4 (± 0.5) x 2.5 (± 0.2)]; secondary ramoconidia cylindrical, 0-1(-2)-septate, (5-)8-16(-36) x (2-)2.5-3(-5) µm [av. (± SD) 12.7 (± 6.5) x 3.0 (± 0.5)], with up to 4 distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.5-2 µm diam.
Cultural characteristics: Colonies on PDA reaching 13-18 mm diam, velvety, olive (3F4) due to profuse sporulation, flat with straight margin. Reverse dark green. Colonies on OA reaching 13-15 mm diam, olive (2F8), of granular appearance due to profuse sporulation; aerial mycelium sparse. Margin regular. Reverse black. Colonies on MEA reaching 8-15 mm diam, olive (2F4), velvety, radially furrowed with undulate white margin. Colonies on MEA with 5 % NaCl growing faster than on other media, reaching 25-27 mm diam, olive (3E6) and granular due to profuse sporulation, either slightly furrowed or heavily wrinkled with regular or undulate margin. Reverse dark green.
Maximum tolerated salt concentration: 17 % NaCl after 14 d.
Cardinal temperatures: Minimum at 4 °C (5 mm diam), optimum and maximum at 25 °C (8-15 mm diam).
Specimen examined: Slovenia, from hypersaline water of
Seovlje salterns, coll. and isol. S. Sonjak, May 1999,
CBS H-19730,
holotype, culture ex-type EXF-391 =
CBS 119412.
Habitats and distribution: Hypersaline water in the Mediterranean basin.
Differential parameters: Growth at 4 °C; maximal NaCl concentration 17 % NaCl, which differentiates it from other species with similar conidia, like C. sphaerospermum, C. halotolerans and C. dominicanum.
Strains examined: EXF-326, EXF-332, EXF-391 (= CBS 119412; ex-type strain), EXF-714.
Cladosporium salinae Zalar, de Hoog & Gunde-Cimerman, sp. nov. MycoBank MB492438. Fig. 11.
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Mycelium partim submersum; hyphae multa rostra lateralia ferentes, hyphae vagina polysaccharidica involutae. Conidiophora vix distincta, lateralia vel terminalia ex hyphis aeriis oriunda; stipes longitudine variabili, (5-)25-50(-60) x (2-)2.5-3(-4) µm, olivaceo-brunneus, levis vel leniter verruculosus, crassitunicatus, irregulariter dense septatus (cellulis 6-29 µm longis), simplex, interdum ramosus. Conidiorum catenae undique divergentes, terminales ad 6 conidia continentes. Cellulae conidiogenae nonnumquam integratae, in summo sequentiam sympodialem denticulorum formantes. Conidia levia, interdum leniter verruculosa, dilute brunnea, unicellularia, plerumque fusiformia, (4.5-)5.5-7.5(-10) x (2-)2.5-3(-3.5) µm, long.: lat. 1.9-2.4; ramoconidia secundaria cylindrica, 0-1(-2)-septata, (7.5-)9.5-13.5(-19) x (2.5-)2.5-3.5(-4.5) µm, ad 5 cicatrices terminales ferentia; cicatrices inspissatae, conspicuae, protuberantes, 0.7-1.8 µm diam.
Mycelium partly superficial partly submerged, with numerous lateral pegs, consistently enveloped in polysaccharide-like material. Conidiophores poorly differentiated, micronematous, stipes (5-)25-50(-60) x (2-)2.5-3(-4) µm, olivaceous-brown, smooth to often minutely verruculose or irregularly rough-walled, thick-walled, irregularly densely septate (length of cells 6-29 µm), arising laterally and terminally from aerial hyphae, unbranched, occasionally branched. Conidial chains branching in all directions, terminal chains with up to 6 conidia. Conidiogenous cells sometimes integrated, producing sympodial clusters of pronounced denticles at their distal ends. Conidia usually smooth, occasionally minutely verruculose, light brown, aseptate, usually oblong ellipsoidal to fusiform, length : width ratio = 1.9-2.4; (4.5-)5.5-7.5(-10) x (2-) 2.5-3(-3.5) µm [av. (± SD) 6.7 (± 1.3) x 2.9 (± 0.4)]; secondary ramoconidia cylindrical, 0-1(-2)-septate, (7.5-)9.5-13.5(-19) x (2.5-)2.5-3.5(-4.5) µm [av. (± SD) 12.1 (± 3.3) x 3.2 (± 0.6)], with up to 5 distal scars. Conidiogenous scars thickened and conspicuous, protuberant, 0.7-1.8 µm diam.
Cultural characteristics: Colonies on PDA reaching 10-27 mm diam, granular, olive (2E4) due to profuse sporulation, with white undulate margin. Aerial mycelium absent. Colonies either heaped or radially furrowed, in the marginal area growing deeply into the agar. Reverse dark brown to dark green. Colonies on OA reaching 7-20 mm diam, olive (3E6), of granular appearance due to profuse sporulation, aerial mycelium present. Margin either undulate or arachnoid, deeply furrowed. Reverse pale brown to dark green. Colonies on MEA reaching 8-19 mm diam, velvety, reseda-green (2E6), heaped. Margin furrowed, growing
lar).
