PCR and sequencing

For nucleotide sequence comparisons, partial regions of SSU, LSU and ITS, as well as part of the ACT, TUB and CHS-1 genes were amplified. The SSU region was amplified with the primers NS1 and NS4 (White et al. 1990) and the LSU region was amplified with the primers LR0R (Rehner & Samuels 1994) and LR7 (Vilgalys & Hester 1990). The ITS and TUB regions were amplified as described by Aveskamp et al. (2009) using the primer pair V9G (de Hoog & Gerrits van den Ende 1998) and ITS4 (White et al. 1990) for the ITS and the BT2Fw and BT4Rd primer pair (Woudenberg et al. 2009) for the TUB locus. The ACT and CHS-1 regions were amplified using the primer pairs ACT-512F / ACT-783R and CHS-354R / CHS-79F (Carbone & Kohn 1999). The amplification reactions were performed and analysed as described by de Gruyter et al. (2009).

Sequencing of the PCR amplicons was conducted using the same primer combinations, although the primer LR5 (Vilgalys & Hester 1990) was used as an additional internal sequencing primer for LSU. The sequence products were purified using Sephadex columns (Sephadex G-50 Superfine, Amersham Biosciences, Roosendaal, Netherlands) and analysed with an ABI Prism 3730XL Sequencer (Applied Biosystems) according to the manufacturer’s instructions. Consensus sequences were computed from both forward and reverse sequences using the Bionumerics v. 4.61 software package (Applied Maths, Sint-Martens-Latem, Belgium) and were lodged with GenBank. All sequences of reference isolates included in this study were obtained from GenBank (Table 1).

Phylogeny of Phoma lingam and Ph. betae, the type species of Phoma sections Plenodomus and Pilosa (Pleosporineae)

The aligned sequence matrix obtained for the SSU and LSU regions had a total length of 2 671 nucleotide characters, 1 367 and 1 304 respectively. In the alignment, an insertion in the SSU at the positions 478-832 was observed for the cultures CBS 216.75, CBS 165.78, CBS 138.96, CBS 331.37 and CBS 674.75. This insertion was excluded from further phylogenetic analyses. The combined dataset used in the analyses included 48 taxa and contained 2 316 characters with 101 and 213 unique site patterns for SSU and LSU, respectively. The tree (Fig. 1) was rooted to Sporormiella minima (CBS 524.50). The Bayesian analysis resulted in 6 5442 trees after 3 272 000 generations, from which the burn-in was discarded and the consensus tree and posterior probabilities were calculated based on 56 028 trees (Fig. 1).

Open in a separate window

Fig. 1.

The phylogeny of Phoma lingam and Phoma betae, the type species of Phoma sections Plenodomus and Pilosa, based on the strict consensus tree from a Bayesian analysis of 48 LSU/SSU sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted to Sporormiella minima (CBS 524.50).

The families that belong to Pleosporineae, represented by the species grouping in clades A-G, clustered in a strongly supported clade (99 % posterior probability). Clade A, representing those species classified in Pleosporaceae, was strongly supported (100 %) and included two subclades. Pleospora betae (anam. Ph. betae), clustered with Pleospora calvescens (anam. Ascochyta caulina), A. obiones and A. hyalospora; all recorded as pathogens on Chenopodiaceae. The generic type species Pleospora herbarum, a plurivorous species, grouped with Cochliobolus sativus, Pyrenophora tritici-repentis and Pleospora typhicola (anam. Ph. typhina), all recorded from Poaceae. Clade B includes Leptosphaeria maculans (anam. Ph. lingam) and clustered with Leptosphaeria biglobosa. In clade B also other important plant pathogens of Phoma section Plenodomus can be found, such as Ph. tracheiphila, Ph. vasinfecta, Ph. drobnjacensis, and Plectophomella visci. Phoma heteromorphospora, type species of Phoma section Heterospora (Boerema et al. 1997) and Ph. dimorphospora also grouped in this Leptosphaeria clade, in congruence with previous findings (de Gruyter et al. 2009, Aveskamp et al. 2010).

Leptosphaeria doliolum (anam. Ph. acuta), type species of the genus Leptosphaeria, is found in Clade D, clustering with L. conoidea and L. slovacica. Leptosphaeria doliolum and its relatives comprise a sister clade C with species classified in Cucurbitariaceae, including Cucurbitaria berberidis, the three Pyrenochaeta species, Py. cava, Py. lycopersici and Py. nobilis, and Pyrenochaetopsis leptospora.

Phaeosphaeria nodorum and its relatives Neosetophoma samarorum, Setophoma terrestris, Chaetosphaeronema hispidulum, Paraphoma radicina and Setomelanomma holmii, represent Phaeosphaeriaceae in clade E as has previously been found (de Gruyter et al. 2009, 2010).

A distinct clade F includes Ph. glycinicola, Ph. carteri, Ph. septicidalis, and the taxonomic confusing species Pyrenochaeta dolichi (Grondona et al. 1997). The position of Coniothyrium palmarum and Neophaeosphaeria filamentosa could not be clarified, but both species are also treated below in a phylogeny including close relatives based on ITS and LSU regions (Fig. 2). Didymella exigua, type species of the genus Didymella, and Ph. herbarum represent Didymellaceae, and clustered in a well-supported clade (G) in congruence with previous studies (de Gruyter et al. 2009, 2010, Aveskamp et al. 2010). The molecular phylogeny of species which group in this analysis outside of Pleosporineae in Montagnulaceae, Massarinaceae and Sporormiaceae were further analysed utilising LSU sequence data of a broader range of taxa (Fig. 5).

Open in a separate window

Fig. 2.

The phylogeny of Phoma section Plenodomus and Leptosphaeria, based on the strict consensus tree from a Bayesian analysis of 87 LSU/ITS sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted to Phoma herbarum (CBS 615.75).

Open in a separate window

Fig. 5.

LSU The phylogeny of phoma-like isolates excluded from the Pleosporineae, based on the strict consensus tree from a Bayesian analysis of 40 LSU sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted to Pseudorobillarda phragmitis (CBS 398.61).

Phoma section Plenodomus and close allies

The aligned sequence matrix obtained for the LSU and ITS regions had a total length of 1 921 nucleotide characters, 1 332 and 589 respectively. The combined dataset used in the analyses included 87 taxa and contained 1921 characters with 298 and 118 unique site patterns for LSU and ITS respectively. The tree (Fig. 2) was rooted to Ph. herbarum (CBS 615.75), the representative isolate of the type species of Phoma (Boerema et al. 2004). The Bayesian analysis resulted in 100 002 trees after 5 000 000 generations, from which the burn-in was discarded and the consensus tree and posterior probabilities were calculated based on 90 930 trees (Fig. 2).

The species currently classified in Leptosphaeria and Phoma section Plenodomus grouped in clades A and B representing Leptosphaeriaceae, including the type species Ph. lingam and Leptosphaeria doliolum, respectively. Isolates of the taxa that represent Cucurbitariaceae, Cucurbitaria berberidis and its related species Pyrenochaeta cava, Py. nobilis, Py. lycopersici and Pyrenochaetopsis leptospora, clustered in a distinct clade D only distantly related to Leptosphaeriaceae. This finding agrees with a recent study (de Gruyter et al. 2010). Phoma pratorum clustered with Pyrenochaetopsis leptospora.

Leptosphaeria biglobosa grouped in a subclade A1 with Ph. wasabiae, the cause of black rot disease on Wasabia japonica (Brassicaceae) and Ph. pimpinellae, a necrotroph on Pimpinella anisum (Apiaceae). Leptosphaeria maculans, considered as closely related to the L. biglobosa complex, proved to be more distantly related in clade A1. In this subclade, other important pathogens can be found, such as Ph. tracheiphila, a quarantine organism on Citrus spp. (Rutaceae), Ph. vasinfecta, a pathogen on Chrysanthemum spp. (Asteraceae), L. lindquistii (anam. Ph. macdonaldii), a worldwide pathogen on Helianthus annuus (Asteraceae) and Ph. lupini, a seed borne pathogen known from Lupinus spp. (Fabaceae). Subclade A1 also comprises both varieties of Ph. enteroleuca, opportunistic pathogens on deciduous trees and shrubs, and the necrotrophic species L. agnita (anam. Ph. agnita), Ph. congesta (both recorded on Asteraceae), Ph. conferta (mainly on Brassicaceae), L. hendersoniae (on Salicaceae), L. fallaciosa, L. collinsoniae (mainly on Lamiaceae) and L. libanotis (on Apiaceae). Plectophomella visci, recorded from leaves of Viscum album (Viscaceae), also clustered in the Leptosphaeriaceae. The genus Plenodomus is re-introduced here to accommodate the species in subclade A1, which are allied to Ph. lingam.

Subclade A2 comprises pathogenic species often causing leaf spots such as Ph. apiicola on Apium graveolens (Apiaceae), Ph. drobnjacensis (on Gentianaceae), Ph. violicola (on Violaceae) as well as the necrotrophic species Ph. valerianae, on Valeriana spp. (Valerianaceae). Phoma apiicola and Ph. valerianae were classified in Phoma section Phoma, and Ph. violicola was classified in Phoma sect. Peyronellaea; however, the relationship of these species in Leptosphaeriaceae is clearly demonstrated (Fig. 2), and therefore the species are transferred to the new genus Subplenodomus. These results are in congruence with a recent study where Ph. violicola, Ph. apiicola and Ph. valerianae clustered in a clade representing both Leptosphaeriaceae and Pleosporaceae (Aveskamp et al. 2010).

Four Leptosphaeria species, L. macrospora (soil) and the necrotrophic species L. nitschkei (on Asteraceae), L. praetermissa, on Rubus idaeus (Rosaceae) and L. dryadis, on Dryas spp. (Rosaceae) grouped in a subclade A3 and are transferred here to a new genus Paraleptosphaeria. Phoma korfii also clustered in this subclade. The European species Ph. heteromorphospora, type species of Phoma section Heterospora, and the American counterpart Ph. dimorphospora, both pathogens on Chenopodiaceae, grouped in a distinct subclade A4. Phoma sect. Heterospora is raised to generic rank to accommodate both species in Leptosphaeriaceae.

Clade B comprises necrotrophic species related to the type species L. doliolum (anam. Ph. acuta). The phylogeny of this species complex, and the closely related species Ph. veronicicola, Ph. macrocapsa and Ph. sydowii, is treated below. The necrotrophic species Ph. sclerotioides, L. conoidea (anam. Ph. doliolum), L. slovacica (anam. Ph. leonuri) and Ph. pedicularis also proved to be related. The species Ph. rubefaciens and Ph. etheridgei also belong to clade B, but these species, both recorded on trees, are more distantly related.

The Phoma species in clades A and B are in majority currently described as anamorphs of the genus Leptosphaeria, or belong to Phoma section Plenodomus. These Phoma anamorphs are only distantly related to the type species Ph. herbarum and its relatives in Didymellaceae, and therefore these species described in section Plenodomus are excluded from the genus Phoma. Clade C is more distantly related to Leptosphaeriaceae and comprises species that are related to Coniothyrium palmarum in Coniothyriaceae. Two subclades are recognised in clade C: Ph. glycinicola, Py. dolichi and Ph. carteri group with the generic type species C. palmarum, whereas two isolates of Ph. septicidalis group with Ph. multipora. The teleomorph Neophaeosphaeria filamentosa clustered basal to this clade. Clade D includes the genera Cucurbitaria, Pyrenochaetopsis and Pyrenochaeta, which represent Cucurbitariaceae. This finding is in congruence with previous studies (de Gruyter et al. 2010).

Phylogeny of the Leptosphaeria doliolum complex

The aligned sequence matrix obtained for the ITS, ACT, TUB and CHS-1 regions had a total length of 1 345 nucleotide characters; ITS 522, ACT 240, TUB 332 and CHS-1 251, respectively. The combined dataset used in the analyses included 18 taxa and contained 1 345 characters with 98 unique site patterns. The tree (Fig. 3) was rooted to “Ph. pedicularis” (CBS 390.80). The Bayesian analysis resulted in 6 002 trees after 30 000 generations, from which the burn-in was discarded and the consensus tree and posterior probabilities were calculated based on 3 341 trees.

Open in a separate window

Fig. 3.

The phylogeny of the Leptosphaeria doliolum complex, based on the strict consensus tree from a Bayesian analysis of 18 ITS/ACT/TUB/CHS-1 sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted to Leptosphaeria pedicularis comb. nov. (CBS 390.80).

The phylogenetic tree revealed two clades with high posterior probabilities, 98 and 99 % respectively, clade A with Ph. acuta subsp. errabunda and Ph. macrocapsa, and clade B with Ph. acuta subsp. acuta (anamorph of Leptosphaeria doliolum) and Ph. acuta subsp. acuta f. sp. phlogis. Phoma sydowii, a necrotroph on Asteraceae, Senecio spp. in particular, proved to be closely related to Ph. acuta subsp. errabunda. The isolate CBS 297.51 preserved as Ph. acuta is similar to Ph. sydowii, a synonym of L. sydowii, see below. Phoma veronicicola, as a necrotroph specifically occurring on Veronica spp. (Scrophulariaceae), also proved to be related to Leptosphaeria doliolum.

Phylogeny of Phoma section Pilosa

The aligned sequence matrix obtained for the ACT region had a total length of 252 nucleotide characters (20 taxa), and contained 165 unique sites. The tree was rooted to Ph. lingam (CBS 147.24 and CBS 260.94). The Bayesian analysis resulted in 34 802 trees after 174 000 generations, from which the burn-in was discarded, and the consensus tree and posterior probabilities were calculated based on 11 728 trees (Fig. 4).

Open in a separate window

Fig. 4.

The phylogeny of phoma-like anamorphs in the Pleosporaceae based on the strict consensus tree from a Bayesian analysis of 20 ACT sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted to Plenodomus lingam (CBS 147.24, CBS 260.94).

The phylogenetic tree representing the Pleosporaceae includes Ph. betae, type species of Phoma section Pilosa. This section is characterised by producing pycnidia that are covered by mycelial hairs. Phoma betae clearly groups with other pycnidial fungi pathogenic on Chenopodiaceae, including Ascochyta obiones, A. hyalospora and A. caulina and Chaetodiplodia sp. All species produce similar hairy pycnidia, but are classified in Ascochyta or Coniothyrium due to conidial septation, or brown pigmentation of conidia, respectively.

A subclade comprises the cosmopolitan Pleospora herbarum and related species. The species involved are associated with various hosts or substrates. The most closely related Ph. incompta is a specific pathogen on Olea europea (Oleaceae). Phoma incompta was classified in Phoma section Sclerophomella because of its thick-walled pycnidia (de Gruyter & Noordeloos 1992, Boerema & de Gruyter 1998). The pycnidial characters of Ph. incompta, pycnidia covered with mycelial hairs and with an indistinct ostiole visible as a pallid spot (de Gruyter & Noordeloos 1992) however, agrees with those of Ph. betae and Ph. typhina.

Phoma fallens proved to be closely related to Ph. glaucispora in keeping with the similar in vitro characters, especially the low growth-rate and the size and shape of its conidia (Boerema et al. 2004). Both species originate from southern Europe, and have been associated with spots on fruits and leaves of Olea europea, or leaf spots on Nerium oleander, respectively. An isolate preserved as Leptosphaeria clavata, CBS 259.51, proved to be closely related. The origin of the isolate, deposited by E. Müller, is unknown; however, it is likely that the isolate was obtained from Poaceae, Triticum vulgare or Dactylis glomerata (Müller 1950). Phoma flavigena, once isolated from water and also recorded from southern Europe, proved to be more distantly related in Pleosporaceae.

Phoma-like anamorphs excluded from the suborder Pleosporineae

Montagnulaceae M.E. Barr, Mycotaxon 77: 194. 2001.

Paraconiothyrium Verkley, Stud. Mycol. 50: 327. 2004.

Type species: Paraconiothyrium estuarinum Verkley & M. da Silva, Stud. Mycol. 50: 327. 2004.

Paraconiothyrium flavescens (Gruyter, Noordel. & Boerema) Verkley & Gruyter, comb. nov. MycoBank MB564786.

Basionym: Phoma flavescens Gruyter, Noordel. & Boerema, Persoonia 15(3): 375. 1993.

Specimen examined: Netherlands, Nagele, from soil, rhizosphere of Solanum tuberosum (Solanaceae), CBS 178.93 = PD 82/1062.

Paraconiothyrium fuckelii (Sacc.) Verkley & Gruyter, comb. nov. MycoBank MB564787.

Basionym: Coniothyrium fuckelii Sacc., Nuovo Giorn. Bot. Ital. 8: 200. 1876; Michelia 1: 207. 1878

• ≡ Clisosporium fuckelii (Sacc.) Kuntze, Revis. Gen. Pl. 3: 458. 1898.

• ≡ Microsphaeropsis fuckelii (Sacc.) Boerema, 2003, Persoonia 18: 160. 2003.

Specimen examined: Denmark, Geelskov, from a dead stem of Rubus sp. (Rosaceae), 1995, A.M. Dahl-Jensen, CBS 797.95.

Notes: Coniothyrium fuckelii var. sporulosum has been redisposed as Paraconiothyrium sporulosum (Verkley et al. 2004) and it is clearly different from Paraconiothyrium fuckelii (Damm et al. 2008).

Paraconiothyrium fusco-maculans (Sacc.) Verkley & Gruyter, comb. nov. MycoBank MB564788.

Basionym: Phoma fusco-maculans Sacc., Michelia 2: 275. 1881

• ≡ Plenodomus fusco-maculans (Sacc.) Coons, J. Agric. Res. 5: 714. 1916.

Specimens examined: Italy, Selva, from decorticated wood of Malus pumila (Rosaceae), Oct. 1880, PAD, holotype of Phoma fusco-maculans Sacc. USA, from wood of Malus sp. (Rosaceae), July 1916, G.H. Coons, epitype designated here CBS H-20825, culture ex-epitype CBS 116.16.

Notes: Plenodomus fusco-maculans was discussed by Boerema & Loerakker (1985) and de Gruyter et al. (2010). The holotype of the basionym Aposphaeria fusco-maculans was studied and considered to be Aposphaeria pulviscula (Boerema et al. 1996). However, the description of A. fusco-maculans given by Boerema et al. (1996) fits the generic concept of Paraconiothyrium, in congruence with the molecular phylogeny of the culture CBS 116.16.

Paraconiothyrium lini (Pass.) Verkley & Gruyter, comb. nov. MycoBank MB564789.

Basionym: Phoma lini Pass., Diagn. Funghi Nuovi 4, No. 81. 1890.

Specimen examined: Netherlands, from Wisconsin tank, 1970, CBS 253.92 = PD 70/998.

Paraconiothyrium maculicutis Verkley & Gruyter, sp. nov. MycoBank MB564796. Fig. 6.

Open in a separate window

Fig. 6.

Paraconiothyrium maculicutis sp. nov. CBS 101461. A-B. Fourteen day old cultures on OA (A) and MA (B). C-D. Pycnidia. E. Phoma-like conidiogenous cells. F-G. Conidia, initially hyaline to pale olivaceous (F), then becoming olivaceous (G). Scale bars: C-D = 20 μm; E = 10 μm; F-G = 5 μm.

Etymology: Latin, cutis = skin; maculae = spots.

Pycnidia in vitro 50-125 μm diam, globose to subglobose, glabrous or with mycelial outgrowth, scattered, non-ostiolate or ostiolate, pycnidial wall made up of 5-7 layers of cells. Conidiogenous cells 1.5-3 × 0.5-2.5 μm, indeterminate or ampulliform to filiform in a later state, up to 10 μm in length. Conidia 1.5-2.5 × 0.5-1.5 μm, ellipsoidal, initially hyaline, then discolouring to olivaceous.

Description in vitro: Colonies on OA 50-52 mm diam after 7 d, margin entire; colony olivaceous buff to greenish olivaceous/grey olivaceous, with greenish olivaceous to pale olivaceous grey, finely floccose to woolly aerial mycelium; reverse smoke-grey to greenish olivaceous, with olivaceous patches. Colonies on MEA 43-44 mm diam after 7 d, margin entire; colony pale olivaceous grey to greenish olivaceous, with isabelline to cinnamon at centre, with compact pale olivaceous grey, finely floccose to woolly aerial mycelium; reverse buff to honey, isabelline to olivaceous near margin. Pycnidia globose to subglobose, olivaceous to brick, finally olivaceous black, scattered, mainly on the agar, 50-125 μm diam, glabrous or with mycelial outgrowth, non-ostiolate or ostiolate, pycnidial wall made up of 5-7 layers of cells. Conidiogenous cells 1.5-3 × 0.5-2.5 μm, ampulliform to filiform in a later state, up to 10 μm in length. Conidia 1.5-2.5 × 0.5-1.5 μm, av. 1 × 2 μm, length/width ratio = 1.5-3.2, av. 2.2, ellipsoidal, initially hyaline, then discolouring to olivaceous. Chlamydospores absent. NaOH spot test: negative. Crystals absent.

Specimen examined: USA, Texas; San Antonio, Fort Sam Houston, from human, cutaneous lesions, 1989, D.P. Dooley, holotype CBS H-20824, culture ex-holotype CBS 101461 =IMI 320754 = UTHSC 87-144.

Notes: Isolate CBS 101461 was identified as Pleurophoma pleurospora (Dooly et al. 1989). However, in vitro data and the molecular phylogeny demonstrate that this isolate does not belong to Pleurophoma pleurospora, see below, and therefore is described as a new species in the genus Paraconiothyrium.

Paraconiothyrium minitans (W.A. Campb.) Verkley, Stud. Mycol. 50: 332. 2004.

Basionym: Coniothyrium minitans W.A. Campb., Mycologia 39: 191. 1947.

Specimens examined: Netherlands, Boskoop, from stem of Clematis sp. (Ranunculaceae), 1999, J. de Gruyter, CBS 122786 =PD 99/1064-1. UK, CBS 122788 =PD 07/03486739.

Paraconiothyrium tiliae (F. Rudolphi) Verkley & Gruyter, comb. nov. MycoBank MB564790.

Basionym: Asteroma tiliae F. Rudolphi, Linnaea 4: 514. 1829.

• ≡ Asteromella tiliae (F. Rudolphi) Butin & Kehr, Mycol. Res. 99: 1193. 1995, nom. inval., Art. 33.4.

Specimen examined: Austria, Amlach, from a leaf of Tilia platyphyllos (Tiliaceae), 10 Sep. 1993, H. Butin, neotype IMI 362854, lectotype designated here CBS H-20826, culture ex-lectotype CBS 265.94.

Pleurophoma pleurospora (Sacc.) Höhn., Sitzungsber. Kaiserl. Akad. Wiss., Math.-Naturwiss. Cl., Abt. 1. 123: 117. 1914. Fig. 7.

Open in a separate window

Fig. 7.

Pleurophoma pleurospora. CBS 130329. A-B. Fourteen day old cultures on OA (A) and MA (B). C. Pycnidia. D-H. Conidiogenous cells, septate conidiophores with acropleurogenous conidiogenesis (D-G) or phoma-like (H). I. Conidia. Scale bars: C = 50 μm; D-G, I = 10 μm; H = 5 μm.

Basionym: Dendrophoma pleurospora Sacc., Michelia 2: 97. 1880.

Description in vitro: Colonies on OA 14-18 mm diam after 7 d (18-28 mm after 14 d), margin entire to undulate; colony greenish olivaceous/olivaceous to rosy-buff and sepia, with white, felty aerial mycelium; reverse olivaceous grey to greenish olivaceous/olivaceous. Colonies on MEA 11-16 mm diam after 7 d (19-29 mm after 14 d), colony margin undulate; colony pale olivaceous grey/olivaceous grey to dark mouse-grey with rosy-buff tinges, with white, floccose, compact aerial mycelium, reverse umber/brown olivaceous to olivaceous/olivaceous black. Pycnidia globose to subglobose,olivaceous to olivaceous black, abundant, scattered, mainly on the agar, 30-120 μm diam, solitary or aggregated, covered by mycelial outgrowths or setae-like hyphae, up to 50 μm, non-papillated, without or with ostiole, walls made up of 2-5 layers of cells, outer layer(s) pigmented; conidial exudate not observed. Conidiogenous cells of two types; ampulliform to doliiform, 4-6.5 × 2-5.5 μm, or filiform, septate, branched, acropleurogenous, up to 60 μm long. Conidia 3.5-5.5 × 1.5-2.5 μm, av. 4.5 × 2 μm, length/width ratio = 1.5-3, av. 2.1, cylindrical to oblong, without or with some minute, polar orientated guttules. Chlamydospores absent. NaOH spot test: a weak reddish discolouring may occur on MA, not specific. Crystals absent.

Specimens examined: France, Perpignan, from leaf of Laurus nobilis (Lauraceae), PAD, holotype of Dendrophoma pleurospora Sacc. Netherlands, from wood of Lonicera sp. (Caprifoliaceae), lectotype designated here CBS H-20626, culture ex-lectotype CBS 130329 =PD 82/371; Molenhoek, Heumense Schans, from twig lesions of Cytisus scoparius (Fabaceae), 23 Aug. 2004, G. Verkley & M. Starink, CBS 116668.

Notes: A specimen derived from isolate CBS 130329 is assigned here as lectotype of Pleurophoma pleurospora, the type species of the genus (von Höhnel 1914). The species is known from branches and bare wood of trees and shrubs (Sutton 1980, Boerema et al. 1996) and the isolate from Cytisus scoparius demonstrates that the species also may occur on green twigs. The isolates showed two types of conidiogenesis characteristic for the genus Pleurophoma; phoma-like, ampulliform to doliiform conidiogenous cells, as well as pyrenochaeta-like branched, filiform, septate, acropleurogenous. As a result, species of the genus Pleurophoma can easily be confused with taxa classified in the genera Phoma, Paraphoma, Pyrenochaeta and Pyrenochaetopsis.

Paraphaeosphaeria michotii (Westend.) O.E. Erikss., Arkiv før Botanik 6: 406. 1967.

Basionym: Sphaeria michotii Westend., Bull. Acad. Roy. Sci. Belgique Ser. 2, 7: 87. 1859.

Specimen examined: Switzerland, Kt. Obwalden, from Typha latifolia (Typhaceae), 18 May 1980, A. Leuchtmann, CBS 652.86 = ETH 9483.

Massarinaceae Munk, Friesia 5: 305. 1956.

Byssothecium circinans Fuckel, Bot. Zeitung (Berlin) 19: 251. 1861.

• • ≡ Leptosphaeria circinans (Fuckel) Sacc., Syll. Fung. 2: 88. 1883.

  • ≡ Passeriniella circinans (Fuckel) Sacc., Syll. Fung. 11: 326. 1895.

  • ≡ Trematosphaeria circinans (Fuckel) G. Winter, Rabenh. Krypt.-Fl., ed 1(2): 277. 1887.

  • ≡ Heptameria circinans (Fuckel) Cooke, Grevillea 18: 30. 1889.

• = Melanomma vindelicorum Rehm, Ber. Nat. Ver. Augsburg: 116. 1881.

  • ≡ Trematosphaeria vindelicorum (Rehm) Sacc., Syll. Fung. 2: 122. 1883.

Specimen examined: USA, South Dakota, from rotten crown of Medicago sativa (Fabaceae), G. Semeniuk, CBS 675.92 = ATCC 52767 = ATCC 52678 = IMI 266220.

Massarina eburnea (Tul. & C. Tul.) Sacc., Syll. Fung. 2: 153. 1883.

Basionym: Massaria eburnea Tul. & C. Tul., Select. Fung. Carpol. (Paris) 2: 239. 1863.

Specimens examined: Switzerland, Zürich, from Fagus sylvatica (Fagaceae), S.K. Bose, CBS 473.64 = ETH 2945. UK, Wales, isolated from dead branch of Fagus sylvatica, HHUF 26621, JCM 14422 = H3953.

Neottiosporina paspali (G.F. Atk.) B. Sutton & Alcorn, Austral. J. Bot. 22: 519. 1974.

Basionym: Stagonospora paspali G.F. Atk., Bull. Cornell Univ. (Science) 3: 33. 1897.

Specimen examined: USA, Florida, from Paspalum notatum (Poaceae), Oct. 1937, R.K. Voorhees, CBS 331.37.

Trematosphaeriaceae Suetrong et al. Cryptogamie Mycol. 32: 347. 2011.

Falciformispora lignatilis K.D. Hyde, Mycol. Res. 96: 27. 1992.

Specimen examined: Thailand, Pinruan Ban Bang, from Elaeis guineensis (Arecaceae), BCC 21118.

Medicopsis Gruyter, Verkley & Crous, gen. nov. MycoBank MB564791.

Etymology: refers to Medi-medica, Latin, -opsis, refers to, Greek. The description of the type species as the cause of a mycetoma suggest this is a human pathogen. However, the mycetoma described was secondary to a wound produced by a thorn of Palito blanco tree, and the species was found later on Hordeum vulgare.

Pycnidia solitary or confluent, on upper surface of the agar, globose to pyriform with elongated neck, setose, ostiolate, olivaceous to olivaceous-black, the wall with pseudoparenchymatal cells. Conidiogenous cells hyaline, phialidic, ampulliform to doliiform, to elongated. Conidia sub-hyaline to yellowish, ellipsoid, aseptate, catenulate.

Type species: Medicopsis romeroi (Borelli) Gruyter, Verkley & Crous (see below).

Medicopsis romeroi (Borelli) Gruyter, Verkley & Crous, comb. nov. MycoBank MB564792.

Basionym: Pyrenochaeta romeroi Borelli, Dermatol. Venez. 1: 326. 1959.

Specimens examined: Venezuela, from human, maduromycosis, no date, D. Borelli, UAMH 2892, holotype of Pyrenochaeta romeroi Borelli, culture ex-holotype CBS 252.60 = ATCC 13735 = FMC 151 = UAMH 10841. Country unknown, from Hordeum vulgare (Poaceae), 1984, M.M.J. Dorenbosch, CBS 122784 =PD 84/1022.

Notes: The species was described as a human pathogen of tropical origin, and it may cause suppurative subcutaneous or deep nonmycetomatous infections, or a subcutaneous phaeohyphomycotic cyst (Badali et al. 2010). However, the species also occurs in plant material.

Trematosphaeria pertusa (Pers.) Fuckel, Jahrb. Nassauischen Vereins Naturk 23-24: 161. 1870.

Basionym: Sphaeria pertusa Pers., Syn. Meth. Fung. 1: 83. 1801.

Specimen examined: France, Deux Sèvres, from bark of a dead stump of Fraxinus excelsior (Oleaceae), 25 Apr. 2004, Jacques Fournier, epitype IFRD 2002, culture ex-epitype CBS 122368.

Note: The epitype IFRD 2002 was designated by Zhang et al. (2008).

Lentitheciaceae Yin. Zhang, C.L. Schoch, J. Fourn., Crous & K.D. Hyde, Stud. Mycol. 64: 93. 2009.

Splanchnonema platani (Ces.) M.E. Barr, Mycotaxon 15: 364. 1982.

Basionym: Sphaeria (Massaria) platani Ces., in Rabenhorst, Klotzschii Herb. Viv. Mycol.: no. 1842. 1854.

Specimen examined: USA, from Platanus occidentalis (Platanaceae), Jan. 1937, C.L. Shear, CBS 221.37.

Note: This taxon was shown by Zhang et al. (2012) to cluster basal to the Lentitheciaceae.

Melanommataceae G. Winter, Rabenh. Krypt.-Fl., ed 1(2): 220 (1885) [as “Melanommeae”]

Aposphaeria corallinolutea Gruyter, Aveskamp & Verkley, sp. nov. MycoBank MB564798. Fig. 8.

Open in a separate window

Fig. 8.

Aposphaeria corallinolutea sp. nov. CBS 131287. A-B. Fourteen day old cultures on OA (A) and MA (B). C-D. Pycnidia. E-H. Conidiogenous cells. I. Conidia. Scale bars: C = 50 μm; D = 20 μm; E-I = 10 μm.

Etymology: The name refers to the coral coloured colony on OA, and the luteous exudate diffusing into the agar medium.

Pycnidia in vitro 65-215 μm diam, solitary or aggregated to confluent, globose to subglobose, ostiolate or non-ostiolate. Conidiogenous cells 7-9 × 2-4 μm, ampuliform to filiform. Conidia 3-5 × 1-2 μm, ellipsoidal to allantoid, eguttulate or with some small, polar guttules.

Description in vitro: Colonies on OA 13-15 mm diam after 14 d, margin entire to somewhat lobated; colony vinaceous to brick, with white at centre, ochraceous near margin due to a diffusible pigment, with white, felty or poorly developed aerial mycelium; reverse cinnamon to brick. Colonies on MEA 15-20 mm diam after 14 d, margin entire to somewhat lobated; colony white with dull green and grey olivaceous sectors and primrose tinges, with white, felty aerial mycelium; reverse sepia to brown olivaceous, greenish grey at centre, white near margin. Pycnidia globose to subglobose, olivaceous to brick, then olivaceous black, solitary or aggregated, 65-215 μm diam, non-setose or with short setae-like outgrowths up to 25 μm long, with or without distinct ostiole, pycnidial wall consisting of 3-5 layers of cells. Conidiogenous cells 7-9 × 2-4 μm, ampulliform to filiform. Conidia 3-5 × 1-2 μm, av. 4 × 1.5 μm, length/width ratio is 1.7-3.3, av. = 2.5, ellipsoidal to allantoid, eguttulate or with some small, polar guttules. Chlamydospores absent, NaOH test negative. Crystals produced in the agar, small, orange coloured.

Specimens examined: Netherlands, from wood of Fraxinus excelsior (Oleaceae), 1983, M.M.J. Dorenbosch, holotype CBS H-20625, culture ex-holotype CBS 131287 =PD 83/831; from wood of Kerria japonica (Rosaceae), 1983, M.M.J. Dorenbosch, CBS 131286 =PD 83/367.

Aposphaeria populina Died., Krypt.-Fl. Brandenburg 9: 206. 1912 (vol. dated “1915”). Fig. 9.

Open in a separate window

Fig. 9.

Aposphaeria populina. CBS 543.70. A-B. Fourteen day old cultures on OA (A) and MA (B). C. Pycnidium with mycelial outgrowths (CBS 130330). D-E. Conidiogenous cells. F. Conidia. Scale bars: C = 20 μm; D-E = 10 μm; F = 5 μm.

Description in vitro: Colonies on OA 21-24 mm diam after 7 d (32-37 mm diam after 14 d), margin entire to undulate; colony grey olivaceous/olivaceous to pale luteous/luteous, with white to pale olivaceous grey, finely felty to woolly aerial mycelium; reverse luteous to orange, greenish olivaceous to olivaceous or grey olivaceous/olivaceous grey to iron-grey, a rosy-buff discolouring near margin may occur. Colonies on MEA 16-20 mm diam after 7 d (30-37 mm diam after 14 d), margin entire to undulate; colony pale olivaceous grey with rosy-vinaceous tinges to peach or olivaceous grey, with white, woolly aerial mycelium; reverse saffron to pale olivaceous/olivaceous grey, sometimes with dark vinaceous tinges, rosy-buff near margin. Pycnidia globose to subglobose, olivaceous to olivaceous black, scattered, 55-305 μm diam, glabrous or with mycelial outgrowths, non-ostiolate or ostiolate, pycnidial wall composed of up to 10 layers of cells. Conidiogenous cells 5-11.5 × 1.5-3 μm, ampulliform to filiform. Conidia hyaline, subglobose to ellipsoidal, with 1-3 minute guttules, 1-2 × 1-1.5 μm, av. 1.5 × 1 μm, length/width ratio is 1.0-2.0, av. = 1.4. Chlamydospores and crystals absent, NaOH test negative.

Specimens examined: Germany, Triglitz, from twigs of Populus canadensis (Salicaceae), Mar. 1904. O. Jaap, B, holotype; from branch scars of Picea abies, (Pinaceae), Feb. 1982, H. von Aufess, CBS 350.82. Netherlands, Valkenswaard, from fallen twig of Populus canadensis (Salicaceae), 23 Mar. 1970, H.A. van der Aa, epitype designated here CBS H-9336, culture ex lectotype CBS 543.70; from wood of Cornus mas (Cornaceae), 1984, M.M.J. Dorenbosch, CBS 130330 =PD 84/221.

Beverwykella pulmonaria (Beverw.) Tubaki, Trans. Mycol. Soc. Japan 16: 139. 1975.

Basionym: Papulaspora pulmonaria Beverw., Antonie van Leeuwenhoek 20: 11. 1954.

Specimen examined: Netherlands, Baarn, from submerged leaf in rain water barrel of Fagus sylvatica (Fagaceae), Apr. 1953, A.L. van Beverwijk, culture CBS 283.53 = ATCC 32983 = IFO 6800.

Herpotrichia juniperi (Duby) Petr., Ann. Mycol. 23: 43. 1925.

Basionym: Sphaeria juniperi Duby, Klotzsch. Herb. Vivum Mycol. Sistems Fungorum German., no. 1833. 1854.

Specimen examined: Switzerland, Andermatt, from Juniperus nana (Cupressaceae), Nov. 1931, E. Gäumann, CBS 200.31.

Melanomma pulvis-pyrius (Pers.) Fuckel, Jahrb. Nassauischen Vereins Naturk. 23-24: 160. 1870.

Basionym: Sphaeria pulvis-pyrius Pers., Syn. Meth. Fung. 1: 86. 1801.

Specimens examined: Belgium, from wood of Fagus sp. (Fagaceae), CBS 400.97. France, Vosges, Bot. Garden Le Chitelet, from unidentified decaying wood, CBS 371.75.

Notes: Phoma-like anamorphs have been reported by Chesters (1938) and Sivanesan (1984), but no anamorphic stage was observed in IFRDCC 2044, CBS 109.77 or CBS 371.75 after culturing 3 mo on PDA (Zhang et al. 2008). CBS 400.97 was preserved as Trematosphaeria pertusa.

Pleomassaria siparia (Berk. & Broome) Sacc., Syll. Fung. 2: 239. 1883.

Basionym: Sphaeria siparia Berk. & Broome, Ann. Mag. Nat. Hist. Ser. 2(9): 321. 1852.

Specimen examined: Netherlands, Uden, from dead branch of Betula verrucosa (Betulaceae), 8 Dec. 1973, W.M. Loerakker, CBS H-258, CBS H-260, culture CBS 279.74.

Sporormiaceae Munk, Dansk Bot. Ark. 17(1): 450. 1957, nom. inval., Art. 36.1.

Preussia funiculata (Preuss) Fuckel, Jahrb. Nassauischen Vereins Naturk. 23-24: 91. 1870 (1869-70).

Basionym: Perisporium funiculatum Preuss, Linnaea 24(1): 143. 1851.

Specimen examined: Senegal, from soil, CBS 659.74.

Sporormiella minima (Auersw.) S.I. Ahmed & Cain, Canad. J. Bot. 50: 449. 1972.

Basionym: Sporormia minima Auersw., Hedwigia 7: 66. 1868.

Specimen examined: Panama, from dung of goat, CBS 524.50.

Westerdykella Stolk, Trans. Brit. Mycol. Soc. 38: 422. 1955.

Type species: Westerdykella ornata Stolk, see below.

Westerdykella capitulum (V.H. Pawar, P.N. Mathur & Thirum) Gruyter, Aveskamp & Verkley, comb. nov. MycoBank MB564801.

Basionym: Phoma capitulum V.H. Pawar, P.N. Mathur & Thirum., Trans. Brit. Mycol. Soc. 50: 261. 1967.

• • ≡ Phoma capitulum V.H. Pawar & Thirum., Nova Hedwigia 12: 502. 1966 (as “capitula”), nom. nud., nom. inval.

• = Phoma ostiolata V.H. Pawar, P.N. Mathur & Thirum., Trans. Brit. Mycol. Soc. 50: 262. 1967, var. ostiolata.

  • ≡ Phoma ostiolata V.H. Pawar & Thirum., Nova Hedwigia 12: 502. 1966, nom. nud., nom. inval.

• = Phoma ostiolata var. brunnea V.H. Pawar, P.N. Mathur & Thirum., Trans. Brit. Mycol. Soc. 50: 263. 1967.

  • ≡ Phoma ostiolata var. brunnea V.H. Pawar & Thirum., Nova Hedwigia 12: 502. 1966, nom. nud., nom. inval.

Specimen examined: India, Bandra, Bombay, from saline soil, 15 Jan. 1958, M.J. Thirumalachar, Isotype CBS H-7602, culture ex-isotype CBS 337.65 = ATCC 16195 = HACC 167 = IMI 113693 = PD 91/1614.

Westerdykella minutispora (P.N. Mathur ex Gruyter & Noordel.) Gruyter, Aveskamp & Verkley, comb. nov. MycoBank MB564793.

Basionym: Phoma minutispora P.N. Mathur ex Gruyter & Noordel., Persoonia 15: 75. 1992 (as “collection name” originally also referred to Thirumalachar; = depositor).

Replaced synonym: Phoma oryzae Cooke & Massee, Grevillea 16: 15. 1887 (not Phoma oryzae Catt., Arch. Triennale Bot. Crittog. Pavia 2-3: 118. 1879, nom. illeg).

• ≡ Phyllosticta oryzae (Cooke & Massee) I. Miyake. J. Coll. Agric. Imp. Univ. Tokyo 2: 252. 1910, nom. illeg.

Specimen examined: India, from saline soil, 1977, M.J. Thirumalachar, CBS H-5941, culture CBS 509.91 = PD 77/920.

Westerdykella ornata Stolk, Trans. Brit. Mycol. Soc. 38: 422. 1955.

Specimen examined: Mozambique, from mangrove mud, CBS 379.55.

Didymosphaeriaceae Munk, Dansk Bot. Ark. 15(2): 128. 1953.

Roussoella hysterioides (Ces.) Höhn., Sitzungsber. Kaiserl. Akad. Wiss., Math.-Naturwiss. Cl., Abt. 1. 128: 563. 1919.

Basionym: Dothidea hysterioides Ces., Atti Accad. Sci. Fis. 8: 24. 1879.

Specimen examined: Japan, Aomori, Shimokita Yagen, from culms of Sasa kurilensis (Poaceae), Y. Ooki, culture CBS 125434 =HH 26988.

The genera Leptosphaeria, Paraleptosphaeria, Plenodomus, Subplenodomus and Heterospora

Plenodomus lingam and L. doliolum, the type species of Plenodomus and Leptosphaeria respectively, were found to be distant genetically, which agrees with findings of previous molecular phylogenetic studies (Jasalavic et al. 1995, Morales et al. 1995, Dong et al. 1998, Câmara et al. 2002, Eriksson & Hawksworth 2003, Wunsch & Bergstrom 2011). In our study the generic type species grouped in sister clades, which represent Leptosphaeria and Plenodomus. Species of Leptosphaeria produce dark brown, 3-septate ascospores, which have been considered the primitive state with more recently evolved species producing ascospores that are paler in colour, longer and narrower, and more than 3-septate (Wehmeyer 1946). This hypothesis is supported by the results obtained in our study. Paraleptosphaeria is distinct but seems to be most closely related to Leptosphaeria producing 3(-5)-septate, yellow/brown or hyaline ascospores. Both genera include only necrotrophic species. Plenodomus and Subplenodomus include necrotrophs and plant pathogens. Ascospores in Plenodomus are 3-7-septate, whereas in Subplenodomus no sexual state has thus far been recorded. The scleroplectenchymatous pycnidial cell wall is typical for Plenodomus, whereas in Subplenodomus the pycnidial cell wall is pseudoparenchymatous. Heterospora is closely allied to Subplenodomus and no sexual state has been recorded for this genus either. The distinctive characterisitics of the genera Heterospora, Leptosphaeria, Paraleptosphaeria, Plenodomus and Subplenodomus are summerised in Table 2. A blast search in GenBank using ITS sequences of five selected species of the Leptosphaeriaceae, namely L. doliolum, L. etheridgei, Plen. lingam, H. dimorphospora and Subplen. drobnjacensis, did not reveal close matches to other teleomorphic or anamorphic genera.

Plectophomella visci grouped in Plenodomus in this study and in the Leptosphaeriaceae in a previous molecular phylogeny of Phoma and allied anamorph genera (de Gruyter et al. 2009). Plectophomella visci is the type species of Plectophomella (Moesz 1922) and three additional species have been described in the genus. Two species were described from the bark of Ulmus spp., viz. Plectophomella ulmi (basionym Dothiorella ulmi) and Plectophomella concentrica (Redfern & Sutton 1981). Dothiorella ulmi is considered the appropriate name for Plectophomella ulmi (Crous et al. 2004). A third species, Plectophomella nypae, was described from Nypa fruticans (Arecaceae) (Hyde & Sutton 1992). As a result of the transfer of the type species Plectophomella visci to Plenodomus, the taxonomy of both Plectophomella concentrica and P. nypae needs to be reconsidered based on the outcome of a molecular study.

Plenodomus chrysanthemi could not be differentiated from Plen. tracheiphilus based on comparison of their LSU and ITS sequences. Plenodomus vasinfecta was proposed by Boerema et al. (1994) for the species originally described as Phoma tracheiphila f. sp. chrysanthemi (Baker et al. 1985). Because these are part of the Plenodomus clade the name Plenodomus chrysanthemi is proposed with P. tracheiphila f. sp. chrysanthemi and P. vasinfecta as synonyms. Plenodomus chrysanthemi and Plen. tracheiphilus are host specific (Chrysanthemum and Citrus, respectively) and the scleroplectenchymatous conidiomatal wall of Plen. tracheiphilus differentiates this species from Plen. chrysanthemi, where only a parenchymatous wall has been observed (Boerema et al. 1994). The results of this molecular study and the production of a Phialophora synanamorph by both species demonstrate the close relationship of both taxa.

Plenodomus enteroleucus and Plen. influorescens have a similar ecological niche as opportunistic pathogens on woody plants in Europe. Both taxa were formerly described as varieties of Ph. enteroleuca, vars. enteroleuca and influorescens, and could be differentiated only by the fluorescence of var. enteroleuca under black light. However, the molecular phylogeny demonstrates the two varieties are only distantly related and they are raised from varietal status to species rank. The close relation of Plen. wasabiae with Plen. biglobosus agrees with the results of a previous study on the production of Phomalignin A and other yellow pigments, as well as ITS sequence analyses (Pedras et al. 1995).

Subplenodomus apiicola, Subplen. drobnjacensis, Subplen. valerianae and Subplen. violicola all produce pycnidia with an elongated neck, resembling Plenodomus. The pycnidial wall remains usually pseudoparenchymatous. Pycnidia with a scleroplectenchymatous wall are only observed in Subplen. drobnjacensis. Subplenodomus apiicolus, Subplen. drobnjacensis and Subplen. valerianae produce relatively small conidia, up to 4.5 × 2 μm (de Gruyter & Noordeloos 1992) in congruence with many of the Plenodomus species described; however, in contrast Subplen. violicola produces relatively large conidia, up to 11 × 3 μm (Boerema 1993).

The grouping of species of Phoma section Plenodomus based on the host being either herbaceous plants or wood of trees and shrubs (Boerema 1982, Boerema et al. 1994) is not supported by the molecular phylogeny. The grouping of the species into two categories based on the production of pseudoparenchymatous pycnidia that become scleroplectenchymatous pycnidia (type I), versus always scleroplectenchymatous pycnidia (type 2) (Boerema et al. 1981), is partly supported by the molecular phylogeny. In the Leptosphaeria clade most species directly develop scleroplectenchymatous pycnidia, whereas in the Plenodomus clade the pycnidia generally are pseudoparenchymatous and become scleroplectenchymatous.

Heterospora is established for two species of Phoma sect. Heterospora that cluster in the Leptosphaeriaceae, viz H. chenopodii and H. dimorphospora. All other species of Phoma sect. Heterospora are in the Didymellaceae (Aveskamp et al. 2010).

The Leptosphaeria doliolum species complex

The taxonomy of the generic type species Leptosphaeria doliolum and Phoma anamorphs is complex with a number of subspecies and varieties described in literature. Leptosphaeria doliolum subsp. doliolum and L. doliolum subsp. errabunda are morphologically very similar, as well as the anamorphs Ph. acuta subsp. errabunda and Ph. acuta subsp. acuta. It has been suggested that both taxa represent originally American and European counterparts (Boerema et al. 1994). Both subspecies of L. doliolum proved to be closely related in a phylogenetic analysis utilising LSU and ITS. A detailed multilocus phylogenetic study including the ITS, ACT, TUB and CHS genes, however, demonstrated that both subspecies could be clearly differentiated, and represent two subclades in the L. doliolum complex. All species allied with L. doliolum and L. errabunda are necrotrophic species. Surprisingly, L. macrocapsa grouped with the L. errabunda isolates. Leptosphaeria macrocapsa is described as a host-specialised necrotroph on Mercurialis perennis (Euphorbiaceae) in Europe (Boerema et al. 1994). The species is characterised by large pycnidia (Grove, 1935), with a conspicuously broad, long cylindrical neck (Boerema et al. 1994). This is different to the sharply delimited papilla or neck of variable length of the pycnidia of L. errabunda. Leptosphaeria sydowii, a necrotroph on Senecio spp. in particular (Asteraceae), proved to be closely related to L. errabunda. It can be concluded that the Leptosphaeria doliolum complex includes several necrotrophic species, with adapted host specificity.

The genus Coniothyrium

Coniothyrium palmarum is the type species of the genus Coniothyrium. Coniothyrium is characterised by ostiolate pycnidial conidiomata, annellidic conidiogenous cells, the absence of conidiophores, and brown, thick-walled, 0- or 1-septate, verrucose conidia. Coniothyrium is similar morphologically to some species in the genus Microsphaeropsis. However, Microsphaeropsis is characterised by the production of phialidic conidiogenous cells with periclinal thickening, and thin-walled, pale greenish brown conidia.

Coniothyrium, Microsphaeropsis and Paraconiothyrium clearly grouped in different clades in a study of the partial SSU nrDNA (Verkley et al. 2004). In a subsequent study utilising SSU and LSU sequences, the generic type species Microsphaeropsis olivacea grouped in Didymellaceae, whereas Coniothyrium palmarum clustered with the genus Leptosphaeria in Leptosphaeriaceae (de Gruyter et al. 2009). In the present study C. palmarum and its relatives grouped in a distinct clade, which represents Coniothyriaceae. Phoma carteri, Ph. glycinicola, Ph. septicidalis and Pyrenochaeta dolichi grouped in this clade and are transferred to the genus Coniothyrium. The inclusion of these species with setose pycnidia and conidiogenesis with elongated conidiophores expands the morphological circumscription of Coniothyrium. Species with those characters are also found in other genera treated in this paper in the Cucurbitariaceae, Didymellaceae, Phaeosphaeriaceae, Leptosphaeriaceae, Montagnulaceae and Sporormiaceae, indicating convergent evolution.

The Coniothyrium species included here are plurivorous or soil-borne, such as C. palmarum, C. septicidalis and C. multiporum, or are associated with a specific host such as C. carteri on Quercus spp. (Fagaceae), C. glycinicola on Glycine max (Fabaceae) and C. dolichii on Dolichos biflorus (Fabaceae). The species also are diverse geographically.

Coniothyrium palmarum was frequently found associated with leaf spots on Phoenix dactylifera (Arecaceae) in India and Cyprus (Sutton 1980). The C. palmarum isolates regularly used in phylogenetic studies are CBS 758.73, from leaf spots on Phoenix dactylifera in Israel, and CBS 400.71, from a dead petiole of Chaemeropsis humulis (Arecaceae) in Italy. The subtropical distribution of these species is similar to that of the most closely allied C. dolichi and C. glycinicola. Coniothyrium multiporum, recorded from marine soil, also is found in warm regions. Coniothyium carteri, in contrast, is reported from North America and Europe.

Coniothyrium dolichi produces setose pycnidia with hyaline conidia (Mohanty 1958). The conidiogenesis was studied in detail later. phoma-like ampulliform conidiogenous cells as well as conidiogenous cells on filiform, septate conidiophores were found in the same pycnidia leading to confusion regarding the classification of this species in Phoma or Pyrenochaeta (Grodona et al. 1997). This study clearly supports the classification in Coniothyrium. Coniothyrium glycinicola was originally placed in the genus Pyrenochaeta as Py. glycines due to its setose pycnidia (Stewart 1957). The conidiogenesis and hyaline conidia are phoma-like and therefore, it was reclassified as Ph. glycinicola in Phoma sect. Paraphoma (de Gruyter & Boerema 2002). However, in the original description it was noted that the conidia were greenish-yellow in mass (Stewart 1957), resembling Microsphaeropsis or coniothyrium-like conidia. This study clearly supports the classification in Coniothyrium. Coniothyrium carteri produces setose pycnidia with hyaline conidia and therefore, the species was classified in Phoma section Paraphoma (de Gruyter & Boerema 2002). In spite of this similarity, C. carteri was determined to be only distantly related to the generic type species Paraphoma radicina (de Gruyter et al. 2010). Coniothyrium multiporum was described in Phoma section Phoma; however, it proved to be unrelated to Phoma in Didymellaceae (Aveskamp et al. 2010). The conidiogenesis may comprise elongated conidiophores (Pawar et al. 1967). Two isolates originally described as Ph. septicidalis are placed here in Coniothyrium telephii. Other strains deposited as Ph. septicidalis proved to be Pyrenochaeta unguis-hominis (de Gruyter et al. 2010).

The anamorph of the genus Neophaeosphaeria was described as coniothyrium-like, producing pigmented, aseptate conidia from holoblastic, percurrently proliferating conidiogenous cells with conspicuous annellations (Câmara et al. 2003). Although Neophaeosphaeria is related to Coniothyrium based on the molecular data, Neophaeosphaeria probably belongs to a separate phylogenetic clade. The grouping of N. filamentosa with the Coniothyrium species included in this study was poorly supported and N. filamentosa proved to be more distantly related in previous molecular phylogenetic studies (Verkley et al. 2004, Damm et al. 2008, de Gruyter et al. 2010).

Both anamorph genera Cyclothyrium and Cytoplea were considered to be related to Coniothyrium and Microsphaeropsis (Sutton 1980) based on morphological similarities. Cyclothyrium also resembles Paraconiothyrium but produces conidiogenous cells that are more elongated than in most species of Paraconiothyrium and the conidia are almost truncate at the base, or at least they are much less rounded at the base than the conidia of Paraconiothyrium (Verkley et al. 2004). The generic type species Cyclothyrium juglandis, the anamorph of Thyridaria rubronotata, proved to be related to Roussoella hysterioides, teleomorph of Cytoplea (Verkley et al. 2004). Based on present results R. hysterioides could not be assigned to familial rank. The clustering of this species in Massariaceae (Zhang et al. 2009) could not be confirmed. Moreover, Roussoella probably is not a monophyletic genus (Tanaka et al. 2009). Thyridaria rubronotata, the teleomorph of Cyclothyrium juglandis, proved to be related to Massariosphaeria phaeospora but was not assigned to familial rank (Schoch et al. 2009).

Coniothyrium-like anamorphs also have been linked to Mycosphaerella in the past. However, these species were subsequently accommodated in Colletogloeopsis (Cortinas et al. 2006), Readeriella/Kirramyces (Crous et al. 2007) and are now known to be species of Teratosphaeria (Crous et al. 2009b).

The genus Pleospora

Pleospora is a large genus in Pleosporaceae, Pleosporales, and includes important pathogens that occur on both monocotyledons and dicotyledons. Anamorphs of Pleospora s. lat. have been described in various genera of coelomycetes and hyphomycetes as summarised by Zhang et al. (2009, 2012). A delimitation of Pleospora into two sections, Pyrenophora and Eu-Pleospora was made based on the size of fruiting bodies and ascospore septation and colour (Munk 1957). The genus Pyrenophora (Drechslera anamorphs) is recognised at the generic rank. However, Pleospora remains heterogenous (Wehmeyer 1961, Berbee 1996) and molecular phylogenetic studies demonstrated that Pleospora is polyphyletic in Pleosporaceae (Kodsueb et al. 2006, Wang et al. 2007, Inderbitzin et al. 2009). Taxa with a Stemphylium anamorph such as Pleospora sedicola and Pleo. tomatonis, as well as Pleo. halophola with no known anamorph, are closely related to Cochliobolus, whereas Pleo. herbarum and Pleo. ambigua were more distantly related in the Pleosporaceae (Kodsueb et al. 2006, Wang et al. 2007). A phylogenetic study of the genus Massariosphaeria demonstrated the polyphyly in the genera Pleospora, Kirschsteiniothelia, Massarina, Melanomma, Trematosphaeria and Massariosphaeria in the Loculoascomycetes (Wang et al. 2007) and the paraphyletic character of the genus Cochliobolus was demonstrated (Kodsueb et al. 2006, Mugambi & Huhndorf 2009). These findings support the previous speculation by several authors that ascomatal and ascospore morphologies have undergone convergent evolution among Pleosporales (Wang et al. 2007).

Pleospora betae groups ambiguously in Pleosporaceae (Dong et al. 1998). SSU nrDNA sequence data supported the affinity of P. betae to Leptosphaeriaceae. Partial LSU nrDNA data supported the affinity of P. betae to Pleosporaceae (Dong et al. 1998), but bootstrap support values in that study were low. In a multigene phylogenetic study Pleo. betae was found as being basal to Pleosporaceae (Zhang et al. 2009). Our results demonstrate the sister group relationship of Pleo. betae and its relatives to the generic type species Pleo. herbarum.

Pleospora betae has been often confused with Pleo. calvescens as was discussed by Boerema et al. (1987). Both species are pathogens of Chenopodiaceae and are morphologically rather similar and therefore, a phylogenetic relation of both species was inferred (Boerema 1984). In addition Ascochyta hyalospora, originally found on the American continent on Chenopodiaceae, also was supposed to be closely related. Our results demonstrate that Pleo. betae and Pleo. calvescens could be recognised at species rank and confirmed that A. hyalospora is related supporting our transfer to Pleospora as Pleo. chenopodii. The delimitation of both halophytic species Pleo. chenopodii and Pleo. calvescens needs further study; both species could not be clearly differentiated based on the ACT sequences alone. Additional studies are underway to elucidate these species boundaries, in which also the recently described halophyte, Ascochyta manawaorae (Verkley et al. 2010), will be included. Pleospora fallens and Pleo. incompta, formerly described in Phoma sect. Phoma and producing mainly glabrous pycnidia, grouped in the Pleo. herbarum clade. Pleospora typhicola, producing pilose pycnidia, also grouped in this clade.

This project, “Strengthening the Plant Health Infrastructure”, was supported by The Dutch Ministry of Economic Affairs, Agriculture and Innovation. We thank Mrs Trix Merkx and Mrs Karin Rosendahl-Peters for providing the strains from the culture collection of CBS and PD respectively and for their assistance in the deposit of strains. Mrs Arien van Iperen kindly helped us with the deposit of herbarium material. Thanks are due to Marjan Vermaas for her assistance in preparing the photoplates. We are indebted to Machiel E. Noordeloos and the reviewers for critical reading of the manuscript.