Highlights of the Didymellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera

Abstract

Fungal taxonomists routinely encounter problems when dealing with asexual fungal species due to poly- and paraphyletic generic phylogenies, and unclear species boundaries. These problems are aptly illustrated in the genus Phoma. This phytopathologically significant fungal genus is currently subdivided into nine sections which are mainly based on a single or just a few morphological characters. However, this subdivision is ambiguous as several of the section-specific characters can occur within a single species. In addition, many teleomorph genera have been linked to Phoma, three of which are recognised here. In this study it is attempted to delineate generic boundaries, and to come to a generic circumscription which is more correct from an evolutionary point of view by means of multilocus sequence typing. Therefore, multiple analyses were conducted utilising sequences obtained from 28S nrDNA (Large Subunit - LSU), 18S nrDNA (Small Subunit - SSU), the Internal Transcribed Spacer regions 1 & 2 and 5.8S nrDNA (ITS), and part of the beta-tubulin (TUB) gene region. A total of 324 strains were included in the analyses of which most belonged to Phoma taxa, whilst 54 to related pleosporalean fungi. In total, 206 taxa were investigated, of which 159 are known to have affinities to Phoma. The phylogenetic analysis revealed that the current Boeremaean subdivision is incorrect from an evolutionary point of view, revealing the genus to be highly polyphyletic. Phoma species are retrieved in six distinct clades within the Pleosporales, and appear to reside in different families. The majority of the species, however, including the generic type, clustered in a recently established family, Didymellaceae. In the second part of this study, the phylogenetic variation of the species and varieties in this clade was further assessed. Next to the genus Didymella, which is considered to be the sole teleomorph of Phoma s. str., we also retrieved taxa belonging to the teleomorph genera Leptosphaerulina and Macroventuria in this clade. Based on the sequence data obtained, the Didymellaceae segregate into at least 18 distinct clusters, of which many can be associated with several specific taxonomic characters. Four of these clusters were defined well enough by means of phylogeny and morphology, so that the associated taxa could be transferred to separate genera. Aditionally, this study addresses the taxonomic description of eight species and two varieties that are novel to science, and the recombination of 61 additional taxa.

Keywords: Boeremia; DNA phylogeny; Didymella; Didymellaceae; Epicoccum; Leptosphaerulina; Macroventuria; Peyronellaea; Phoma; Pleosporales; Stagonosporopsis; coelomycetes; taxonomy.

Fig. 1.

(p. 15) Fifty percent majority rule consensus tree from a BI analysis of Large and Small subunit sequences of Phoma and related genera (n = 76). At the nodes the BI Posterior Probabilities are presented above the branch, and bootstrap percentages of the ML analysis are given below the branch. Branches that were less than 50 % supported in the ML analyses are indicated with a hyphen. The bar indicates the number of substitutions per site. The tree is rooted with Pseudorobillarda phragmitis (CBS 398.61).

Fig. 2.

Fifty percent majority rule consensus tree from a BI analysis of LSU, ITS and TUB sequences of Didymellaceae (n = 274). At the nodes the BI Posterior Probabilities are presented above the branch, and bootstrap percentages of the analysis are given below the branch. Branches that were less than 50 % supported in the ML analyses are indicated with a hyphen. The bar indicates the number of substitutions per site. The tree is rooted with Ascochyta hordei var. hordei (CBS 544.74) and Phoma paspali (CBS 560.81 & CBS 561.81).>

Fig. 2.

Fifty percent majority rule consensus tree from a BI analysis of LSU, ITS and TUB sequences of Didymellaceae (n = 274). At the nodes the BI Posterior Probabilities are presented above the branch, and bootstrap percentages of the analysis are given below the branch. Branches that were less than 50 % supported in the ML analyses are indicated with a hyphen. The bar indicates the number of substitutions per site. The tree is rooted with Ascochyta hordei var. hordei (CBS 544.74) and Phoma paspali (CBS 560.81 & CBS 561.81).>

Fig. 2.

Fifty percent majority rule consensus tree from a BI analysis of LSU, ITS and TUB sequences of Didymellaceae (n = 274). At the nodes the BI Posterior Probabilities are presented above the branch, and bootstrap percentages of the analysis are given below the branch. Branches that were less than 50 % supported in the ML analyses are indicated with a hyphen. The bar indicates the number of substitutions per site. The tree is rooted with Ascochyta hordei var. hordei (CBS 544.74) and Phoma paspali (CBS 560.81 & CBS 561.81).>

Fig. 2.

Fifty percent majority rule consensus tree from a BI analysis of LSU, ITS and TUB sequences of Didymellaceae (n = 274). At the nodes the BI Posterior Probabilities are presented above the branch, and bootstrap percentages of the analysis are given below the branch. Branches that were less than 50 % supported in the ML analyses are indicated with a hyphen. The bar indicates the number of substitutions per site. The tree is rooted with Ascochyta hordei var. hordei (CBS 544.74) and Phoma paspali (CBS 560.81 & CBS 561.81).>

Fig. 3.

Phoma saxea (CBS 419.92). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia. E–F. Conidiogenous cells. G. Conidia. H. Chain of unicellular chlamydospores. Scale bars: D = 100 μm; E–G = 5 μm; H = 20 μm.

Fig. 4.

Phoma pedeiae (CBS 124517). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia. E. Section of the pycnidial wall. F. Conidia. Scale bars: D = 100 μm; E–F = 10 μm.

Fig. 5.

Phoma dimorpha (CBS 346.82). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia on stem of Urtica dioica. E. Pycnidia. F. Pycnidial wall. G–H. Conidia in vitro (G) and in vivo (H). Scale bars: D–E = 100 μm; F = 20 μm; G–H = 10 μm.

Fig. 6.

Phoma brasiliensis (CBS 120105a). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D–E. Pycnidia. F. Section of the pycnidial wall. G. Conidia. Scale bars: D = 200 μm; E = 100 μm; F–G = 10 μm.

Fig. 7.

Globose chlamydospores of Epicoccum spp. A–B. E. nigrum (CBS 173.73). C–E. E. sorghi (CBS 246.60). F–H. E. pimprinum (CBS 179.80). Scale bars: A–B = 50 μm; C–H = 20 μm.

Fig. 8.

Boeremia gen. nov. A. Ostiole configuration of B. exigua var. exigua (CBS 431.74). B. Pycnidial wall and conidiogenous cells of B. telephii (CBS 760.73) C. Aseptate and septate conidia of B. lycopersici (CBS 378.67). Scale bars: A = 20 μm; B–C = 10 μm.

Fig. 9.

Boeremia exigua var. gilvescens (CBS 101150). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia. E. Chains of wollen cells. F. Conidia. Scale bars: D = 100 μm; E = 100 μm; F = 10 μm.

Fig. 10.

Boeremia exigua var. pseudolilacis (CBS 101207). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia. E. section of young pycnidium. F. Conidia. Scale bars: D = 100 μm; E = 20 μm; F = 5 μm.

Fig. 11.

Phoma minor (CBS 325.82). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D–E. Pycnidia. F. Section of the pycnidial wall. G. Conidia. Scale bars: D = 200 μm; E = 100 μm; F–G = 10 μm.

Fig. 12.

Conidial dimorphism in three species of Stagonosporopsis. A. S. actaeae (CBS 106.96). B. S. lupini (CBS 101494). C. S. cucurbitacearum (CBS 109171). Scale bars: A = 20 μm; B–C = 10 μm.

Fig. 13.

Phoma bulgarica (CBS 357.84). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D. Pycnidia in vivo, isolated from manually infected sterilised stems of Urtica dioica. E. Pycnidial section. F. Pycnidium. G. Crystals. H. Hyphal strand. Scale bars: D, F = 100 μm; E, H = 50 μm; G = 10 μm.

Fig. 14.

Phoma dactylidis (CBS 124513). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D-E. Pycnidia. F. Section of the pycnidial wall. G. Conidia. Scale bars: D–E = 100 μm; F–G = 5 μm.

Fig. 15.

Phoma longicolla (CBS 124514). A–C. Fourteen-day-old colonies on OA (A), MEA (B) and CHA (C). D–F. Pycnidia. G. Section of the pycnidial wall. H. Conidia. Scale bars: E–G = 100 μm; H = 50 μm; I–J = 10 μm.