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Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002 South Africa
Z. Wilhelm de Beer
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002 South Africa
Thomas Kirisits
Institute of Forest Entomology, Forest Pathology and Forest Protection (IFFF), Department of Forest and Soil Sciences, BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Hasenauerstrasse 38, A-1190 Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The genus Ophiostoma includes numerous species of primarily insect-vectored, wood-staining fungi. Several anamorph genera that differ in their micronematous or macronematous conidiogenous cells have been associated with Ophiostoma species. Among the former group, Sporothrix is associated with many species and is characterized by conidiogenous cells that arise laterally or terminally from any place on the hyphae and produce nonseptate conidia on sympodially developing denticles. The purpose of this study was to characterize ophiostomatoid isolates with Sporothrix anamorphs recently collected in Austria and Azerbaijan. The isolates were characterized based on comparisons of rDNA and ß-tubulin sequence data. Morphology, growth in culture, and sexual reproductive mode were also considered. Phylogenetic analyses of the combined sequence data showed that the isolates formed two distinct groups, one including isolates from Austria and the other isolates from Austria and Azerbaijan. Growth at 25 C and morphology revealed some differences between the two groups, and supported the view that they represent two new species, which we describe here as Ophiostoma fusiforme sp. nov. and Ophiostoma lunatum sp. nov. Both these groups phylogenetically were related to, but distinct from, Ophiostoma stenoceras.
Key words: DNA sequences, fusiforme, lunatum, nigrocarpum, phylogeny, sap staining, stenoceras
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Upadhyay 1993, Wingfield et al 1993). The genus Ophiostoma H. & P. Sydow includes numerous species that are associated with bark beetles, and some of these are forest pathogens. Others cause gray, black or brown discoloration of wood, which can lead to substantial losses in lumber value (Seifert 1993). Of the plant pathogenic species of Ophiostoma, the Dutch elm disease fungi are the most important. Ophiostoma ulmi and O. novoulmi have been responsible for two pandemics of Dutch elm disease and O. ulmi later was replaced by O. novoulmi, a more aggressive pathogen (Brasier 2000, Pipe et al 2000).
Ophiostoma has many anamorphs that have been classified in at least four genera: Pesotum Crane & Schoknecht, Leptographium Lag. & Melin, Hyalorhinocladiella Upadh. & Kendr. and Sporothrix Hekt. & Perk. These genera are distinguished based on differences in conidiophore structure and in patterns of conidial development (Seifert and Okada 1993, Benade et al 1995, 1997, Okada et al 1998, Jacobs and Wingfield 2001). Pesotum accommodates anamorphs producing synnemata (Okada et al 1998), while Leptographium is characterized by dark mononematous conidiophores that give rise to a series of branches, terminating in conidiogenous cells (Jacobs and Wingfield 2001). Hyalorhinocladiella species lack the distinct denticles on conidiogenous cells that are typical for Sporothrix species (Benade et al 1996), however, the distinction between Hyalorhinocladiella and Sporothrix based on morphology is not clear because intermediate forms between the two genera exist (Mouton et al 1992, Benade et al 1996, 1997).
De Hoog (1974) described Sporothrix as an artificial genus for species morphologically similar to the type species, S. schenckii Hekt. & Perk. Based on his description, conidiogenous cells that arise laterally or terminally from any place on the hypha, bearing one-celled conidia on sympodially developing denticles, characterize the genus. However, it is generally accepted that the genus is paraphyletic and includes species of both ascomycetes and basidiomycetes (de Hoog 1993). Thus, Sporothrix species associated with species of Ophiostoma represent only one of a number of morphologically similar but phylogenetically different lineages (Middelhoven et al 2000).
Recent collections of ophiostomatoid fungi from Azerbaijan and Austria include a number of Ophiostoma isolates with Sporothrix anamorphs. Preliminary morphological obser vations of these fungi have shown that they are broadly similar to O. stenoceras (Robak) Melin & Nannf. and O. nigrocarpum (Davidson) de Hoog. The aim of this study was to characterize these isolates based on morphology and DNA sequence comparisons and thereby determine their phylogenetic relationships.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, de Beer et al 2003) (TABLE I). The ex-type cultures of both species were included (TABLE I). Isolates (CMW7612, CMW7614, CMW7615) of S. schenckii, one isolate (CMW1468) previously described as belonging to the O. nigrocarpum-complex (de Beer et al 2003) and two isolates (CMW109, CMW110) morphologically similar to the isolate CMW1468, were included. In total, 20 isolates from both hardwoods and conifers from different geographic locations were included in this study. These isolates are all maintained in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Representative isolates also have been deposited in the collection of the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Herbarium specimens linked to key cultures have been deposited in the herbarium of the National Collection of Fungi, Pretoria, South Africa (PREM).
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). Part of the ß-tubulin gene was amplified using primers Bt2a and Bt2b (Glass and Donaldson 1995). The reaction mixture (50 µ L final volume) contained 2.5 U Super-Term DNA Polymerase mixture (Hoffmann-La Roche, U.S.A.), 1x PCR reaction buffer, 2.5 mM mMgCl2, 0.2 mM of each dNTPs and 0.2 mM of each primer. PCR reactions were performed in a thermal cycler (Hoffmann-La Roche). PCR conditions were: one cycle of 2.5 min at 95 C, followed by 40 cycles of 30 s at 95 C, 30 s at 55 C and 1 min at 72 C, and a final cycle of 8 min at 72 C. PCR products were electrophoresed on a 1% (w/v) agarose gel stained with ethidium bromide and the amplicons visualized under UV light. PCR fragments were purified using the High Pure PCR Product Purification Kit (Roche). Both strands of the PCR products were sequenced using the above-mentioned primers and the ABI Prism® BigDyeTM Terminator version 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems). DNA sequencing reactions were analyzed on the ABI PRISM® 3100 Genetic Analyzer or the ABI PRISMTM 377 DNA sequencer.
The sequences were aligned manually using Sequence Navigator and analyzed with Phylogenetic Analysis Using Parsimony (PAUP) 4.0b (Swofford 1998). Phylogenetic analysis was done first for each gene region separately, followed by an analysis of the combined data set of the ITS and ß-tubulin sequences. A partition homogeneity test in PAUP was performed to determine the congruence and combinability of the two sequence data sets. Uninformative characters were excluded, and a heuristic search, using TBR branch swapping (MULPAR on), was used to determine the most parsimonious trees. Trees were rooted using sequences of S. schenckii (de Beer et al 2003). Confidence intervals of the branch points were determined by 1000 bootstrap analyses.
Morphological studies. –
Isolates were grown 10 d on 2% MEA. To stimulate sexual sporulation, 2% water agar (WA: 20 g agar, 1L dH2O) amended with debarked oak and pine twigs (40 mm long x 5 mm wide) or with milled elm and spruce sapwood (25 g milled sapwood, 7.5 g agar, 250 mL water), as described by Brasier (1981), was used. Cultures were incubated at room temperature for 10 d. Colony colors were determined using the color charts of Rayner (1970).
Fifty measurements were made of each taxonomically informative structure in isolates that produced perithecia. Three-day-old slide cultures, mounted in lactophenol, were prepared to study the anamorph structures (Riddell 1950).
Growth in culture. –
Agar disks (5 mm diam) bearing mycelium of selected isolates (TABLE I
) were transferred from the actively growing margins of 1 wk old cultures and placed at the center of Petri dishes containing 20 mL 2% MEA. Isolates were grown at temperatures of 5–35 C at 5 C intervals for 10 d in the dark. Six measurements of colony diameter were made for each isolate, by taking two measurements from each of three replicate cultures. Results were computed as an average of these six measurements. Isolates that did not grow at high temperatures were subsequently maintained at room temperature to test their viability.
Mode of sexual reproduction. – Ten single ascospore and 10 single conidial cultures were prepared for each isolate (CMW9968, CMW8281, CMW8285, CMW7131, CMW10565, CMW10563, CMW10564, CMW651, CMW3202, CMW2344, CMW2524) that produced perithecia. For isolates that did not produce perithecia, 10 single conidial cultures were prepared. Single-spore cultures were incubated at room temperature on MEA. Those producing perithecia were considered homothallic. The perithecia were observed using light microscopy to confirm the presence of typical ascospores. Isolates that did not produce perithecia were considered heterothallic. Crosses in all possible combinations were made between these cultures to confirm this observation. Each culture was crossed against itself as a control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PCR products obtained with primers Bt2a and Bt2b were approximately 286 bp in size for 15 of the isolates. The fragments obtained for the two O. nigrocarpum isolates were 517 bp long, and those for the three S. schenckii isolates were 434 bp long. Sequences of the 15 isolates, excluding O. nigrocarpum and S. schenckii, started with an incomplete 5' end of an exon approximately 99 bp in length, followed by an intron with a variable number of base pairs: 84 in O. nigrocarpum, 61 in O. stenoceras, 79 in S. schenckii and 57 bp in the other 10 Ophiostoma isolates. This intron was followed by an incomplete 3' end of an exon of 130 bp in most of the isolates.
Ophiostoma nigrocarpum and S. schenckii differed from the other isolates in that they contained additional introns of about 165 bp and 86 bp, respectively, within the first exon. Sequences for the O. nigrocarpum isolates, as well as the additional intron of the S. schenckii isolates, were excluded from the analysis because they could not be aligned unambiguously with those of the other isolates. Without the intron, the remaining sequences of the S. schenckii isolates could be aligned with the sequences of the other isolates in the study. Although sequences of the O. nigrocarpum isolates did not align well with other isolates, a BLAST search with the ITS sequences (data not shown) confirmed that these isolates belong to the genus Ophiostoma.
Separate analyses of the rDNA and ß-tubulin gene regions resulted in trees with similar topologies. The partition homogeneity test for the combined data set confirmed a significant congruence between the data sets (P = 1.0). A phylogenetic analysis thus was performed on the combined ITS and ß-tubulin data set containing 860 characters, of which 696 were constant, two parsimony uninformative and 162 parsimony informative. S. schenckii sequences were used as outgroup, and a heuristic search resulted in 3568 rearrangements. Two most-parsimonious trees were retained, with a length of 196 steps (CI = 0.980, RI = 0.020, HI = 0.993). One of these trees is presented in FIG. 1.
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) based on the combined data. Two of the clades represented isolates of O. stenoceras (Clade II) and S. schenckii (Clade III) respectively. Clade I contained three subclades. The first subclade included Ophiostoma isolates from both Azerbaijan and Austria (CMW9968, CMW8281, CMW8285, CMW7131, CMW10565) and was supported by a bootstrap value of 99%. The second subclade contained two Ophiostoma isolates from Austria (CMW10563, CMW10564) and also had 99% bootstrap support. The third sub-clade represented the isolate from Canada previously assigned to the O. nigrocarpum complex (de Beer et al 2003) and two other isolates from the United States. These data suggest that there are three distinct taxa in Clade I.
Morphological studies. –
Morphologically similar perithecia were produced by isolates from Austria and Azerbaijan in Clade I. However, those in the first sub-clade had longer necks and wider base diameters than those in the second subclade (TABLE II). The isolates in the third subclade (CMW1468, CMW109, CMW110) did not produce perithecia. Only three O. stenoceras (CMW3202, CMW2344, CMW2524) and one O. nigrocarpum isolate (CMW651) formed perithecia. In the O. stenoceras isolates, protoperithecia were observed after 2 wk but these matured very slowly. Perithecial necks of the O. stenoceras isolates were longer and narrower than those in the other groups of isolates. Perithecia of O. nigrocarpum isolates were considerably smaller than those in the other species.
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). In isolates in the first subclade, the heads of the conidiogenous cells were generally swollen with distinct denticles and conidia were guttuliform to fusiform (FIG. 2D, E). The heads of conidiogenous cells of isolates in the second subclade were slightly swollen and denticles were small, inconspicuous and rarely long or cylindrical (FIG. 3D, E, G). Conidia were clavate or crescent shaped and thus very different to those of isolates in the first subclade. In isolates residing in the third subclade of Clade I, conidiogenous cells were similar to those of O. stenoceras (Clade II) and S. schenckii (Clade III) but conidia were cylindrical with pointed bases and narrower than those of the latter species. In O. stenoceras the conidia were broadly ellipsoidal with pointed bases. Conidiogenous cells of O. nigrocarpum usually are shorter and slightly rough. Conidia of the O. nigrocarpum isolate (CMW650) were broadly ellipsoidal but basally rounded and smaller than those of the isolates in the third subclade of Clade I and of the conidia of O. stenoceras.
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Isolates from Austria and Azerbaijan (first and second subclades of Clade I) differed in culture morphology from isolates in clades II and III but were indistinguishable from each other and those from Canada and the United States (third subclade of Clade I). O. stenoceras isolates were white, later slightly yellowish ("buff " 19d), flat with sparse aerial hyphae in comparison with the isolates residing in Clade I. The O. nigrocarpum isolates produced gray, brownish colonies ("honey" 19"), with obvious aerial mycelium. The colonies of S. schenckii were "smoke grey" (21""f) and 40 d old cultures showed "Isabella color" (19"i) toward the margins, first smooth, later undulating at the centers of 3 wk old cultures. Tufts of mycelium arising from different parts of the 40 d old cultures and exudates were common in these cultures (TABLE II).
Mode of sexual reproduction. –
All single conidial and single ascospore cultures obtained from the seven isolates from Austria and Azerbaijan were self fertile, producing perithecia with viable ascospores. The three O. stenoceras isolates and one O. nigrocarpum isolate (CMW651) also were self fertile (TABLE I). Single conidial cultures obtained from the remaining isolates did not produce perithecia, even when crossed with other cultures. These included the ex-type O. nigrocarpum isolate (CMW650), the Canadian isolate (CMW1468) previously assigned to the O. nigrocarpum-complex by de Beer et al (2003), two North American isolates (CMW109, CMW110) and the S. schenckii isolates.
Taxonomy. – Morphological and molecular characteristics support the placement of the isolates from Azerbaijan and Austria into two phylogenetic groups which were different from the Sporothrix isolates placed in the third subclade of Clade I. We consider subclades 1 and 2 to represent new taxa and herein described them as new species of Ophiostoma. Isolates in the third subclade did not produce perithecia in culture and, therefore, we have not provided a name for this fungus but refer to it as an undescribed species of Sporothrix.
Ophiostoma fusiforme D.N. Aghayeva & M. J. Wingfield sp. nov. FIG . 2
Anamorph. Sporothrix sp.
Coloniae in vitro in MEA post 10 dies in 25 C diametrum medium 33.5 mm attingentes; infra 10 C et supra 30 C non crescunt. Mycelium aereum primo laeve, mox floccosum cum orbibus concentricis incrementi. Incrementum in cultura parce rapidum, album, postea cinerascens vel post perithecia facta sunt nigrescens, superficie ima incolore vel sub-lutescente in coloniis veteris, aroma absente. Perithecia post 30 dies evoluta, superficialia vel in agaro semiimmersa. Bases globosae, nigrae, 121.5–273.8 µm diametro, trichomatibus hyphalibus laete brunneis vel brunneis, septatis, non ramosis, 16.6–94.5(–142.5) x 1.7–2.2(–2.6) µm, parietibus tenuibus vel crassis, ornatae. Colla interdum duo, ergo approximata, basin versus nigra, apicem versus laete brunnea, recta vel aliquando curvata, 301.8–985(–1168) µm longa, basi 21.8–33.7(44.9) µm, apice 9.1–13.5(–18) µm lata. Hyphae ostiolares multae, hyalinae, laeves, rectae vel subtortuosae, divergentes, 23.4–68.4(96.4) µm, basi 1.2–2.5(–3) µm latae. Ascosporae hyalinae, unicellulares, lateraliter visae allantoideae, 3.4–4.3(–5.4) x 0.8–1.3(–1.6) µm. Cellulae conidiogenae micronematosae, mononematosae, hyalinae, septatae, 14.3–53.9(–72) x (0.9–)1.2–1.8(–2) µm; parte apicale conidia formanti instar fasciculi globosi tumidi, cum denticulis acutis 0.3–1.2 µm longis. Conidia proxime in denticulis efferta, holoblastica, hyalina, unicellularia, clavata, plerumque fusiformia, interdum guttuliformia, basibus acutis, apicibus rotundatis, 3.2–5.9(–8.0) x 1.1–1.9 (–2.1) µm, singuli efferta, in massis mucosis aggregantes.
Colonies in vitro on MEA attaining an average diameter of 33.5 mm in 10 d at 25 C. No growth below 10 C or above 30 C. Aerial mycelium at first smooth, soon becoming floccose with concentric circles of growth. Growth in culture moderately rapid, white, later becoming dull white or blackish after perithecia form, reverse uncolored or slightly yellowish, ("Buff yellow— 19d") in old cultures, aroma absent. Perithecia (FIG. 2A) developing after 30 d superficial or partly embedded in agar. Bases globose, black, 121.5–273.8 µm diam; ornamented with pale brown to brown, septate, thin to thick-walled, unbranched hyphal hairs of variable length, 16.6–94.5(–142.5) x 1.7–2.2(–2.6) µm. Necks sometimes two, in this case close to each other, black at base, pale brown at apex, straight or occasionally curved, 301.8–985(–1168) µm long, 21.8–33.7(44.9) µm wide at base 9.1–13.5 (–18) µm at tip. Ostiolar hyphae (FIG. 2B) numerous, hyaline, smooth, straight or rather tortuous, divergent 23.4–68.4(96.4) µm, and 1.2–2.5(–3) µm wide at base. Ascospores hyaline (FIG. 2C), 1-celled, allantoid in side view, 3.4–4.3(–5.4) x 0.8–1.3(–1.6) µm. Conidiogenous cells (FIG. 2D, E) micronematous, mononematous, hyaline, septate, 14.3–53.9(–72) x (0.9–)1.2–1.8(–2) µm, apical part forming conidia consists of a globose, swollen cluster bearing sharp denticles 0.3–1.2 µm long. Conidia (FIG. 2F) produced directly on denticles, holoblastic, hyaline, 1-celled, clavate, guttuliform to fusiform (mostly fusiform, sometimes guttuliform) with pointed bases and rounded apices 3.2–5.9(–8.0) x 1.1–1.9(–2.1) µm, formed single, becoming aggregated in slimy masses.
Etymology.. The Latin fusiforme refers to fusiform shaped conidia.
Specimens examined.. AZERBAIJAN: Baku-Rostow highway. Wood of living Populus nigra, 03-VI-2000, D.N. Aghayeva PREM57486 (culture CMW9968) (HOLOTY PE). AUSTRIA. LOWER AUSTRIA: Hochleitenwald. Twig of living Quercus petraea, 07-IX-1992, E. Halmschlager PREM57487 (culture CMW7131). AUSTRIA. STYRIA: Kindberg, Kind-talgraben near Troiseck. Galleries of the bark beetle Ips cembrae in the bark of Larix decidua, VII-1995, T. Kirisits PREM 57488 (culture CMW10565). Herbarium specimens of holotype and paratypes have been deposited in the National Collection of Fungi (PREM), Pretoria, South Africa.
Ophiostoma lunatum D.N. Aghayeva & M. J. Wingfield sp. nov. FIG . 3
Anamorph. Sporothrix sp.
Coloniae in vitro in MEA post 10 dies in 25 C diametrum medium 31.5 mm attingentes; infra 10 C et supra 30 C non crescunt. Mycelium aereum primo laeve, mox floccosum cum orbibus concentricis incrementi, album, postea cinerascens vel post perithecia facta sunt subnigrescens, superficie ima incolore vel sublutescente, aroma absente. Perithecia post 40 dies evoluta, superficialia vel in agaro semiimmersa. Bases globosae, interdum pyriformae, nigrae, 59.5–178.3(–204.5) µm diametro, trichomatibus hyphalibus laete brunneis vel brunneis, septatis, non ramosis, longitudine variabili, 11.9–77.9(–106.8) x 0.69–2.6(–3.58) µm, parietibus tenuibus vel crassis, ornatae. Collum plerumque 1, interdum 2, basin versus nigrum, apicem versus laete brunneum, subundulatum vel curvatum, (162.4–)248.4 x 554.2(–700) µm longum, basi 15.3–33.4(– 40.5) µm, apice 7.5–10.4(–13.8) µm latum. Hyphae ostiolares multae, hyalinae, laeves, plerumque rectae, interdum curvatae, 13.6–56.9(–61.7) µm, basi 1.01–1.7(–2.7) µm latae. Ascosporae hyalinae, unicellulares, lateraliter visae allantoideae, 3.1–3.9 (–4.3) x 0.7–1.2 µm. Cellulae conidiogenae micronematosae, mononematosae, hyalinae, septatae, 11.3–35.2 (–59.4) x 0.8–1.3(–1.5) µm crassae, parte apicale cum denticulis, subincrassata. Denticula brevia, rotundata, interdum inconspicua, 0.3–0.6 µm longa, apicem versus cylindracea, usque ad 4.5 µm longa. Conidia proxime in denticulis efferta, et sympodialiter et lateraliter in hyphis unicis, holoblastica, hyalina, unicellularia, clavata, valde curvata, subfalcata, 2.3–4.8(–6.2) x 0.8–1.5(–1.6) µm, singuli efferta, in massis mucosis aggregantes. Blastoconidia lateralia copiosa.
Colonies in vitro on MEA attaining 31.5 mm diam in 10 d at 25 C. No growth below 10 C or above 30 C. At first smooth, soon becoming floccose with concentric circles. White, later becoming dull white or blackish after perithecial formation, reverse uncolored or slightly yellowish (19d and 19"b), aroma absent. Perithecia (FIG. 3A) developing after 40 d superficial or partly embedded in the agar. Bases globose, occasionally pyriform, black, 59.5–178.3 (–204.5) µm diam; ornamented with pale brown to brown, septate, thin- to thick-walled, unbranched hyphal hairs of variable length, 11.91–77.9(–106.84) x 0.69–2.6(–3.58) µm. Neck usually 1, rarely 2, black at base, pale brown at apex, slightly waved or curved, (162.4–)248.4 x 554.2(– 700) µm long, 15.3–33.4 (– 40.5) µm wide at base, 7.5–10.4(– 13.8) µm at tip. Ostiolar hyphae (FIG. 3B) numerous, hyaline, smooth, curved or rather straight, 13.6–56.9(–61.7) µm, and 1.01–1.7(–2.7) µm wide at the base. Ascospores (FIG. 3C) hyaline, 1-celled, allantoid in side view, 3.1–3.9(–4.3) x 0.7–1.2 µm. Conidiogenous cells (FIG. 3D, E, G) micronematous, mononematous, hyaline, septate, 11.3–35.2(–59.4) x 0.8–1.3(–1.5) µm thick, apical part bearing denticles, slightly swollen. Denticles short, rounded, sometimes inconspicuous 0.3–0.6 µm long, and cylindrical in apical region, up to 4.5 µm long. Conidia (FIG. 3F) produced directly on denticles, sympodially as well as laterally on single hyphae, holoblastic, hyaline, 1-celled, clavate, distinctly curved, somewhat crescent shaped 2.3–4.8(–6.2) x 0.8–1.5(–1.6) µm, formed singly, becoming aggregated in slimy masses. Lateral blastoconidia abundant.
Etymology.. The Latin lunatum, refers to the lunate shaped conidia.
Specimens examined.. AUSTRIA. VIENNA: Lainzer Tiergarten forest reservation Johannser Kogel. Inner bark of wind broken Carpinus betulus, 07-V-1995, T. Kirisits PREM57489 (culture CMW10563)(HOLOTY PE). AUSTRIA. STYRIA: Kindberg, Kindtalgraben near Troiseck. Pupa of the bark beetle Ips cembrae on Larix decidua, 01-VII-1995, T. Kirisits PREM57490 (culture CMW10564). Herbarium specimens of holotype and paratype have been deposited in the National Collection of Fungi (PREM), Pretoria, South Africa.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, de Beer et al 2003). An interesting outcome of this study is that two authentic isolates of O. nigrocarpum, including the ex-type, did not group in what has been referred to previously as the O. nigrocarpum complex (de Beer et al 2003). This, as well as clear morphological and cultural differences, suggests that O. nigrocarpum, represented in this study by the two authentic strains of Davidson, is more distantly related to O. stenoceras and S. schenckii than previously realized.
The teleomorph of O. fusiforme resembles that of O. stenoceras, although the descriptions of O. stenoceras vary (Robak 1932, Davidson 1942, Aoshima 1965, Mariat 1971, Upadhyay 1981, Kowalski and Butin 1989). The ascospores produced by the ex-type culture of O. stenoceras are shorter than those of O. fusiforme. The new species also differs from O. stenoceras in culture morphology and the size and shape of its conidia. For example O. fusiforme gives rise to floccose cultures, different to the sparse and flat mycelium of O. stenoceras. Conidia in O. fusiforme are mostly fusiform and easily distinguished from the broadly ellipsoidal conidia of O. stenoceras. The anamorph of O. fusiforme is similar to Sporothrix inflata de Hoog (de Hoog 1974), which has conidiophores scattered, arising orthotropically or plagiotropically from undifferentiated hyphae, often in a terminal position or integrated in short straight or curved side branches. However, S. inflata is gray to olivaceous in culture, which is different from the white cultures of O. fusiforme.
The teleomorph of O. lunatum shares some similarity with O. stenoceras and O. fusiforme. The most important character distinguishing this species from the others is the conidia that are flattened at one side or curved with a blunt base. This is different from O. fusiforme, which has fusiform conidia, and O. stenoceras, with ellipsoidal conidia. Conidiogenous cells of O. lunatum arising orthotropically from undifferentiated hyphae sometimes are integrated in short branches. The anamorph stage of O. lunatum is reminiscent of Sporothrix curviconia de Hoog. However, the conidia of O. curviconia are substantially larger than those of S. lunatum (de Hoog 1974).
The three Sporothrix isolates from Canada and the U.S.A. were different morphologically from the anamorphs of Ophiostoma fusiforme, O. lunatum and O. stenoceras. Conidia in O. fusiforme are guttuliform to fusiform, those in O. lunatum are clavate or crescent shaped, and conidia of O. stenoceras are broadly ellipsoidal. This is in contrast to conidia in the three Sporothrix isolates from Canada, which were cylindrical. De Beer et al (2003) included the Sporothrix isolate from Canada that also was used in this study. They assigned this isolate to the O. nigrocarpum-complex. Comparisons of our sequence data, however, show that the two authentic O. nigrocarpum isolates are distinct from all other species in our study. The colony morphology of the three Sporothrix isolates from Canada and the U.S.A. was different from that of the O. nigrocarpum isolates. Colonies of O. nigrocarpum were brownish-gray to blackish after perithecia formation, while colonies of these three isolates were white. The conidiogenous cells and conidia are similar in shape, but those of O. nigrocarpum are smaller and wider than those of the three Sporothrix isolates. Conidia of O. nigrocarpum also are rounded basally with inconspicuous scars, but in these isolates they are pointed. Our results suggest that the group that de Beer et al (2003) referred to as the O. nigrocarpum complex is not that species but an undescribed taxon (third sub-clade, Clade I). Because the teleomorph could not be obtained, we have chosen not to provide a name for this fungus but rather to treat it as an undescribed Sporothrix sp.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Permanent address: Institute of Botany, ANAS, Patamdar sh. 40, Baku 370073, Republic of Azerbaijan. 
1 Corresponding author. E-mail: adilzara{at}hotmail.com
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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