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Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa
Martin P. A. Coetzee
Brenda D. Wingfield
Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa
Karin Jacobs
Michael J. Wingfield
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phialocephala was established for species in the Leptographium complex that produce conidia from phialides at the apices of dark mononematous conidiophores. Some species previously included in Phialocephala were re-allocated to Sporendocladia because they resembled Thielaviopsis in having ring-wall-building conidial development and conidia with two attachment points that emerge in false chains. Despite this significant realignment of the genus, a great deal of morphological heterogeneity remains in Phialocephala. The objective of this study was to consider the heterogeneity among Phialocephala spp. based on comparisons of sequence data derived from the large and small subunits (LSU and SSU) of the rRNA operon of species in Phialocephala. Phialocephala dimorphospora, the type species of the genus, and P. fortinii grouped with genera of the Helotiales in phylogenetic trees generated based on the LSU and SSU datasets. Phialocephala xalapensis and P. fusca clearly are unrelated to Phialocephala sensu stricto and should represent a new genus in the Ophiostomatales. Phialocephala compacta resides with representatives of the Hypocreales, and we believe that it represents a distinct genus. Phialocephala scopiformis and P. repens are not closely related to the other Phialocephala species and group within the Dothideales. The morphological heterogeneity among species of Phialocephala clearly is reflected by phylogenetic analysis of sequence data from two conserved rRNA gene regions. Appropriate genera now need to be found to accommodate these fungi.
Key words: Leptographium, morphology, Phialophora, phylogeny
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This distinguishes them from Leptographium spp. that are characterized by percurrent or sympodial proliferation of the conidiogenous cells (Jacobs and Wingfield 2001). Phialocephala spp. are further characterized by having dark mononematous conidiophores that branch penicillately at their apices (Crane 1971) and thus resemble Phialophora Medlar (Gams 2000). Hyaline ameroconidia accumulate in slimy masses around the sporogenous heads (Kendrick 1961, 1963). In addition, some species produce solitary phialides that are formed directly on the mycelium (Onofri and Zucconi 1984). The so-called "stalked spore drop" structure, as described by Ingold (1961), suggests an adaptation to insect dispersal, although insect associations are not known for most species of Phialocephala (Jacobs and Wingfield 2001).
Phialocephala spp. occupies a diverse range of ecological niches (Wang and Wilcox 1985, Kowalski and Kehr 1995). Phialocephala dimorphospora W.B. Kendrick, P. fortinii C.J.K. Wang & H.E. Wilcox, P. compacta T. Kowalski & R.D. Kehr and P. scopiformis T. Kowalski & R.D. Kehr are readily isolated from plants growing in cool or cold environments, such as those encountered in alpine, subalpine and boreal regions (Wang and Wilcox 1985, Hambleton and Currah 1997, Stoyke and Currah 1990). Phialocephala trigonospora R. Kirschner & F. Oberwinkler was isolated from bark beetle tunnels in Pinus sylvestris L. and Picea abies L. Karst., while P. scopiformis and P. compacta are endophytes of Pinus and Picea spp. (Kowalski and Kehr 1995, Kirschner and Oberwinkler 1998). Most species are not associated with disease, but P. virens A.L. Siegfried & K.A. Seifert was isolated from root rot on Tsuga and Picea spp. (Siegfried et al 1992). Phialocephala fortinii also has been reported as a weak pathogen of container-grown conifers (Wilcox and Wang 1987). The latter fungus is well known as Mycelium radicis atrovirens Melin, which colonizes tree roots (Wang and Wilcox 1985).
No teleomorph associations have been determined for species of Phialocephala, although a connection to the Leotiales has been proposed (Currah et al 1993). This was based on the morphology of apothecium-like structures with cells resembling immature asci, produced in some cultures (Currah et al 1993). Previously, the anamorph of Ophiostoma francke grosmanniae R.W. Davidson also was suggested to represent a Phialocephala species. However, the presence of closely packed annellations, observed in an ultrastructural study, showed that the anamorph of this fungus rather should reside in Leptographium (Mouton et al 1992), an assignment that was confirmed in a recent phylogenetic study of Leptographium spp. based on DNA sequence data (Jacobs et al 2001).
The morphologically heterogeneous nature of Phialocephala was emphasised when the species with inconspicuous collarettes and ring-wall-building conidial development were moved to Sporendocladia G. Arnaud ex Nag Raj & W.B. Kendr. (Wingfield et al 1987). However, based on morphological and physiological variability, the remaining Phialocephala spp. still represents a heterogeneous group. The variable morphological characteristics include a wide diversity of conidial forms and variously structured collarettes at the apices of conidiogenous cells. Furthermore, the variable presence of rhizoids at the base of conidiophores and sterile outgrowths on the stipes suggest that many of these fungi phylogenetically are unrelated.
Phialocephala fusca W.A. Kendrick is the only Phialocephala sp. that forms rhizoids at the base of conidiophore stipes (Kendrick 1963). Likewise, P. canadensis W.A. Kendrick and P. fluminis C.A. Shearer, J.L. Crane & M.A. Miller are unique in that they have sterile outgrowths on stipes (Kendrick 1963, Shearer et al 1976). Collarette morphology in Phialocephala spp. varies from being broadly flared in P. fusca to deeply set in P. dimorphospora and inconspicuous in P. humicola Jong & E. Davis (= P. gabalongii Sivasith.) (Kendrick 1961, 1963, Jong and Davis 1972). Conidial shapes in Phialocephala spp. range from ellipsoidal to globose and subglobose. Some species have two distinct forms of conidia. The first-formed conidium that develops fully inside a very long collarette is larger than the second and subsequent conidia. This dimorphism is present in P. dimorphospora, P. fortinii, P. compacta and P. scopiformis, while one species, P. trigonospora, has uniquely triangular spores (Kirschner and Oberwinkler 1998).
Phialocephala spp. varies in tolerance to the antibiotic cycloheximide. This tolerance to cycloheximide might indicate connections to the Ophiostomatales (Harrington 1988, Jacobs and Wingfield 2001). Species such as P. dimorphospora displays 84%, P. fortinii 55% and P. humicola 60% reduction in growth in the presence of 0.5 g/L of cycloheximide. Phialocephala fusca, P. repens and P. xalapensis will not grow in the presence of the antibiotic (Jacobs 2000).
Very limited molecular data are available from which to infer phylogenetic relationships for Phialocephala spp. The only species that have been considered at this level are P. fortinii and P. dimorphospora (Rogers et al 1999). Based on ITS sequence comparisons, these two species appear to be closely related. This relationship also is supported strongly by morphological and ecological characteristics.
The aim of this study was to consider phylogenetic relationships between Phialocephala spp. for which cultures are available. In addition, we evaluated the placement of Phialocephala spp. within orders of the Ascomycota. These objectives were achieved by means of analyzing partial sequences of the SSU and LSU genes of the ribosomal RNA operon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Mycelium was ground to a fine powder in liquid nitrogen, to which 1 mL extraction buffer (200 mM Tris-HCl, pH 8; 25 mM EDTA, pH 8; 150 mM NaCl; and 0.5% SDS) was added. This was followed by further homogenization and incubation (1 h, 60 C). Cell debris was precipitated by centrifugation (ca 15 700 x g, 30 min). A series of phenol:chloroform (0.5 v/v) extractions were performed until the interface was clean. Nucleic acids were precipitated in cold 100% ethanol (2:1 v/v) and incubated at -20 C for 24 h. The mixture subsequently was centrifuged (15 700 x g, 30 min) and washed in 70% ethanol. The pellet was resuspended in 300 µL sterile water.
PCR –
Extracted DNA was used as template in a PCR reaction to amplify regions of the nuclear LSU and the SSU genes of the ribosomal RNA (rRNA) operon. The SSU gene was amplified using primer sets 2F (5'-ATCTGGTTGATCCTGCCAGTAG-3') and 1794R (5'-GATCCTTCCGCAGGTTCACC-3') (Okada et al 1997). The ITS 2 region and a portion of the LSU gene were amplified using the primer set CS3 (5'-CGAATCTTTGAACGCACATTG-3') (Visser et al 1995) and LR3 (5'-CCGTGTTTCAAGACGGG-3') (White et al 1990). The PCR reaction mixture included MgCl2 (2.5 mM), Expand HF buffer without MgCl2, dNTPs (0.2 mM each), primers (0.025 mM), template DNA (25 ng) and ExpandTM High Fidelity PCR System (1.75 U) (Roche Pharmaceuticals, Germany). The PCR reaction conditions for the amplification of the LSU were an initial denaturation at 94 C for 2 min, annealing at 48 C for 1 min, ramping at 5 C/s to 72 C for 2 min. This was repeated for 40 cycles, and a final elongation step was included at 72 C for 8 min. The SSU was amplified following the same PCR reaction conditions but only for 25 cycles. The resulting PCR amplicons were purified with a QIAquick PCR Purification kit (QIAGEN, Germany), according to specifications of the manufacturer.
DNA sequencing –
DNA sequences were determined with the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase (Applied Biosystems, UK), using primer sets CS3, LR3 and 2F, 1794R. Two internal primers 404X (5'-CCCTTTCAACAATTTCAC-3') (Witthuhn et al 1999) and 1332R (5'- AAGGTCTCGTTCGTTATCG-3') (Okada et al 1997) were included for the large and small subunit, respectively. Sequences generated in this study have been deposited in GenBank (Table I).
Sequence analysis –
Alignments of the LSU and SSU datasets were obtained by means of the Clustal X (Thompson et al 1994) program, and the inserted gaps were treated as "new state". Ambiguously aligned regions and parsimony-uninformative characters were excluded from the datasets. The remaining characters were reweighted according to the mean consistency index (CI). Phylogenetic analysis was based on parsimony using PAUP 4.0* (Phylogenetic Analysis Using Parsimony* and Other Methods version 4 (Swofford 1998). Heuristic searches were conducted with random addition of sequences (100 replicates), tree bisection-reconnection (TBR) branch swapping and MULPAR effective and MaxTrees set to auto-increase. Phylogenetic signal in the datasets (g1) was assessed by evaluating tree length distributions over 100 randomly generated trees (Hillis and Huelsenbeck 1992). The CI and retention indexes (RI) were determined for all datasets. Phylogenetic trees were rooted with Xylaria curta as a monophyletic sister outgroup to the rest of the taxa. Bootstrap analyses were performed to determine confidence in branching points (1000 replicates) for the most-parsimonious (MP) trees generated from the SSU and LSU data. The combinability of the SSU and LSU datasets were tested using the partition-homogeneity test and the Templeton Nonparametric Wilcoxon Signed Ranked test in PAUP 4.0 (Farris et al 1994, Kellogg 1996). The datasets were submitted to Treebase (SN904–3213).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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SSU sequence data – Parsimony analysis of the SSU sequence data was done to determine the phylogenetic placement of Phialocephala species in relation to representatives of different orders in the Ascomycetes. Alignment by inserting gaps resulted in a total of 423 characters used in the comparison of the different species. The inserted gaps were treated as "new state" and all parsimony-uninformative and ambiguous characters were excluded. The remaining characters were reweighted according to the mean CI value. A total of 100 parsimony-informative characters were obtained. Heuristic searches on the dataset generated 100 MP trees and a single tree is presented in Fig. 1.
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LSU sequence data – Alignment of the LSU gene sequences was achieved by inserting gaps. These gaps were treated as "new state", and all ambiguous and parsimony-uninformative characters were excluded. The remaining characters were reweighted according to the mean CI value. A total of 100 parsimony-informative characters were used in the comparison of the different species. Heuristic searches on the dataset generated a single MP tree. The tree obtained is presented in Fig. 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phialocephala dimorphospora is the type species of the genus. The fungus has characteristic deeply set conidiogenous loci with tubular collarettes (Kendrick 1961). This is very similar to species of Cystodendron Bubák, and the relatedness of Phialocephala and Cystodendron should be considered in future studies. Cystodendron is characterized by dark, densely penicillate and more or less sporodochial conidiophores. The phialides have pronounced tubular collarettes. Phialocephala fortinii has conidiophores and conidia similar to those of P. dimorphospora, but its sporulation is scanty and occurs only at low temperatures, and the two fungi share similar ecological niches. It was not surprising, therefore, that the two fungi are found to be phylogenetically related. Furthermore, our results support those of a previous study that has suggested that these fungi probably reside in the Leotiales (Rogers et al 1999, Currah et al 1993). The low bootstrap values obtained for the relatedness of the two P. dimorphospora isolates suggest that there is variability in isolates of this fungus and this matter deserves further study. The relationship, however, is supported strongly by the more variable LSU dataset.
In many respects, Phialocephala spp. is morphologically similar to Leptographium species. Species in both genera have erect conidiophores with conidia produced in slimy masses at the apices of branched conidiogenous cells. In Leptographium, this morphological form is known to facilitate an association with insect vectors (Jacobs and Wingfield 2001). Thus it is not surprising that two Phialocephala species (P. fusca and P. xalapensis) included in this study were found to be related to Leptographium in the Ophiostomatales. However, both species lack tolerance to 0.5 g/L cycloheximide, which is unlike typical Leptographium spp. (Jacobs and Wingfield 2001) and might represent a new genus associated with Ophiostomatoid fungi.
Loss of collarettes in a number of Phialocephala spp., including P. fusca, after repeated transfers also suggests that this character might not be stable (Vujanovic et al 2000).
Analysis of SSU sequence data indicates a phylogenetic affinity between P. scopiformis, P. repens and representatives of the Dothideales. These Phialocephala spp. species clearly are unrelated to other Phialocephala spp. studied and also are distinctly different from each other. The relatedness of P. scopiformis, P. repens and representatives of the Dothideales, as well as between P. compacta and representatives of the Hypocreales in this study, was not supported by the LSU data and remains unclear. Morphological evidence to support these affiliations also is lacking. Species characterized by brown conidiophores becoming paler toward the apex are not included in the Dothideales.
In this study we included an isolate of Sporendocladia bactrospora, a species that previously was accommodated in Phialocephala, as P. bactrospora (Kendrick 1961). Based on a study of conidiogenesis and the presence of ring-wall-building conidial development in this fungus, Wingfield et al (1987) transferred it to Sporendocladia. Conidial production through ring-wall building makes this fungus morphologically similar to Thielaviopsis anamorphs of Ceratocystis, in which conidia typically are produced in this manner (Nag Raj and Kendrick 1975, Paulin and Harrington 2000). Thus it was anticipated that the isolate of S. bactrospora included in this study would group together with Ceratocystis in the Microascales.
This study has enabled us to suggest appropriate phylogenetic placements for a number of Phialocephala spp., namely P. dimorphospora, P. fortinii, P. scopiformis, P. repens, P. compacta, P. fusca and P. xalapensis. Thus we confirm previous contentions that the genus is heterogeneous and that most species are unrelated. Phialocephala should be restricted to species that are similar to P. dimorphospora, based on sequence data, namely P. fortinii. Alternative generic names will be needed for other species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication December 23, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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