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Unitat de Microbiologia, Facultat de Medicina i Cièncias de la Salut, Universitat Rovira i Virgili, 43201 Reus, Tarragona, Spain
Correspondence
Josepa Gené
josepa.gene{at}urv.cat
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin (two loci), calmodulin and the internal transcribed spacer region of the 5.8S rRNA, confirmed our proposal. Both the phylogenetic analysis and morphological studies excluded Pseudallescheria africana and Pseudallescheria fimeti from the genus Pseudallescheria. The former is proposed as a member of the new genus Petriellopsis, and the latter has been accommodated in Lophotrichus. The type strains of Parascedosporium tectonae gen. nov., comb. nov., Petriellopsis africana gen. nov., comb. nov. and Lophotrichus fimeti comb. nov. are respectively CBS 127.84T, CBS 311.72T and CBS 129.78T.
The GenBank/EMBL/DDBJ accession numbers for the gene sequences determined in this study are listed in Table 1.
Figures showing phylogenetic analyses of sequences of the ITS region and TUB, BT2, CAL and SSU sequences of strains examined in this study and a table comparing sequence dataset characteristics and characteristics inferred from the datasets using maximum-parsimony analyses are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; de Hoog et al., 2000). They show a teleomorph (sexual state) characterized by closed ascomata (cleistothecia), a peridium (ascomata wall) of ‘textura epidermoidea’, asci that are broadly clavate or spherical, and ellipsoidal or fusiform ascospores which are symmetrical or nearly so. In addition, they can develop two anamorphs (asexual states), Scedosporium, characterized by hyaline, cylindrical conidiogenous cells arising from undifferentiated hyphae which produce obovoidal, hyaline, sticky conidia, and Graphium, characterized by large, erect bundles of hyphae terminating in a dense aggregate of conidiogenous cells. Both anamorphs produce conidia from a short extension of the conidiogenous cells with annellidic development. Although not the case for all strains, the three forms can be found in a single culture.
Until recently, the genus Pseudallescheria comprised the following species: Pseudallescheria africana, Pseudallescheria angusta, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Pseudallescheria desertorum, Pseudallescheria fimeti and Pseudallescheria fusoidea (von Arx et al., 1988). These species are morphologically very similar and the differences between them are in the size of the cleistothecia and ascospores. Pseudallescheria boydii is the most common species of the genus, but at the same time it has been proven to be genetically highly variable (de Hoog et al., 1994; Rainer et al., 2000; Gilgado et al., 2005; Rainer & de Hoog, 2006). Gilgado et al. (2005), based on multilocus analysis, described two novel species within the Pseudallescheria boydii complex (Pseudallescheria minutispora and Scedosporium aurantiacum), although other phylogenetic species remain undescribed. Similar conclusions were reached by Rainer & de Hoog (2006) based on sequence analysis of the rRNA genes. In addition, recent molecular studies revealed that Pseudallescheria africana and Pseudallescheria fimeti are phylogenetically very distant from the other Pseudallescheria species, suggesting that they may belong to other genera of the Microascaceae (Issakainen et al., 1999, 2003; Rainer & de Hoog, 2006), but unfortunately only one strain of each of the two species exists (CBS 311.72T and CBS 129.78T, respectively).
Although these fungi can infect animals, including humans, most of them are geophilic. Considering that one of the limitations of the Microascaceae is that only a very reduced number of strains are known for the different species, the soil can be an important source of fresh isolates useful for performing phylogenetic studies. In a recent soil survey of different geographical regions, we recovered a fungus (CBS 120338) which developed two types of anamorph, but the teleomorph was not observed. It produced a Graphium anamorph similar to that developed by Pseudallescheria species and a second type of anamorph characterized by solitary conidiophores that produced conidia from denticulate conidiogenous cells similar to those of the anamorph genus Sporothrix. This fungus was morphologically similar to Graphium tectonae (Booth, 1964), an uncommon and poorly studied phytopathogenic fungus. The present paper focuses on its morphological and molecular characterization using a multilocus sequence analysis and determines its phylogenetic relationships with Pseudallescheria and relatives.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. A total of 38 isolates were studied; most of them were reference strains provided by different culture collections and seven were isolated by the authors from soil of different geographical regions. One hundred and six sequences of different species of Pseudallescheria, Scedosporium and other morphologically close species of the family Microascaceae were obtained from GenBank and a total of 24 sequences [six each of two regions of the -tubulin gene (BT2 and TUB), six of the calmodulin gene (CAL), five of the rRNA internal transcribed spacer region (ITS) and one of the 18S rRNA gene] were newly generated for the present study. The techniques and culture media used for isolation of the soil isolates were described previously (Gilgado et al., 2005).
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-tubulin gene (BT2 and TUB) were described previously (Gilgado et al., 2005). A partial region of the 18S (SSU) rRNA gene was amplified by using the primers NS1 and NS24UCB and sequenced with primers NS19UCB and NS22UCB (Gargas & Taylor, 1992). The protocol used for amplification and sequencing was that described by Gargas & DePriest (1996).
Phylogenetic analysis.
Sequences were aligned with CLUSTAL_X (version 1.8) (Thompson et al., 1997) followed by manual adjustments with a text editor. Ambiguously aligned regions were excluded from the analysis (Supplementary Table S1 in IJSEM Online). The phylogenetic analysis was performed using PAUP* version 4.0b10 (Swofford, 2002). Briefly, the most-parsimonious tree was obtained after 100 heuristic searches with random sequence addition and tree bisection–reconnection branch-swapping algorithms, collapsing zero-length branches and saving all minimal-length trees (MulTrees). Support for internal branches was assessed using a heuristic parsimony search of 1000 bootstrapped datasets. Combined datasets of ITS, CAL, BT2 and TUB were tested for incongruence with the partition homogeneity test, as implemented in PAUP*. Kishino–Hasegawa tests were performed in order to determine whether trees were significantly different. The trees obtained were rooted at the midpoint with the exception of the ITS, which was rooted with a sequence of an isolate of Scopulariopsis chartarum as outgroup.
Morphological study.
The isolates were subcultured on potato dextrose agar (PDA; Difco) and on oatmeal agar (OA; 30 g oat flakes, 1 g MgSO4 . 7H2O, 1.5 g KH2PO4, 15 g agar, 1 litre tap water) at 25 °C in darkness. Microscopic features were examined by direct observation in wet mounts with 85 % lactic acid and by slide cultures on OA, the medium in which these fungi showed the best sporulation. In descriptions, colour notations in parentheses were from Kornerup & Wanscher (1984). Photomicrographs were obtained under a Leitz Dialux 20 light microscope, using phase-contrast and Nomarski differential interference. Growth rates were obtained on PDA at 25, 37, 40, 42 and 45 °C in the dark.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This analysis again confirmed the close relationship of Pseudallescheria africana with Petriella sordida and Scedosporium prolificans. The clade formed by these species received 100 % bootstrap support. The clear separation of isolate CBS 120338 from the other Pseudallescheria and Scedosporium isolates was proved.
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). One showed the features typical of Graphium, similar to those observed in the other species studied here. The second one was characterized by solitary conidiophores with sympodial conidia emerging from denticulate conidiogenous cells, as mentioned above. The latter type of conidiogenesis is clearly different from that of Scedosporium and similar to that showed by Graphium tectonae. Comparisons of the ITS sequences (485 bp) of the Argentinian isolate CBS 120338 with those of two reference strains of Graphium tectonae (the type strain CBS 127.84T and strain CBS 118694) showed 100 % similarity and confirmed the identification of this isolate as Graphium tectonae.
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), we noted the presence of clavate asci and asymmetrical ascospores with inconspicuous or absent germ pores. In Pseudallescheria species, the asci are globose to ovoid and the ascospores are symmetrical or nearly so and with a conspicuous germ pore at each pole (von Arx et al., 1988). The absence of ostiolate ascomata differentiates Pseudallescheria africana from the other species of Petriella.
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). In addition, no anamorph was observed. These features are typical of Lophotrichus, a genus that also belongs to the Microascaceae. To confirm this, a comparative partial sequence analysis of the SSU gene of the type strain of Pseudallescheria fimeti and other sequences from different taxa of the Microascaceae retrieved from GenBank was performed. In the phylogram obtained (Supplementary Fig. S5), Pseudallescheria fimeti was nested with the two Lophotrichus species (Lophotrichus plumbescens and Lophotrichus indicus) included in the study, and was placed very far from the species of Pseudallescheria and Petriella. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, numerous taxonomic studies on the Microascaceae have been published, based mainly on different molecular analyses (de Hoog et al., 1994; Guého & de Hoog, 1991; Issakainen et al., 1999, 2003; Rainer et al., 2000; Rainer & de Hoog, 2006), and several novel species have been proposed (Gilgado et al., 2005). However, none of the species of these genera shows the type of conidiogenesis observed in the Argentinian fungus. Given the genotypic and phenotypic uniqueness of this fungus, the name Parascedosporium tectonae gen. nov., comb. nov. is proposed.
The results of the combined loci dataset demonstrated that Pseudallescheria africana was clearly separated from the other species of Pseudallescheria. In a recent study, the phylogenetic position of this species was ambiguous, since, on the basis of large subunit (LSU) rRNA gene sequence data, it was placed in the Petriella clade, and, based on ITS sequences, in the Pseudallescheria branch (Rainer & de Hoog, 2006). Judging from its non-ostiolate ascomata, these authors suggested that this species should be classified in Pseudallescheria. However, our study revealed that this species was phylogenetically distant from the Pseudallescheria clade. Issakainen et al. (1999, 2003), based on the analysis of partial LSU rRNA gene sequences, had already demonstrated that Pseudallescheria africana was phylogenetically distant from the remaining Pseudallescheria species. Therefore, on the basis of its phenotypic and molecular characteristics, the new combination Petriellopsis africana gen. nov., comb. nov. is proposed.
The present study also revealed that Pseudallescheria fimeti was placed outside the Petriella and Pseudallescheria clades, which agrees with Issakainen et al. (1999) and Rainer & de Hoog (2006). We examined the type strain of this species and noticed that its peridium has a ‘textura angularis’, a feature typical of the genus Lophotrichus (von Arx et al., 1988). In the analysis of part of the SSU rRNA gene sequence, Pseudallescheria fimeti clustered in the same clade as the two species of Lophotrichus included in the study. These results confirm that Pseudallescheria fimeti should be reclassified in Lophotrichus, and consequently the new combination Lophotrichus fimeti comb. nov. is proposed.
Latin diagnosis of Parascedosporium Gilgado, Gené, Cano et Guarro gen. nov.
Ad fungos conidiales hyphomycetes pertinens. Conidiophora erecta, simplicia vel ramosa. Cellulae conidiogenae polyblasticae, terminales, sympodiales, cum denticulis cylindricis manifestis praeditae. Conidia solitaria, sicca, unicellularia, subhyalina vel brunnea, laevia et crassitunicata. Synanamorphosis: Graphium sp. Teleomorphosis ignota. Typus: Parascedosporium tectonae (C. Booth) Gilgado, Gené, Cano et Guarro.
Description of Parascedosporium Gilgado, Gené, Cano & Guarro gen. nov.
Parascedosporium (Pa'ra.sce.do.spo'ri.um. Gr. prep. para beside; N.L. neut. n. Scedosporium a genus name; N.L. neut. n. Parascedosporium beside the genus Scedosporium, because the morphology is similar to that of Scedosporium).
Conidial fungi, hyphomycetes. Conidiophores erect, simple or irregularly branched. Conidiogenous cells polyblastic, usually terminal, forming conidia by sympodial growth on conspicuous cylindrical denticles. Conidia solitary, dry, single-celled, subhyaline to brown, thick-walled. A Graphium synanamorph is present. Teleomorph unknown. The type species is Parascedosporium tectonae (C. Booth) Gilgado, Gené, Cano & Guarro.
Description of Parascedosporium tectonae (C. Booth) Gilgado, Gené, Cano & Guarro comb. nov.
Parascedosporium tectonae (tec.to'nae. N.L. fem. adj. tectonae referring to the plant taxon from where the fungus was first isolated).
Basionym: Graphium tectonae C. Booth, Mycol Pap 94, 5 (1964).
Since the original description of the species is quite incomplete, the following description is provided.
Colonies on PDA at 25 °C are 49–50 mm after 14 days at 25 °C. They are velvety, radially folded, grey (2D–E1), slightly granular at the centre due to the presence of numerous synnemata, smooth and dark grey (2F1) at the periphery, with an entire and slightly fimbriate margin; reverse dark grey (2F1) to black at the centre, pale grey (2B–C1) at the periphery. Colonies on OA are zonate and olive–grey (2F2–4), with aerial mycelium practically absent and granular at the centre due to the abundance of synnemata; the margin is diffuse and the reverse colourless. Conidiophores are solitary and emerge from aerial mycelium or form synnemata (Graphium) mainly from surface mycelium. Solitary conidiophores are simple, and are often reduced to conidiogenous cells, or irregularly branched, with branches often bearing two to three conidiogenous cells. Conidiogenous cells are cylindrical to flask-shaped, 6–20x1.5–2.5 µm, hyaline, thin-walled, usually terminating in a cluster of two to five cylindrical denticles, up to 1 µm long. Conidia are often obovate, 5–6x3–4 µm, smooth and more or less thick-walled. Synnemata are erect, 325–450 µm long, consisting of a cylindrical stipe, 12.5–25 µm wide, dark grey, smooth-walled, slightly roughened apically, and they terminate in a slimy head of conidia, 50–87x80–138 µm. Conidiogenous cells are percurrent, with conspicuous annellations, lateral or terminal, subhyaline, smooth-walled, cylindrical, 10–37x1.5–2.5 µm, less frequently intercalary as a lateral projection on hyphae, and up to 5x2 µm. Conidia are (sub)cylindrical or clavate, 5.5–7.5x2.5–3.5 µm. Conidia developing from undifferentiated hyphae of the substratum are also observed. They are produced scarcely, lateral, usually sessile, brown, smooth- and thick-walled, subglobose to obovate, 6–8x4.5–6 µm. The optimum growth temperature is 25–30 °C. Maximum temperature for growth is 37 °C; does not grow at 40 °C. Phylogenetic placement, as deduced by analysis of the combined data of four loci, is shown in Fig. 1. Relevant morphological features are shown in Fig. 2.
The type strain is CBS 127.84T.
Latin diagnosis of Petriellopsis Gilgado, Cano, Guarro et Gené gen. nov.
Ascomata sphaerica, non ostiolata, fusca. Asci unitunicati, clavati, structure apicalis absens, evanescentes. Ascosporae uniseriate, unicellulares, ellipsoideae, asymmetricae, poris germinationis inconspicuus vel absentibus. Conidia modo Scedosporii et Graphii. Typus: Petriellopsis africana (Arx et G. Franz) Gilgado, Cano, Guarro et Gené.
Description of Petriellopsis Gilgado, Cano, Guarro & Gené gen. nov.
Petriellopsis (Pe.tri.ell.op'sis. N.L. fem. n. Petriella a genus name; N.L. fem. n. opsis from Gr. fem. n. opsis appearance; N.L. fem. n. Petriellopsis like the genus Petriella in appearance, because the morphology is similar to Petriella).
Ascomata are spherical, non-ostiolate, immersed to nearly superficial, blackish brown, with a membranaceus peridium, composed of several layers of cells, ‘textura epidermoidea’ in surface. Asci are unitunicate, clavate, without distinct apical structures, evanescent. Ascospores are uniseriate, single-celled, broadly ellipsoidal, asymmetrical, with inconspicuous or absent germ pores, orange to greenish coloured. Produces a Scedosporium and a Graphium anamorph. Its phylogenetic placement, as deduced by analysis of the combined data of four loci, is shown in Fig. 1. Relevant morphological features are shown in Fig. 3(a, b). The type species is Petriellopsis africana (Arx & G. Franz) Gilgado, Cano, Guarro & Gené.
Description of Petriellopsis africana (Arx & G. Franz) Gilgado, Cano, Guarro & Gené comb. nov.
Petriellopsis africana (a.fri.ca'na. L. fem. adj. africana pertaining to the African continent).
Basionym: Pseudallescheria africana (Arx & G. Franz) McGinnis, A. A. Padhye & Ajello, Mycotaxon 14, 97 (1982).
Synonym: Petriellidium africanum Arx & G. Franz (1973).
A detailed description of this species was reported by von Arx (1973). The relevant morphological features that distinguish this fungus from other related genera are its clavate asci and the asymmetrical ascospores with inconspicuous or absent germ pores (Fig. 3a, b). The type strain is CBS 311.72T.
Description of Lophotrichus fimeti (Arx, Mukerji & N. Singh) Gilgado, Guarro, Gené & Cano comb. nov.
Lophotrichus fimeti (fi.me'ti. L. gen. n. fimeti of a dung-hill, referring to its coprophilous habitat).
Basionym: Pseudallescheria fimeti (Arx, Mukerji & N. Singh) McGinnis, A. A. Padhye & Ajello, Mycotaxon 14, 98 (1982).
Synonym: Petriellidium fimeti Arx, Mukerji & N. Singh (1978).
This species was described by von Arx (1978). Analysis of the SSU sequence proved that it clustered together with members of Lophotrichus (Supplementary Fig. S5). The morphological characters that distinguish this fungus from other related genera are its non-ostiolate ascomata with a peridium of ‘textura angularis’, its large ascospores with wide germ pores (Fig. 3c, d) and the absence of Scedosporium and Graphium anamorphs. The type strain is CBS 129.78T.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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de Hoog, G. S., Marvin-Sikkema, F. D., Lahpoor, G. A., Gottschall, J. C., Prins, R. A. & Guého, E. (1994). Ecology and physiology of Pseudallescheria boydii, an emerging opportunistic fungus. Mycoses 37, 71–78.[Medline]
de Hoog, G. S., Guarro, J., Gené, J. & Figueras, M. J. (2000). Atlas of Clinical Fungi, 2nd edn. Utrecht, The Netherlands/Reus, Spain: Centraalbureau voor Schimmelcultures/Universitat Rovira i Virgili.
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Gilgado, F., Cano, J., Gené, J. & Guarro, J. (2005). Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species. J Clin Microbiol 43, 4930–4942.
Guého, E. & de Hoog, G. S. (1991). Taxonomy of the medical species of Pseudallescheria and Scedosporium. J Mycol Med 1, 3–9.
Issakainen, J., Jalava, J., Saari, J. & Campbell, C. K. (1999). Relationship of Scedosporium prolificans with Petriella confirmed by partial LSU rDNA sequences. Mycol Res 103, 1179–1184.[CrossRef]
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Kornerup, A. & Wanscher, J. H. (1984). Methuen Handbook of Colour, 3rd edn. London: Methuen.
Rainer, J. & de Hoog, G. S. (2006). Molecular taxonomy and ecology of Pseudallescheria, Petriella and Scedosporium prolificans (Microascaceae) containing opportunistic agents on humans. Mycol Res 110, 151–160.[CrossRef][Medline]
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Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
von Arx, J. A. (1973). The genera Petriellidium and Pithoascus (Microascaceae). Persoonia 7, 367–375.
von Arx, J. A. (1978). Notes on Microascaeae with the description of two new species. Persoonia 10, 23–31.
von Arx, J. A., Figueras, M. J. & Guarro, J. (1988). Sordariaceous Ascomycetes without Ascospore Ejaculation (Nova Hedwigia Beihefte 94). Stuttgart: E. Schweizerbart'sche Verlagsbuchhandlung.
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