DNA isolation

Mycelium from actively growing fungal cultures was scraped from the surface of MEA or PDA plates using a sterile scalpel blade. Harvested mycelium was ground to a fine powder using liquid nitrogen and DNA was isolated using the CTAB extraction protocol as outlined by Crous et al. (2009d) or the UltraClean™ Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Solana Beach, CA, USA) following the manufacturers’ protocols. Isolated DNA was visualised by electrophoresis in 1 % agarose gels (w/v) stained with ethidium bromide and viewed under near ultra-violet light. DNA concentrations were determined by measuring electrophoresed DNA samples against a HyperLadder™ I molecular marker (BIOLINE) or alternatively by a NanoDrop quantification as outlined by the manufacturer.

PCR amplification

DNA isolated from fungal isolates was used as template for further Polymerase Chain Reaction (PCR) amplifications. Four nuclear gene regions were targeted for PCR amplification and subsequent sequencing. These regions included the Internal Transcribed Spacer regions ITS-1, ITS-2 and the 5.8S nrRNA gene regions (ITS), the first 900 bp of the Large Subunit (28S, LSU) (domains D1-D3) of the rDNA operon and partial gene regions of the translation elongation factor 1-alpha (EF-1α) and the actin (ACT) genes.

The ITS region was amplified using primers ITS-1 or ITS-5 and ITS-4 (White et al. 1990) while primers used for amplification of the LSU region were LR0R (Rehner & Samuels 1994) or LSU1Fd (Crous et al. 2009b) and LR5 or LR7 (Vilgalys & Hester 1990). Primers employed for the amplification of EF-1α included EF1-728F and EF1-986R (Carbone & Kohn 1999) or EF-2 (O’Donnell et al. 1998) while ACT-512F and ACT-783R (Carbone & Kohn 1999) were used to amplify a portion of the ACT gene. All PCR reaction mixtures and conditions followed those outlined by Hunter et al. (2006b). Following PCR amplification, amplicons were visualized on 1.5 % agarose gels stained with ethidium bromide and viewed under ultra-violet light and sizes of amplicons were determined against a HyperLadder™ I molecular marker (BIOLINE). The PCR amplicons for the four loci were subsequently diluted 1 to 10 times in preparation for further DNA sequencing reactions.

DNA sequencing and phylogenetic inference

PCR amplicons of the four gene regions targeted in this study served as templates for DNA sequencing reactions with the BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) following the protocol of the manufacturer. DNA sequencing reactions used the same primers as those for the PCR reactions. However, additional internal primers LR3R (http://www.biology.duke.edu/fungi/mycolab/primers.htm), LR16 (Moncalvo et al. 1993) and LR5 were used to sequence the LSU in order to obtain reliable sequences spanning the entire D1-D3 region. DNA sequencing amplicons were purified through Sephadex® G-50 Superfine columns (Sigma Aldrich, St. Louis, MO) in MultiScreen HV plates (Millipore, Billerica, MA). Purified sequence reactions were run on an ABI Prism 3730xl DNA Sequencer (Life Technologies, Carlsbad, CA, USA).

Generated DNA sequence electropherograms were analysed using MEGA (Molecular Evolutionary Genetics Analysis) v. 4.0 (Tamura et al. 2007), 4Peaks v. 1.7.2 (http://www.mekentosj.com/) and SeqMan v. 8.0.2. from the DNASTAR Lasergene® software package. Consensus sequences were generated and imported into MEGA for initial alignment and the construction of sequence datasets. DNA sequences representing isolates of closely allied genera, for which material could not be obtained were downloaded from the NCBI GenBank nucleotide database (www.ncbi.nlm.nih.gov) and added to the DNA sequence datasets generated in this study. Sequence datasets for the four genomic loci were aligned in MAFFT (“Multiple alignment program for amino acids or nucleotide sequences”) v. 6.0 (Katoh & Toh 2006, Katoh et al. 2005; http://mafft.cbrc.jp/alignment/server/index.html) using the Auto alignment strategy with the 200PAM/ K=2 scoring matrix and a gap opening penalty of 1.53 with an offset value of 0.0. Resulting sequence alignments were manually evaluated and adjusted in MEGA, MacClade v.4.08 (Maddison & Maddison 2000) or Sequence Alignment Editor v. 2.0a11 (Rambaut 2002).

A phylogenetic re-construction was conducted for the aligned LSU data set to determine generic relationships using MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Subsequently, a species level phylogeny was derived from the combined ITS, ACT and EF-1α alignment of Pseudocercospora s. str. sequences using PAUP v. 4.0b10 (Swofford 2003). For the LSU alignment, MrModeltest v. 2.2 (Nylander 2004) was used to determine the best nucleotide substitution model settings for MrBayes. Based on the results of the MrModeltest, a phylogenetic analysis was performed with MrBayes v. 3.1.2 applying a general time-reversible (GTR) substitution model with inverse gamma rates and dirichlet base frequencies and a heating parameter set at 0.3. The Markov Chain Monte Carlo (MCMC) analysis of 4 chains started in parallel from a random tree topology and had 8 000 000 generations. Trees were saved each 1 000 generations, resulting in 8 001 saved trees in each of the two tree files. Burn-in was set at 2 000 000 generations after which the likelihood values were stationary. For parsimony analysis of the combined ITS, ACT and EF-1α alignment, alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum parsimony analysis was performed in PAUP using the heuristic search option with 100 random taxon additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally most parsimonious trees were saved. The robustness of the trees was evaluated by 1 000 bootstrap replicates (Hillis & Bull 1993). Tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) were calculated and the resulting trees were printed with Geneious v. 5.5.4 (Drummond et al. 2011). Sequences derived in this study were deposited in GenBank (Table 1), the alignments in TreeBASE (www.treebase.org/treebase/index.html), and taxonomic novelties in MycoBank (www.MycoBank.org; Crous et al. 2004b).

Taxonomy

Isolates representing 146 species of Pseudocercospora were subjected to DNA analysis and morphological comparison. Phylogenetic analyses based on the LSU gene resolved a total of 14 clades in the Pseudocercospora complex.

Clade 1 represented Strelitziana (pseudocercospora-like but with a separating cell between conidia and conidiogenous cells) and Cyphellophora (pseudocercospora-like but phialides with flaring collarettes, situated directly on hyphae). Thedgonia ligustrina (pseudocercosporella-like, but conidia in chains) represented Clade 2. Clade 3 included several isolates of Pseudocercospora cantuariensis, which represents a novel genus, distinguished from Pseudocercospora based on its broad conidial hila and scars, as well hyaline mycelium, and the presence of hyphopodia-like structures. Xenostigmina zilleri, characterised as being stigmina-like, but also having sympodial proliferation of the conidiogenous cells, clustered in Clade 4, which was basal to Cladosporium (Cladosporiaceae; Clade 5). Clade 6 represented several members of Teratosphaeriaceae, known to have a wide range of anamorphs, including Microcyclospora. Clade 7 represented species of Dissoconium (Dissoconiaceae), distinct due to their dimorphic conidia that are actively discharged. Clade 8 remains unresolved, and was represented by disjunct elements appearing Zasmidium- and pseudocercospora-like in morphology, including Microcyclosporella. Clade 9 was represented by several Mycosphaerella species such as M. laricina (anamorph Pseudocercospora sp.), and Paracercospora egenula. Paracercospora was separated from Pseudocercospora based on a combination of characters, including pale olivaceous conidia, and a minute thickening along the rim of its conidial hila and scars. Clade 10 included a diverse assemblage of genera. Two genera that differ mainly based on their conidiomatal structure, Pseudocercosporella and Septoria, clustered in this clade. Miuraea, a genus intermediate between Cercospora and Pseudocercospora, also resided within this clade. Clade 11 was represented by two coelomycetous species of Sonderhenia that clustered basal to Clade 12. The latter included a new genus with pseudocercospora-like anamorphs, mostly distinguished from Pseudocercospora s. str. by having species with smooth, pale brown conidia, and the frequent production of red crystals in agar (previously referred to in literature as the Mycosphaerella heimii complex). Scolecostigmina (based on S. mangiferae), which is characterised by verruculose conidia and percurrently proliferating conidiogenous cells, clustered alongside to Trochophora, characterised by brown sickle-shaped conidia with three thick, dark septa. Passalora eucalypti formed a separate lineage in Clade 13 that was adjacent to Pseudocercospora s. str. in Clade 14. This clade included the type species, P. vitis that is basal in this cluster. Although there was structure within the clade, we regard it as representing a single genus, including Stigmina platani, the type of Stigmina, Phaeoisariopsis (P. griseola), and Pseudophaeoramularia (P. angolensis). Several isolates identified from different countries as representing the same species based on host, disease symptoms and general morphology, clustered apart from one another. These collections were found to represent novel cryptic species.

Treatment of species

Several novel taxa were identified in this study on the basis of phylogenetic analyses of the various gene regions together with morphological examination of the specimens and isolates. Recognised clades, as well as novel species and genera, are described and discussed below. Where descriptions of known taxa are freely available online in MycoBank or journals, they are not repeated here, other than their generic circumscriptions.

Clade 1: Strelitziana and Cyphellophora

Strelitziana M. Arzanlou & Crous, Fungal Planet No. 8: 2006.

Conidiophores erect, solitary, arising from aerial and submerged mycelium, subcylindrical, straight to geniculate-sinuous, pale brown. Conidiogenous cells terminal, integrated, rejuvenating percurrently, proliferating apically via several short, conspicuous denticles; conidiogenesis holoblastic with rhexolytic conidial secession. Conidia solitary, pale brown, smooth, long obclavate, multi-euseptate; microcyclic conidiation present in culture.

Type species: Strelitziana africana M. Arzanlou & Crous, Fungal Planet No. 8. 2006.

Notes: The genus Strelitziana presently accommodates four species that are primarily distinguished based on their conidial dimensions. These include S. africana, S. australiensis, S. eucalypti and S. mali (Arzanlou & Crous 2006, Cheewangkoon et al. 2009, Zhang et al. 2009, Crous et al. 2010).

Cyphellophora G.A. de Vries, Mycopathol. Mycol. Appl. 16: 47. 1962.

Colonies (on OA) with moderate to rapid growth, velvety to lanose, in various shades of grey; reverse black. Fertile hyphae pale brown, sometimes with constrictions at the septa. Conidiogenous cells phialidic, intercalary, sometimes on short side branches, each with a short, lateral or terminal collarette. Conidia sickle-shaped, brown, smooth-walled, transversely septate, adhering in small bundles (from de Vries 1962).

Type species: Cyphellophora laciniata G.A. de Vries, Mycopathol. Mycol. Appl. 16: 47. 1962.

Notes: The genus Cyphellophora, which is based on C. laciniata (isolated from human skin; De Vries et al. 1986), appears to be heterogeneous (Decock et al. 2003, Crous et al. 2007a, 2009a, Cheewangkoon et al. 2009) and requires further study.

Clade 2: Thedgonia

Thedgonia B. Sutton, Trans. Brit. Mycol. Soc. 61: 426. 1973.

Foliicolous, phytopathogenic, causing discrete leaf spots. Conidiomata fasciculate, punctiform. Mycelium internal, hyphae subhyaline, septate, branched, forming substomatal stromata, hyaline to pale brown. Conidiophores fasciculate, arising from stromata, simple, rarely branched, subcylindrical, straight to geniculate-sinuous, continuous to septate, smooth, hyaline to pale yellowish green. Conidiogenous cells integrated, terminal, occasionally conidiophores reduced to conidiogenous cells, holoblastic-thalloblastic, sympodial, conidiogenous loci more or less planate, unthickened, non-pigmented. Conidia in disarticulating chains, rarely in branched chains, subcylindrical to obclavate, with one to several transverse eusepta, hyaline or almost so, apex rounded to truncate, base truncate, hila flat, unthickened, hyaline (Crous et al. 2009a).

Type species: Thedgonia ligustrina B. Sutton, Trans. Brit. Mycol. Soc. 61: 426. 1973.

Thedgonia ligustrina (Boerema) B. Sutton, Trans. Brit. Mycol. Soc. 61: 428. 1973.

Basionym: Cercospora ligustrina Boerema, Tijdschr. Plantenziekten 68: 117. 1962.

• ≡ Cercoseptoria ligustrina (Boerema) Arx, Genera of Fungi Sporulating in Pure Culture, ed. 3: 306, Lehre 1981.

Specimens examined: Asia, on Ligustrum sp., H. Evans, CPC 4296 = W2072, CPC 4297 = W 2073, CPC 4298 = W 1877. Netherlands, Eefde, on Ligustrum ovalifolium, 23 Mar. 1959, G.H. Boerema, holotype L, ex-type culture CBS 148.59; Bilthoven, on L. ovalifolium, 2003, P.W. Crous, CPC 10530 = CBS 124332, CPC 10532, 10533. South Korea, Namyangju, on L. ovalifolium, 9 Oct. 2002, leg. H.D. Shin, isol. P.W. Crous, CBS H-20204, CPC 10019, 10861-10863; Suwon, on L. obtusifolium, 2 Oct. 2007, leg. H.D. Shin, isol. P.W. Crous, CBS H-20207, CPC 14754-14756.

Notes: Contrary to the earlier hypothesis that Thedgonia belonged to the Mycosphaerellaceae (Kaiser & Crous 1998), Crous et al. (2009a) showed that it resides in Helotiales. Consequently, thedgonia-like anamorphs that occur in the Mycosphaerellaceae must be accommodated elsewhere.

Clade 3: Xenostigmina

Xenostigmina Crous, Mycol. Mem. 21: 154. 1998.

Foliicolous, phytopathogenic, causing discrete leaf spots. Mycelium internal, consisting of hyaline to pale brown, septate, branched, smooth hyphae. Conidiomata sporodochial, brown to black. Conidiophores densely aggregated, arising from the upper cells of a pale brown stroma, finely verruculose, hyaline to pale brown, multiseptate, subcylindrical, straight to variously curved, branched. Conidiogenous cells terminal and intercalary, hyaline to pale brown, finely verruculose, doliiform to subcylindrical, tapering to flat tipped loci, mono- to polyblastic, proliferating sympodially and percurrent; loci not thickened or conspicuous. Conidia solitary, pale to medium brown, with pale brown apical and basal regions, finely verruculose, mostly straight, ellipsoidal, apex subobtuse, frequently extending into a beak; base truncate at dehiscence, inner part extending later to form a short, subobtuse basal appendage; septation muriform; basal marginal frill present (Crous et al. 2009a).

Type species: Xenostigmina zilleri (A. Funk) Crous, Mycol. Mem. 21: 155. 1998.

Specimens examined: Canada, British Columbia, 15 km east of Sardis, on living leaves of Acer macrophyllum, 22 Oct. 1985, A. Funk & C.E. Dorworth, holotype DAVFP 23272; British Columbia, on living leaves of Acer sp., 2002, leg. K.A. Seifert, isol. P.W. Crous, CBS 115686 =CPC 4010, CBS 115685 =CPC 4011; Victoria BC, 48°30’25.63”N, 123°30’46.99”W, 115 m, fallen leaves of A. macrophyllum, 6 Sep. 2007, leg. B. Callan, isol. P.W. Crous, epitype designated here CBS H-20208, cultures ex-epitype CPC 14376 = CBS 124108, CPC 14377, 14378 (Xenostigmina zilleri), CPC 14379 = CBS 124109, CPC 14380, 14381 (Mycopappus aceris).

Notes: Xenostigmina with its Mycopappus synanamorph is distinct from Stigmina s. str., which is a synonym of Pseudocercospora s. str. (Crous et al. 2006, Braun & Crous 2006, 2007). The genus Xenostigmina (Crous 1998) appears related to Seifertia (Seifert et al. 2007) in the Dothideomycetes (Crous et al. 2009b).

Clade 4: Phaeomycocentrospora

Phaeomycocentrospora Crous, H.D. Shin & U. Braun, gen. nov. MycoBank MB564813.

Etymology: Name reflects the pale brown appearance of conidia and the superficial similarity to Mycocentrospora.

Foliicolous, phytopathogenic, causing discrete leaf spots. Mycelium internal and external, consisting of hyaline, septate, branched, smooth, 3-5 μm diam hyphae; hyphopodium-like structures present. Caespituli amphigenous. Conidiophores in loose fascicles, arising from a poorly developed stroma, or from superficial hyphae emerging from stomata, or erumpent through the cuticle; erect on superficial hyphae, olivaceous-brown, straight to slightly curved, unbranched, not geniculate, obconically truncate at the apex; conidiogenous cells integrated, terminal or conidiophores reduced to conidiogenous cells, mono- to polyblastic, proliferating sympodially, transversely septate; conidiogenous loci broad, more or less planate, neither thickened nor darkened. Conidia solitary, filiform to cylindrical, straight to moderately curved, subhyaline to pale olivaceous, transversely euseptate, usually not constricted at septa, tapering somewhat towards an obtuse apex, truncate at the base; hilum unthickened, not darkened, broad.

Type species: Phaeomycocentrospora cantuariensis (E.S. Salmon & Wormald) Crous, H.D. Shin & U. Braun, comb. nov.

Notes: Phaeomycocentrospora is similar to Pseudocercospora in that its conidia and conidiophores appear to be pigmented and its conidiogenous loci are unthickened and not darkened. It is distinct from Pseudocercospora in that its mycelium is hyaline, hyphopodia-like structures are present, and conidia are hyaline with a pale brown inner wall layer, giving the impression of pigmented conidia. This fungus also has extremely broad conidial loci and scars that are untypical of Pseudocercospora. Chupp (1954) commented that Cercospora cantuariensis represented an unusual species that should be transferred to a genus of its own. Based on its unique phylogenetic placement (Fig. 4) and morphology, Phaeomycocentrospora gen. nov. is established for this taxon. Deighton (1971, 1972) assigned this species to Mycocentrospora, but the type species M. acerina is phylogenetically distinct from other genera morphologically similar to it and differs in having conidia with filiform appendages and often with strongly swollen intercalary cells.

Phaeomycocentrospora cantuariensis (E.S. Salmon & Wormald) Crous, H.D. Shin & U. Braun, comb. nov. MycoBank MB564814. Fig. 6.

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Fig. 6.

Phaeomycocentrospora cantuariensis (CPC 11691-11693). A. Leaf spots on upper and lower leaf surface. B, C. Sporulation of leaf surface. D-I. Conidiophores and conidiogenous cells. J-M. Conidia. Scale bars = 10 μm.

Basionym: Cercospora cantuariensis E.S. Salmon & Wormald, J. Bot. (London) 61: 134. 1923.

• ≡ Centrospora cantuariensis (E.S. Salmon & Wormald) Deighton, Mycol. Pap. 124: 8. 1971.

• ≡ Mycocentrospora cantuariensis (E.S. Salmon & Wormald) Deighton, Taxon 21: 716. 1972.

• ≡ Pseudocercospora cantuariensis (E.S. Salmon & Wormald) U. Braun, Mycotaxon 48: 281. 1993.

Leaf spots amphigenous, scattered, often confluent, subcircular to irregular, 1-5 mm diam, becoming up to 10 mm diam when confluent, greyish to white, centre reddish brown with yellowish brown zone on upper surface; greyish brown to grey on lower surface. Caespituli amphigenous, but predominantly hypophyllous. Mycelium internal and external; internal hyphae hyaline, septate, branched, smooth, 3-4 μm diam; external hyphae plagiotropous, branched, septate, smooth, hyaline, 3-5 μm diam. Conidiophores in loose fascicles, arising from a poorly developed stroma, or from superficial hyphae emerging from stomata, or erumpent through the cuticle; erect on superficial hyphae, olivaceous-brown, straight to slightly curved, unbranched, not geniculate, obconically truncate at the apex, proliferating sympodially, 0-3-septate, 30-140 × 7-20 μm. Conidiogenous cells terminal, unbranched, pale brown, smooth, tapering to flat-tipped apical loci, with scars neither thickened nor darkened, 4-7 μm diam; at times proliferating percurrently, with 1-3 percurrent proliferations at the apex, 12-45 × 5-8 μm. Conidia solitary, filiform to cylindrical, straight to moderately curved, subhyaline to pale olivaceous, smooth, 3-15(-21)-septate, usually not constricted at septa, tapering somewhat towards obtuse apex, truncate at the base, or long obconically subtruncate, (100-)140-200(-500) × (5-)7-12(-20) μm; hilum unthickened, not darkened, 4-7 μm diam; conidia appear to have an inner wall layer that is pale brown when studied in culture (adapted from Shin & Kim 2001).

Specimens examined: South Korea, Hoengseong, on Humulus scandens (= H. japonicus), 4 Sep. 2005, H.D. Shin, CBS H-20830; Suwon, Acalypha australis, 5 Nov. 2004, H.D. Shin, cultures CPC 11691-11693; Suwon, H. scandens, 5 Nov. 2004, H.D. Shin, CBS H-20831, cultures CPC 11694-11696; Hoengseong, on H. scandens, 11 Oct. 2004, H.D. Shin, CBS H-20832, cultures CPC 11646, 11647; Wonju, on H. scandens, 18 Oct. 2002, H.D. Shin, CBS H-20833, cultures 10157, 10158; Namyangju, on Luffa aegyptica (= L. cylindrica), 22 Oct. 2003, H.D. Shin, CBS H-20834, cultures CPC 10762-10766.

Clade 5: Cladosporium (Cladosporiaceae)

Cladosporium Link, Ges. Naturf. Freunde Berlin Mag. Neuesten Entdeck. Gesammten Naturk. 7: 37. 1816.

Teleomorph: Davidiella Crous & U. Braun, Mycol. Progr. 2: 8. 2003.

Saprobic or phytopathogenic. Ascomata pseudothecial, black to red-brown, globose, inconspicuous and immersed beneath stomata to superficial, situated on a reduced stroma, with 1(-3) short, periphysate ostiolar necks; periphysoids frequently growing down into cavity; wall consisting of 3-6 layers of textura angularis. Asci fasciculate, short-stalked or not, bitunicate, subsessile, obovoid to broadly ellipsoid or subcylindrical, straight to slightly curved, 8-spored. Pseudoparaphyses frequently present in mature ascomata, hyaline, septate, subcylindrical. Ascospores bi- to multiseriate, hyaline, obovoid to ellipsoid-fusiform, with irregular luminar inclusions, mostly thick-walled, straight to slightly curved; frequently becoming brown and verruculose in asci; at times covered in mucoid sheath (from Schubert et al. 2007). Mycelium superficial, loosely branched, septate, sometimes constricted at septa, hyaline, subhyaline to pale brown, smooth or almost so to verruculose or irregularly rough-walled, sometimes appearing irregular in outline due to small swellings and constrictions, walls unthickened to somewhat thickened. Conidiophores both macro- and micronematous, arising laterally from plagiotropous hyphae or terminally from ascending hyphae. Macronematous conidiophores erect, straight to flexuous, somewhat geniculate-sinuous, nodulose or not, unbranched or occasionally branched, pluriseptate, pale to medium brown, older ones almost dark brown, walls thickened, sometimes even two-layered. Conidiogenous cells integrated, terminal or intercalary, mono- to usually polyblastic, nodulose to nodose or not, proliferation sympodial, with several conidiogenous loci, mostly situated on small lateral shoulders, more or less protuberant, characteristically coronate (SEM), i.e. with a convex central dome surrounded by a low to distinctly raised rim, appearing to be thickened and somewhat darkened-refractive. Micronematous conidiophores hardly distinguishable from hyphae, sometimes only as short lateral outgrowth with a single apical scar, short, conical to almost filiform or narrowly cylindrical, pluriseptate, usually short, subhyaline to pale brown, almost smooth to minutely verruculose or irregularly rough-walled, 0-3-septate. Conidiogenous cells integrated, terminal or conidiophores reduced to conidiogenous cells, narrowly cylindrical or filiform, with a single or two loci. Conidia solitary (in heterosporium-like species) to usually catenate, in unbranched or loosely branched chains, straight to slightly curved; small terminal conidia without distal hilum, obovoid to ellipsoid to subcylindrical, aseptate, subhyaline to pale brown; intercalary conidia with a single or sometimes up to three distal hila, limoniform, ellipsoid to subcylindrical, 0-1-septate; secondary ramoconidia with up to four distal hila, ellipsoid to cylindrical-oblong, 0-1(-2)-septate, pale greyish brown or brown to medium brown, smooth to minutely verruculose to verrucose, walls slightly to distinctly thickened, apex obtuse or slightly truncate, towards the base sometimes distinctly attenuated with hila situated on short stalk-like prolongations, hila slightly to distinctly protuberant, coronate structure as in conidiogenous loci, somewhat thickened and darkened-refractive; microcyclic conidiogenesis occurring; primary ramoconidia similar to secondary ramoconidia, except base truncate, uniform with conidiogenous cell, and more subcylindrical in shape (adapted from Schubert et al. 2007).

Type species: Cladosporium herbarum (Pers.: Fr.) Link, Ges. Naturf. Freunde Berlin Mag. Neuesten Entdeck. Gesammten Naturk. 7: 37. 1816.

Notes: Cladosporium is well-defined by having Davidiella teleomorphs and conidiophores that give rise to conidial chains with unique coronate scars (David 1997, Braun et al. 2003a, Schubert et al. 2007, Bensch et al. 2010, 2012), which easily distinguish it from a range of other morphologically similar genera (Crous et al. 2007a, b; Braun & Crous, in Seifert et al. 2011).

Clade 6: Teratosphaeriaceae

Teratosphaeria Syd. & P. Syd., Ann. Mycol. 10: 39. 1912.

Phytopathogenic, commonly associated with leaf spots, but also on fruit, or causing cankers on stems. Ascomata pseudothecial, superficial to immersed, frequently situated in a stroma of brown pseudoparenchymatal cells, globose, unilocular, papillate, ostiolate, canal periphysate, with periphysoids frequently present; wall consisting of several layers of brown textura angularis; inner layer of flattened, hyaline cells. Pseudoparaphyses frequently present, subcylindrical, branched, septate, anastomosing. Asci fasciculate, 8-spored, bitunicate, frequently with multi-layered endotunica. Ascospores ellipsoid-fusoid to obovoid, 1-septate, hyaline, but becoming pale brown and verruculose, frequently covered in mucoid sheath (from Crous et al. 2007a).

Type species: Teratosphaeria fibrillosa Syd. & P. Syd., Ann. Mycol. 10: 40. 1912.

Notes: Teratosphaeria accommodates a group of plant pathogenic fungi that can cause serious leaf spot, blotch and canker diseases of a range of hosts (Crous 2009, Crous et al. 2007a, 2009b, Hunter et al. 2009, 2011). The Teratosphaeriaceae remains to be clearly resolved, and several different genera are presently recognised in the family. Some are plant-associated such as Batcheloromyces, Baudoinea, Capnobotryella, Catenulostroma, Davisoniella, Devriesia, Hortea, Penidiella, Phaeothecoidea, Pseudotaeniolina, Readeriella, Staninwardia, and Stenella s. str. (Crous et al. 2007a, 2009a, 2011b), and others including Cystocoleus, Racodium, Friedmanniomyces, Elasticomyces, Recurvomyces (Selbmann et al. 2008) and Xanthoriicola (Ruibal et al. 2011) are lichenicolous or rock inhabiting.

Microcyclospora Jana Frank, Schroers & Crous, Persoonia 24: 99. 2010.

Epiphytic and endophytic, occurring on leaves and fruit. Mycelium consisting of branched, septate, pale brown, smooth, 2-3 μm wide hyphae. Conidiophores reduced to conidiogenous cells, integrated in hyphae, giving rise to peg-like lateral protuberances, 1 μm wide, 1-2 μm tall, mono- to polyblastic. Conidia scolecosporous, cylindrical, straight to variously curved, flexuous, apex obtuse, base truncate, 1-multi-septate, somewhat constricted at septa, smooth, pale brown, guttulate, aggregated in mucoid masses; hila not thickened or darkened; microcyclic conidiation observed in culture.

Type species: Microcyclospora pomicola Jana Frank, B. Oertel, Schroers & Crous, Persoonia 24: 100. 2010.

Notes: Microcyclospora was recently introduced in Teratosphaeriaceae for three taxa associated with sooty blotch of apple (Frank et al. 2010). The species described here resembles others presently known in Microcyclospora by having pigmented structures and undergoing microcyclic condiation. Other than having distinct conidial dimensions, it differs from other genera in that its conidiogenous cells are annellidic (not mono- to polyblastic), and its conidia are darker brown and verruculose to warty, not pale brown and smooth.

Microcyclospora quercina Crous & Verkley, sp. nov. MycoBank MB564815. Figs ​Figs7,7, ​,88.

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Fig. 7.

Microcyclospora quercina (CPC 10712). Line drawing showing conidiogenous cells and conidia formed in culture. Scale bar = 10 μm.

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Fig. 8.

Microcyclospora quercina (CPC 10712). A, B. Colony on oatmeal and potato-dextrose agar, respectively. C-E. Conidiogenous cells giving rise to conidia (arrows). F. Microcyclic conidiation. G, H. Conidia. Scale bars = 10 μm.

Etymology: Name reflects its host, Quercus.

Foliicolous, endophytic. Mycelium consisting of branched, septate, brown, 1.5-3 μm diam hyphae, guttulate, smooth to verruculose or warty, with or without mucoid sheath. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lateral on hyphae, brown, solitary, not aggregated, 1.5-2 μm diam, with 1-4 percurrent proliferations and flaring collarettes. Conidia solitary, subcylindrical (rarely obclavate), gently curved, apex obtuse (rarely subobtuse), base truncate or long obconically truncate, with slight basal taper to hilum that is 2 μm diam, unthickened, nor darkened, frequently with small marginal frill, brown, guttulate to granular, smooth, appearing warty or roughened due to external mucoid layer which is sometimes present, transversely (1-)3-4(-11)-euseptate, becoming constricted at septa with age, (12-)30-45(-70) × (2-) 2.5-3 μm; microcyclic conidiation commonly observed.

Culture characteristics: Colonies after 2 wk in the dark up to 15 mm diam, with sparse aerial mycelium, folded surface and uneven to somewhat feathery, lobate margins, exuding copious amounts of slime on PDA, but less so on MEA and OA; colonies olivaceous-black on all media.

Specimen examined: Netherlands, endophytic in leaves of Quercus robur, Sep. 2003, G.J.M. Verkley, holotype CBS H-20835, culture ex-type CPC 10712 = CBS 130827.

Clade 7: Dissoconium (Dissoconiaceae)

Dissoconium de Hoog, Oorschot & Hijwegen, Proc. K. Ned. Akad. Wet., Ser. C, Biol. Med. Sci. 86(2): 198. 1983.

Hyperparasitic, but also reported to be phytopathogenic. Ascomata pseudothecial, immersed, globose, unilocular, papillate, ostiolate, canal periphysate; wall consisting of 3-4 layers of brown textura angularis; inner layer of flattened, hyaline cells. Pseudoparaphyses absent. Asci fasciculate, 8-spored, bitunicate. Ascospores ellipsoid-fusoid, 1-septate, hyaline, with or without mucoid sheath. Mycelium internal and external, consisting of branched, septate, smooth, hyaline to pale brown hyphae. Conidiophores separate, arising from hyphae, subcylindrical, subulate or lageniform to cylindrical, tapering to a bluntly rounded or truncate apex, straight to once geniculate, smooth, medium brown, 0-multi-septate; conidiogenous cells polyblastic, with terminal and lateral conidiogenous loci, visible as slightly thickened, darkened scars on a rachis. Conidia solitary, pale olivaceous-brown, smooth, ellipsoid to obclavate or globose, 0-1-septate; hila somewhat darkened. Secondary conidia present or absent; developing adjacent to primary conidia, pale olivaceous to subhyaline, aseptate, pyriform; conidium discharge active or passive (from Crous et al. 2009b).

Type species: Dissoconium aciculare de Hoog, Oorschot & Hijwegen, Proc. K. Ned. Akad. Wet., Ser. C, Biol. Med. Sci. 86(2): 198. 1983.

Notes: Dissoconium has mycosphaerella-like teleomorphs (Crous 1998, Crous et al. 2004c) and was recently shown to represent a distinct family, Dissoconiaceae (Crous et al. 2009b). Species are different from other taxa in Capnodiales in that they form primary and secondary conidia that are actively discharged and anastomose on the agar surface shortly after germination (De Hoog et al. 1991).

Clade 8: Microcyclosporella and zasmidium-like

Microcyclosporella Jana Frank, Schroers & Crous, Persoonia 24: 101. 2010.

Epiphytic on leaves and fruit. Mycelium consisting of pale brown, smooth to finely verruculose, branched, septate, 2-3.5 μm wide hyphae, at times covered in a mucoid layer, with integrated, lateral, truncate conidiogenous loci. Conidiophores mostly reduced to conidiogenous cells. Conidiogenous cells integrated, intercalary on hyphae, rarely terminal, cylindrical to doliiform, pale brown, but hyaline if occurring in yeast-like sectors of colonies, smooth, mono- or polyblastic, proliferating sympodially; loci inconspicuous, truncate, unthickened, not darkened, pale brown to hyaline. Conidia hyaline, smooth, subcylindrical to narrowly obclavate or narrowly fusoid with acutely rounded apex and obconically truncate base, guttulate, transversely 0-6-septate; microcyclic conidiation common.

Type species: Microcyclosporella mali Jana Frank, Schroers & Crous, Persoonia 24: 101. 2010.

Notes: Microcyclosporella was treated as part of the Pseudocercosporella generic complex (Batzer et al. 2005), but has since been shown to be polyphyletic within Mycosphaerellaceae (Crous 2009, Crous et al. 2003, 2009b, c, Frank et al. 2010). The clade accommodating Microcyclosporella contains many disjunct elements that vary in morphology from Microcyclosporella s. str. (hyaline structures) to pigmented structures, namely zasmidium-like (verrucuclose conidia) to pseudocercospora-like (smooth conidia) (see Crous et al. 2009b). We suspect that these groups may eventually be recognised as distinct genera, but more taxa need to be examined to resolve this issue.

Clade 9: Paracercospora and pseudocercospora-like

Paracercospora Deighton, Mycol. Pap. 144: 47. 1979.

Foliicolous, phytopathogenic, causing leaf spots. Mycelium internal, hyaline to pale olivaceous. Stromata absent to poorly developed. Conidiophores fasciculate, smooth, subhyaline to pale olivaceous. Conidiogenous cells integrated, terminal, mono- to usually polyblastic, proliferating sympodially; conidiogenous loci moderately conspicuous, with narrow thickening along the rim. Conidia solitary, subcylindrical to obclavate-cylindrical, smooth, subhyaline to pale olivaceous, with a narrow thickening along the rim of the hilum.

Type species: Paracercospora egenula (Syd.) Deighton, Mycol. Pap. 144: 48. 1979.

Specimens examined: Japan, Shimane, on leaves of Solanum melongena, 5 Aug. 1998, T. Mikami, CNS-415, cultures MUCC 883, MAFF 237766. South Korea, Hongcheon, on leaves of S. melongena, 26 Oct. 2005, H.D. Shin, CBS H-20836, culture CPC 12537.

Notes: Stewart et al. (1999) conducted the first phylogenetic analysis of the Mycosphaerellaceae and concluded that the marginal thickening that occurs along the rims of conidial scars and hila, originally thought to be the main character to distinguish Paracercospora from Pseudocercospora, was not taxonomically significant and suggested that Paracercospora be reduced to synonymy with Pseudocercospora. The current study provides new evidence that Paracercospora is not a synonym of Pseudocercospora, but no consistent morphological characters that distinguish it from Pseudocercospora s. str. have been identified. Conidia of Paracercospora egenula are subhyaline to pale olivaceous with minimal marginal thickening of the conidiogenous loci (Fig. 9). Conidial scars and hila of Ps. fijiensis (Arzanlou et al. 2008) and Ps. basiramifera (Crous 1998) are marginally thickened. Both of the latter species, which belong to Pseudocercospora s. str., have pale to medium brown conidia. At present Paracercospora may be defined by a combination of the minimal marginal thickening of the conidiogenous loci and its subhyaline conidia.

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Fig. 9.

Paracercospora egenula (CPC 12537). A. Leaf spots on upper and lower leaf surface. B. Close-up of lesion. C-F. Fascicles with conidiogenous cells. G. Conidia. Scale bars = 10 μm.

The taxonomic placement of Paracercospora is complicated by two other taxa that resolve in the clade together with it. These are Passalora brachycarpa (pale olivaceous, catenate conidia, and prominent, thickened, darkened scars; also visible when sporulating in culture), and Pseudocercospora tibouchinigena described below, which has subhyaline conidia, and unthickened hila and scars. This indicates that it is neither a species of Pseudocercospora s. str. (subhyaline conidia), nor Paracercospora (lacking any form of scar thickening). As a temporary solution, the species on Tibouchina is described in Pseudocercospora, although taxa in this subclade may eventually be shown to represent a distinct genus.

Pseudocercospora tibouchinigena Crous & U. Braun, sp. nov. MycoBank MB564816. Fig. 10.

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Fig. 10.

Pseudocercospora tibouchinigena (CBS 116462). A. Leaf spots on upper and lower leaf surface. B, C. Close-up of lesions. D-G. Fascicles with conidiogenous cells. H. Conidia. Scale bar = 10 μm.

Etymology: Name is derived from Tibouchina, the host on which it was collected.

Leaf spots amphigenous, angular to irregular, 1-3 mm diam, up to 10 mm long, medium brown, with raised, dark brown border. Mycelium internal, hyaline, smooth, consisting of septate, branched, smooth, 1.5-2 μm diam hyphae. Caespituli fasciculate, predominantly hypophyllous, hyaline to pale olivaceous on leaves, up to 60 μm wide and 40 μm high. Conidiophores aggregated in dense fascicles, arising from the upper cells of a hyaline to subhyaline stroma, up to 50 μm wide and 20 μm high; conidiophores subcylindrical to ampulliform, 0-3-septate, straight to variously curved or geniculate-sinuous, unbranched, 15-25 × 3-5 μm. Conidiogenous cells terminal, unbranched, hyaline, smooth, tapering to flat-tipped apical loci, proliferating sympodially, 5-10 × 2.5-3.5 μm. Conidia solitary, subhyaline, smooth, guttulate or not, subcylindrical or narrowly obclavate, apex subobtuse, base obconically truncate, straight to variously curved, 3-10-septate, (15-)30-40(-60) × (1.5-)2-2.5(-3) μm; hila unthickened, not darkened nor refractive, 1-1.5 μm diam; prominent microcyclic conidiation observed in vivo.

Culture characteristics: Colonies after 1 mo at 24 °C in the dark on MEA; erumpent, spreading, with moderate aerial mycelium, and smooth, lobate margins. Surface pale olivaceous-grey; reverse olivaceous-grey. Colonies reaching 30 mm diam.

Specimen examined: New Zealand, Auckland, Princes Street, Auckland University Campus, on leaves of Tibouchina sp. (Melastomataceae), 9 Aug. 2004, C.F. Hill 1061, holotype HAL 2359F, culture ex-type CBS 116462.

Notes: Pseudocercospora tibouchinigena was initially reported from New Zealand as P. tibouchina (Braun et al. 2006), which is hitherto known only from Brazil. It differs from P. tibouchinae in that the latter species has narrowly subcylindrical conidia that are larger, 40-120 × 2-3 μm (Viégas 1945), than those of P. tibouchinigena. The subhyaline conidia of P. tibouchinigena are not typical of Pseudocercospora s. str., but for the present, we choose to name it in Pseudocercospora until the clade in which it resides has been more fully resolved (Fig. 5).

Clade 10: Cercospora, Miuraea, Phloeospora, Pseudocercosporella, Septoria, Xenocercospora

Cercospora Fresen., in Fuckel, Hedwigia 1(15): 133. 1863 and in Fuckel, Fungi Rhen. Exs., Fasc. II, No. 117. 1863.

Mostly phytopathogenic producing conspicuous lesions, but also including saprobes. Mycelium internal, rarely also external; hyphae colourless or almost so to pigmented, branched, septate, smooth to faintly rough-walled. Stromata lacking to well-developed, subhyaline to usually pigmented, substomatal to intraepidermal. Conidiophores mononematous, macronematous, solitary to fasciculate, arising from internal hyphae or stromata, emerging through stomata or erumpent, very rarely arising from superficial hyphae, erect, continuous to pluriseptate, subhyaline to pigmented, smooth to faintly rough-walled, thin- to moderately thick-walled. Conidiogenous cells integrated, terminal or intercalary or conidiophores reduced to conidiogenous cells, monoblastic, determinate to usually polyblastic, sympodial, rarely with a few enteroblastically percurrent rejuvenations which are not connected with conidiogenesis; conidiogenous loci (scars) conspicuous, thickened and darkened, planate. Conidia solitary, very rarely catenate, scolecosporous, obclavate, cylindrical-filiform, acicular, hyaline or subhyaline (with a pale greenish tinge), mostly pluriseptate, euseptate, rarely with 0-1 or few septa, smooth or almost so, hila thickened and darkened, planate (from Crous & Braun 2003).

Type species: Cercospora penicillata (Ces.) Fresen., Beiträge zur Mykologie 3: 93. 1863. [= C. depazeoides (Desm.) Sacc.].

Cercospora sojina Hara, Nogyo Sekai, Tokyo 9: 28. 1915. Fig. 11.

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Fig. 11.

Cercospora sojina (CPC 12322). A. Leaf spots on upper and lower leaf surface. B. Close-up of lesion. C-G. Fascicles with conidiophores and conidiogenous cells. H. Conidia. Scale bars = 10 μm.

• ≡ Passalora sojina (Hara) H.D. Shin & U. Braun, Mycotaxon 58: 163. 1996.

Specimen examined: South Korea, Hongcheon, on Glycine soja (= G. max subsp. soja), 20 Jul. 2004, H.D. Shin, CBS H-20837, culture CPC 12322.

Notes: Despite sparingly septate and broadly obclavate-cylindrical conidia that tend to be subhyaline, this species is better accommodated in Cercospora than Passalora (Shin & Braun 1996) based on phylogenetic analysis.

Cercospora eucommiae Crous, U. Braun & H.D. Shin, sp. nov. MycoBank MB564817. Fig. 12.

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Fig. 12.

Cercospora eucommiae (CPC 10047). A. Leaf spots on upper and lower leaf surface. B. Close-up of lesion. C-G. Fascicles with conidiophores and conidiogenous cells. H, I. Conidia. Scale bars = 10 μm.

Etymology: Name derived from Eucommia, the host on which it occurs.

Leaf spots amphigenous, irregular to subcircular, 2-5 mm diam; surface grey-brown to brown with diffuse border; reverse olivaceous-brown with diffuse border. Mycelium internal, hyaline, consisting of septate, branched, smooth, 2-3 μm diam hyphae. Caespituli fasciculate, pale brown, amphigenous, up to 40 μm diam and 50 μm high (conidial mass white on leaf surface). Conidiophores aggregated in loose fascicles arising from the upper cells of a weakly developed brown stroma, up to 30 μm diam and 20 μm high, conidiophores pale brown, smooth, 1-3-septate, subcylindrical, straight to variously curved, unbranched, 20-50 × 4-5 μm. Conidiogenous cells terminal, unbranched, pale brown, smooth, tapering to flat-tipped apical loci that are thickened, somewhat darkened, slightly refractive, 2 μm diam, 15-25 × 4-5 μm, proliferating sympodially at the apex. Conidia solitary, or in unbranched short chains, hyaline to pale olivaceous (with age), smooth, guttulate, obclavate, apex obtuse to subobtuse or clavate, base obconically subtruncate, straight to mildly curved, 3-8-septate, (35-)60-75(-80) × (4-)5-6(-8) μm; hila thickened along the rim, but not darkened or planate, 1.5-2 μm diam.

Culture characteristics: Colonies after 2 wk at 24 °C in the dark on MEA; erumpent, spreading, with sparse aerial mycelium, and smooth, lobate margins. Surface folded, dark mouse-grey with patches of dirty white; reverse fuscous black becoming greyish sepia at margin. Colonies reaching 12 mm diam.

Specimens examined: South Korea, Chuncheon, on Eucommia ulmoides, 7 Oct. 2003, H.D. Shin, holotype CBS H-20839, cultures ex-type CPC 10802 = CBS 131932, CPC 10803, 10804; Chuncheon, on E. ulmoides, 11 Oct. 2002, H.D. Shin, CBS H-20838, culture CPC 10047.

Notes: In the Korean material C. eucommiae occurred in mixed infections with a Pseudocercospora species (conidia 22-160 × 4-7 μm) that resembles P. eucommiae (conidia 15-75 × 2-4 um), which is known from this host in China (Guo & Hsieh 1995). The description of C. eucommiae reveals the genus Cercospora to be paraphyletic. Morphologically C. eucommiae is distinct from other species in Cercospora in that the conidial hila and conidiogenous scars are different (thickened along the rim, not darkened and planate), and conidia also tend to occur in unbranched chains, which is not typical of Cercospora. Interestingly, it does not cluster with C. eremochloae, which also forms conidia in chains (Crous et al. 2011a). Although this species is not part of Cercospora s. str., we name it in this genus until further taxa are collected and studied to resolve the status of this subclade in relation to Cercospora s. str.

Miuraea Hara, Byochugai-Hoten (Manual of Pests and Diseases): 779. 1948.

Synonyms: See Braun (1995).

Foliicolous, phytopathogenic, causing leaf spots. Mycelium internal and external, consisting of septate, branched, hyaline to subhyaline hyphae. Conidiophores semi-macronematous, mononematous, reduced to a single conidiogenous cell, integrated on hyphae, with small lateral peg-like protuberances; conidiogenesis holoblastic, monoblastic, determinate, occasionally polyblastic, proliferation sympodial or percurrent; conidiogenous loci more or less truncate, inconspicuous, unthickened, not darkened. Conidia solitary, ellipsoid-ovoid, subcylindrical-vermiform, obclavate, subclavate, somewhat asymmetrical, euseptate, transversely pluriseptate to muriformly septate, hyaline to faintly pigmented, thin-walled; hila truncate to somewhat convex, unthickened, not darkened (adapted from Braun 1995).

Type species: Miuraea degenerans (Syd. & P. Syd.) Hara, Byochugai-Hoten (Manual of Pests and Diseases): 260. 1948.

Notes: Morphologically Miuraea is intermediate between Pseudocercospora and Pseudocercosporella, which explains its phylogenetic position in this clade (Fig. 4). It differs from Pseudocercosporella in having superficial mycelium, and very broad, muriformly septate conidia.

Miuraea persicae (Sacc.) Hara, Byochugai-Hoten (Manual of pests and diseases): 224. 1948. Fig. 13.

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Fig. 13.

Miuraea persicae (CPC 10069). A. Leaf spots on upper and lower leaf surface. B, C. Close-up of fruiting (rather inconspicuous). D, E. Fascicles with conidiophores and conidiogenous cells. F. Conidia (note septation). Scale bars = 10 μm.

Basionym: Cercospora persicae Sacc., Hedwigia 15: 119. 1876.

Teleomorph: “Mycosphaerella” pruni-persicae Deighton, Trans. Brit. Mycol. Soc. 50: 328. 1967.

Specimens examined: South Korea, Chuncheon, Prunus persica, 11 Oct. 2002, H.D. Shin, CBS H-20841, culture CPC 10069; Chuncheon, 7 Oct. 2003, P. armeniaca, H.D. Shin, CBS H-20840, CPC 10828-10830.

Phloeospora Wallr., Flora Cryptogamica Germaniae 2: 176. 1833.

Phytopathogenic, commonly associated with leaf spots, occurring on leaves and fruit. Mycelium immersed, consisting of hyaline, septate, branched hyphae. Conidiomata acervular, subepidermal, erumpent; wall of thin-walled textura angularis, opening by means of an irregular split. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, cylindrical, discrete, indeterminate, proliferating via percurrent proliferations, or sympodially, formed from the upper cells of the acervulus. Conidia solitary, hyaline, smooth, septate, cylindrical, apex subobtuse to obtuse, base truncate, straight to curved.

Type species: Phloeospora ulmi (Fr.) Wallr., Flora Cryptogamica Germaniae 2: 177. 1833.

Specimens examined: Austria, Ulmus sp., H.A. van der Aa, CBS 613.81; Ulmus glabra, G. Verkley, CBS 344.97. Netherlands, Ulmus sp., H.A. van der Aa, CBS 101564.

Notes: Phloeospora is distinguished from Septoria by the production of conidia in acervuli, whereas conidiomata in the latter genus are pycnidial. Both genera are known to be polyphyletic (Verkley & Priest 2000, Quaedvlieg et al. 2011) and require further revision.

Pseudocercosporella Deighton, Mycol. Pap. 133: 38. 1973.

Foliicolous, phytopathogenic, causing discrete leaf spots. Mycelium mostly consistently internal, in some species with internal as well as external hyphae, hyaline to pale brown, septate, branched, smooth or almost so; stromata lacking or weakly to well-developed, substomatal to intraepidermal, usually colourless. Conidiophores solitary to fasciculate, emerging through stomata or erumpent through the cuticle, arising from inner hyphae or from stromata, sometimes formed as lateral branches of superficial hyphae, or aggregated in crustose to subglobose sporodochia; conidiophores simple, rarely branched, straight and subcylindrical to geniculate-sinuous, hyaline, occasionally faintly pigmented at the base, rarely throughout, one-celled or septate. Conidiogenous cells integrated, terminal, or reduced to conidiogenous cells, mono- to polyblastic, sympodial; conidiogenous loci inconspicuous, unthickened, neither darkened nor conspicuously refractive. Conidia formed singly, rarely in simple or branched chains, subcylindrical, filiform, somewhat obclavate, 1-multi-euseptate, hyaline, thin-walled, mostly smooth, apex obtuse to subacute, base subtruncate, hilum unthickened, neither darkened, nor refractive (adapted from Braun 1995).

Type species: Pseudocercosporella ipomoeae Deighton, Mycol. Pap. 133: 39. 1973. [= P. bakeri (Syd. & P. Syd.) Deighton, Mycol. Pap. 133: 41. 1973].

Note: Pseudocercosporella is polyphyletic (see Frank et al. 2010, Crous et al. 2011b) and new taxonomically useful morphological features will need to be determined to delineate all the genera presently accommodated in this clade.

Pseudocercosporella arcuata S.K. Singh, P.N. Singh & Bhalla, Mycol. Res. 101: 542. 1997. Fig. 14.

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Fig. 14.

Pseudocercosporella arcuata (CPC 10050). A. Leaf spots on upper and lower leaf surface. B-D. Close-up of lesions. E, F. Fascicles with conidiophores and conidiogenous cells. G. Conidia. Scale bars = 10 μm.

Specimen examined: South Korea, Chuncheon, on Rubus oldhamii (≡ R. pungens var. oldhamii), 11 Oct. 2002, H.D. Shin, CBS H-20842, culture CPC 10050.

Pseudocercosporella capsellae (Ellis & Everh.) Deighton, Mycol. Pap. 133: 42. 1973.

Basionym: Cylindrosporium capsellae Ellis & Everh., J. Mycol. 3(11): 130. 1887.

Additional synonyms in Braun (1995).

Teleomorph: “Mycosphaerella” capsellae A.J. Ingman & Sivan., Mycol. Res. 95: 1339. 1991.

Specimen examined: South Korea, Namyangju, Raphanus sativus, 22 Oct. 2007, H.D. Shin, CBS H-20843, cultures CPC 14773 = CBS 131896.

Pseudocercosporella chaenomelis (Y. Suto) C. Nakash., Crous, U. Braun & H.D. Shin, comb. nov. MycoBank MB564818. Fig. 15.

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Fig. 15.

Pseudocercosporella chaenomelis (CPC 14795). A. Leaf spots on upper and lower leaf surface. B. Close-up of leaf spot with white fruiting (rather inconspicuous). C-F. Fascicles with conidiophores and conidiogenous cells. G. Conidia. Scale bars = 10 μm.

Basionym: Cercosporella chaenomelis Y. Suto, Mycoscience 40: 513. 1999.

• = Mycosphaerella chaenomelis Y. Suto, Mycoscience 40: 513. 1999.

Leaf spots amphigenous, irregular to angular, 5-20 mm diam, brown, delimited by leaf veins. Mycelium internal, hyaline, consisting of septate, branched, smooth, 1.5-2 μm diam hyphae. Caespituli fasciculate to sporodochial, white, predominantly epiphyllous, up to 200 μm diam and 120 μm high. Conidiophores aggregated in dense fascicles, arising from the upper cells of a hyaline stroma, up to 180 μm diam and 100 μm high; conidiophores hyaline, smooth, subcylindrical to ampulliform, straight to variously curved, unbranched, reduced to conidiogenous cells, 5-12 × 3-4 μm, proliferating sympodially at the apex. Conidia solitary, hyaline, smooth, guttulate to granular, subcylindrical to obclavate, apex subobtuse, base obconically truncate, straight to variously curved, 1-4-septate, (10-)30-38(-50) × (2-)2.5-3(-4) μm; hila unthickened, not darkened nor refractive, 1.5-2 μm diam; undergoing microcyclic conidiation on the host. Description based on CPC 14795.

Culture characteristics: Colonies after 1 mo at 24 °C in the dark on MEA; Colonies erumpent, spreading, with aerial mycelium sparse to absent, margins smooth, lobate. Surface irregularly folded, with a prominent network of ridges; folds appearing cinnamon, with surrounding areas and border brown-vinaceous; reverse sepia to chestnut, reaching up to 35 mm diam.

Specimens examined: Japan, Shimane Pref., Matsue, on leaves of Chaenomeles sinensis, Y. Suto, 6 Nov. 1983, holotype SFH-917, in Herbarium of SPFRC; Mie Pref., Tsu, on leaves of C. sinensis, C. Nakashima, 29 Oct. 2011, epitype designated here TFM: FPH-8101, culture ex-epitype MUCC 1510 = CBS 132131. South Korea, Kimhae, C. speciosa (= C. lagenaria), 14 Nov. 2007, H.D. Shin, CBS H-20844, culture CPC 14795 = CBS 131897.

Notes: Suto (1999) established the connection between Pseudocercosporella chaenomelis (as Cercosporella) and Mycosphaerella chaenomelis, which is the cause of a serious leaf spot disease referred to as frosty mildew on Chaenomeles sinensis in Japan. The fungus was found to overwinter by means of ascomata on fallen leaves, which provided the primary inoculum for new infections (April to June). Since the disease was previously known in Japan to be caused by a species of Cercosporella, Suto (1999) chose the latter genus to accommodate the anamorph. The hyaline conidia with unthickened conidial hila indicate that the fungus is better placed in Pseudocercosporella, and hence a new combination is proposed. Based on DNA sequence data from the ITS and ACT gene regions, strains from Japan and Korea appear identical (unpubl. data).

Pseudocercosporella chaenomelis occurs in mixed infections with Pseudocercospora cydoniae. Pseudocercosporella chaenomelis is morphologically comparable only with Ps. gei, known on Geum spp. in North America and the Far East of Russia (Braun 1995). The latter species differs in having smaller stromata (20-45 μm diam) and much longer filiform-acicular conidia, 20-120 × 1-3(-4) μm (Braun 1995). Pseudocercosporella crataegi on Crataegus spp. in North America is distinct, forming superficial hyphae with solitary conidiophores, and its much smaller stromata and much longer conidia, and Ps. potentillae on Potentilla sp. in Russia also differs by having very long conidia (Braun 1995).

Pseudocercosporella koreana Crous, U. Braun & H.D. Shin, sp. nov. MycoBank MB564819. Fig. 16.

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Fig. 16.

Pseudocercosporella koreana (CPC 11414). A. Leaf spots on upper and lower leaf surface. B. Close-up of leaf spot with white fruiting. C. Substomatal stroma. D-H. Fascicles with conidiophores and conidiogenous cells. I. Conidia. Scale bars = 10 μm.

Etymology: Name derived from the country where it was collected.

Leaf spots amphigenous, indistinct, irregular, chlorotic, up to 6 mm diam. Mycelium internal, hyaline, consisting of septate, branched, smooth, 1.5-2.5 μm diam hyphae. Caespituli fasciculate, white, amphigenous, up to 60 μm diam and 90 μm high. Conidiophores aggregated in dense fascicles, on the upper cells of a pale brown to hyaline, usually substomatal stroma, up to 45 μm diam and 20 μm high; conidiophores hyaline or pale brown at the base, smooth, 0-2-septate, but frequently reduced to conidiogenous cells, subcylindrical, straight to variously curved or geniculate-sinuous, unbranched or branched below, 15-25 × 4-5 μm, proliferating sympodially at the apex. Conidia solitary, hyaline, smooth, prominently guttulate, narrowly obclavate, apex obtuse to subobtuse, base obconically subtruncate, straight to variously curved, 3-13-septate, (40-)60-80(-130) × (2.5-)3(-4) μm; hila unthickened, neither darkened nor refractive, 2 μm diam.

Culture characteristics: Colonies after 2 wk at 24 °C in the dark on MEA; surface folded with a prominent network of ridges, erumpent, spreading, with sparse aerial mycelium, and smooth, lobate margins. Surface olivaceous-grey to iron-grey; reverse iron-grey to greenish black. Colonies reaching 6 mm diam.

Specimen examined: South Korea, Hoengseong, on Vicia amurensis, 4 Aug. 2004, H.D. Shin, holotype CBS H-20845, isotype HAL 1850 F, culture ex-holotype CPC 11414.

Notes: Braun (1995) listed several species of Pseudocercosporella on Fabaceae. None of these occur on Vicia, and only one, Ps. tephrosiae (on Tephrosia, Africa), has conidia of similar length (40-110 × 3-4.5 μm), although they are wider, subcylindrical-acicular, and have 3-6 septa.

Pseudocercosporella oxalidis (Goh & W.H. Hsieh) U. Braun, Nova Hedwigia 55: 218. 1992.

Basionym: Pseudocercospora oxalidis Goh & W.H. Hsieh, Bot. Bull. Acad. Sinica 30: 127. 1989.

Specimen examined: Taiwan, Taipei, Wulai, on living leaves of Oxalis debilis (= O. corymbosa), R. Kirschner, 2258, 22 Feb. 2005, culture CBS 118758.

Septoria Sacc., Syll. Fung. 3: 474. 1884.

Synonyms: See Sutton (1980).

Phytopathogenic and endophytic, occurring on leaves, fruit and stems, causing discrete lesions. Conidiomata pycnidial, immersed, separate or aggregated, globose, papillate or not, brown, with a thin wall of brown textura angularis. Ostiole single, circular, central, sometimes papillate. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, ampulliform, doliiform or lageniform to short cylindrical, holoblastic, determinate or indeterminate, proliferating sympodially and/or percurrently; conidiogenous loci unthickened. Conidia solitary, hyaline, multiseptate, guttulate or not, thin-walled, filiform, smooth, continuous or constricted at the septa; hila unthickened.

Type species: Septoria cytisi Desm. Ann. Sci. Nat., Bot., Sér. 3, 8: 24. 1847.

Note: Septoria is polyphyletic (Quaedvlieg et al. 2011).

Clade 11: Sonderhenia

Sonderhenia H.J. Swart & J. Walker, Trans. Brit. Mycol. Soc. 90: 640. 1988.

Foliicolous, phytopathogenic, causing discrete leaf spots. Leaf spots amphigenous, round to confluent and irregular, surrounded by a purple border when young, which becomes dark red to brown and raised with age. Ascomata pseudothecial, amphigenous, on one side of each lesion, often 1-3, intermingled with conidiomata, immersed, black, punctiform, globose to subglobose; apical ostiole substomatal; wall olive-brown, of 3-4 layers of textura angularis, subhymenium of 1-2 layers of colorless cells. Asci fasciculate, bitunicate, subsessile, 8-spored, ovoid to obclavate, straight to incurved. Ascospores 2-3-seriate, hyaline, guttulate, straight or slightly curved, fusiform, 1-septate, widest just above median septum, slightly constricted at septum. Conidiomata pycnidial, amphigenous, subepidermal with central non-projecting ostiole, scattered, black, globose; wall of 2-3 layers of brown cells. Conidiogenous cells minute, olivaceous, proliferating enteroblastically and percurrently, lining the inner pycnidial wall layer. Conidia ellipsoid to cylindrical or ovoid, straight or bent, brown, 3-distoseptate, not constricted, verruculose, apex obtuse, base truncate with marginal frill (adapted from Crous 1998).

Type species: Sonderhenia eucalyptorum H.J. Swart & J. Walker, Trans. Brit. Mycol. Soc. 90: 640. 1988.

Notes: Sonderhenia includes taxa with mycosphaerella-like teleomorphs and pycnidial anamorphs that form brown, transversely distoseptate conidia on brown, percurrently proliferating conidiogenous cells. Only two species, S. eucalypticola and S. eucalyptorum are known.

Clade 12: Pallidocercospora, Scolecostigmina, Trochophora and pseudocercospora-like

Pallidocercospora Crous, gen. nov. MycoBank MB564820. Fig. 17.

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Fig. 17.

Pallidocercospora heimii (CPC 1395). Asci, ascospores, germinating ascospores (after 24 h on malt extract agar), hyphae with conidiogenous loci, and conidia. Scale bar = 10 μm.

Etymology: The name reflects the pale brown cercospora-like conidia in this genus.

Foliicolous, phytopathogenic, causing discrete leaf spots. Ascomata single, black, immersed, globose, glabrous; wall of 3-4 layers of medium brown textura angularis. Asci fasciculate, bitunicate, aparaphysate, subsessile, 8-spored, ellipsoid to obclavate or cylindrical, straight or curved, numerous. Ascospores 2-multi-seriate, oblique, overlapping, straight ellipsoidal to obovoid, colourless, smooth, 1-septate. Mycelium predominantly immersed, consisting of olivaceous-brown hyphae, smooth, branched, septate, 2-4 μm diam. Conidiophores in vivo fasciculate, or occurring singly on superficial mycelium as lateral projections, unbranched or branched, septate, cylindrical, straight to geniculate-sinuous, olivaceous-brown. Conidiogenous cells integrated, terminal, cylindrical, straight to geniculate-sinuous, olivaceous-brown, proliferating sympodially or percurrently; conidiogenous loci unthickened, not darker than the surrounding conidiogenous cell. Conidia solitary, straight to irregularly curved, guttulate, pale olivaceous to olivaceous-brown, subcylindrical to narrowly obclavate, multiseptate; hila neither thickened nor darkened.

Type species: Pallidocercospora heimii (Crous) Crous, comb. nov.

Notes: Species of Pallidocercospora have pale olivaceous, smooth conidia (generally referred to as the Mycosphaerella heimii complex; Crous et al. 2004c), and form red crystals when cultivated in agar (on WA, SNA, PDA, MEA), which distinguishes them from Pseudocercospora. Pseudocercospora has several synonyms (see Seifert et al. 2011). Cercoseptoria with its mostly acicular conidia, was correctly treated as synonym of Pseudocercospora by Deighton (1976). Other synonyms include Ancylospora Sawada (based on A. costi), now treated as P. costina; Cercocladospora G.P. Agarwal & S.M. Singh (based on C. adinae, nom. non rite publ.), now treated as P. adinicola; and Helicominia L.S. Olive (based on H. caperonia), now P. caperoniae, and Pantospora Cif. (based on P. guazumae) (see Ellis 1971, Deighton 1976), the muriformly septate conidia of the latter are similar to those of Pseudocercospora pseudostigmina-platani, though Pantospora has been shown to be a genus in its own right (Minnis et al. 2011).

Pallidocercospora acaciigena (Crous & M.J. Wingf.) Crous & M.J. Wingf., comb. nov. MycoBank MB564821.

Basionym: Pseudocercospora acaciigena Crous & M.J. Wingf., Stud. Mycol. 50: 464. 2004.

Teleomorph: “Mycosphaerella” acaciigena Crous & M.J. Wingf., Stud. Mycol. 50: 463. 2004.

Specimen examined: Venezuela, Acarigua, on leaves of Acacia mangium, May 2000, M.J. Wingfield, CBS H-9873, holotype of M. acaciigena and P. acaciigena; cultures ex-type CBS 115432, 112515, 112516 = CPC 3836-3838.

Pallidocercospora crystallina (Crous & M.J. Wingf.) Crous & M.J. Wingf., comb. nov. MycoBank MB564822.

Basionym: Pseudocercospora crystallina Crous & M.J. Wingf., Mycologia 88: 451. 1996.

Teleomorph: “Mycosphaerella” crystallina Crous & M.J. Wingf., Mycologia 88: 451. 1996.

Specimens examined: South Africa, Kwazula-Natal Province,Umvoti, on leaves of Eucalyptus bicostata, Oct. 1994, M.J. Wingfield (holotypes PREM 51922, teleomorph; PREM 51923, anamorph, cultures ex-type CPC 800-802); Kwazula-Natal Province, leaf litter of E. grandis × camaldulensis, Jun. 1995, M.J. Wingfield (PREM 51937, cultures CPC 1178-1180).

Pallidocercospora heimii (Crous) Crous, comb. nov. MycoBank MB564823. Fig. 17.

Basionym: Pseudocercospora heimii Crous, S. African For. J. 172: 4. 1995.

Teleomorph: “Mycosphaerella” heimii Crous, S. African For. J. 172: 2. 1995.

• ≡ “Mycosphaerella” heimii Bouriquet, Encycl. Mycol. 12: 418. 1946, nom. nud.

Specimens examined: Madagascar, Moramanga, on leaves of Eucalyptus sp., Apr. 1994, P.W. Crous, PREM 51749, holotype of teleomorph; PREM 51748, holotype of anamorph, cultures ex-type CPC 760-761 = CBS 110682.

Pallidocercospora heimioides (Crous & M.J. Wingf.) Crous & M.J. Wingf., comb. nov. MycoBank MB564824.

Basionym: Pseudocercospora heimioides Crous & M.J. Wingf., Can. J. Bot. 75: 787. 1997.

Teleomorph: “Mycosphaerella” heimioides Crous & M.J. Wingf., Can. J. Bot. 75: 787. 1997.

Specimens examined: Indonesia, N. Sumatra, Lake Toba area, leaves of Eucalyptus sp., Mar. 1996, M.J. Wingfield, holotype of teleomorph PREM 54966; holotype of anamorph PREM 54967; cultures ex-type CPC 1311, 1312 = CBS 111190).

Pallidocercospora holualoana (Crous, Joanne E. Taylor & M.E. Palm) Crous, comb. nov. MycoBank MB564825.

Basionym: “Mycosphaerella” holualoana Crous, Joanne E. Taylor & M.E. Palm, Mycotaxon 78: 458. 2001.

Specimen examined: USA, Hawaii, Kona district, Holualoa, on a living leaf of Leucospermum sp., P.W. Crous & M.E. Palm, 17 Nov. 1998, holotype PREM 56926, cultures ex-type CPC 2126-2128).

Pallidocercospora irregulariramosa (Crous & M.J. Wingf.) Crous & M.J. Wingf., comb. nov. MycoBank MB564826.

Basionym: Pseudocercospora irregulariramosa Crous & M.J. Wingf., Can. J. Bot. 75: 785. 1997.

Teleomorph: “Mycosphaerella” irregulariramosa Crous & M.J. Wingf., Can. J. Bot. 75: 785. 1997.

Specimens examined: South Africa, Northern Province, Tzaneen, on leaves of Eucalyptus saligna, Mar. 1996, M.J. Wingfield, holotype of teleomorph PREM 54964; holotype of anamorph PREM 54965; cultures ex-type CPC 1360 = CBS 114777).

Pallidocercospora konae (Crous, Joanne E. Taylor & M.E. Palm) Crous, comb. nov. MycoBank MB564827.

Basionym: “Mycosphaerella” konae Crous, Joanne E. Taylor & M.E. Palm, Mycotaxon 78: 459. 2001.

Specimen examined: USA, Hawaii, Kona district, Holualoa, on a living leaf on Leucadendron cv. Safari Sunset, 17 Nov. 1998, P.W. Crous & M.E. Palm, holotype PREM 56921; ex-type cultures CPC 2123-2125.

Scolecostigmina U. Braun, N. Z. J. Bot. 37: 323. 1999. Fig. 18.

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Fig. 18.

Scolecostigmina mangiferae (CBS 125467). A. Leaf spots on upper and lower leaf surface. B. Close-up of leaf spot with fruiting. C-E. Fascicles with conidiophores and conidiogenous cells (note rough percurrent proliferations). F. Conidia. Scale bars = 10 μm.

Foliicolous, phytopathogenic, associated with leaf spots. Mycelium immersed, consisting of septate, branched, pigmented hyphae. Sporodochia immersed to erumpent; stromata subglobose to applanate, composed of brown, angular to subglobose cells. Conidiophores numerous, densely aggregated, arising from stroma, subcylindrical or somewhat tapered towards the apex, occasionally ampulliform, continuous or septate, pigmented, wall somewhat thickened, usually verruculose; conidiogenous cells integrated, terminal or at times conidiophores reduced to conidiogenous cells, holoblastic, proliferating percurrently via conspicuous annellations. Conidia solitary, scolecosporous, usually subcylindrical-obclavate, transversely pluriseptate, occasionally with few longitudinal or oblique septa, euseptate, rarely with few intermixed distosepta, thick-walled, pigmented, dark, smooth to verrucose, apex obtuse to subacute, base truncate or obconically truncate; secession schizolytic (adapted from Braun et al. 1999).

Type species: Scolecostigmina mangiferae (Koord.) U. Braun & Mouch., N. Z. J. Bot. 37: 323. 1999.

Specimen examined: Australia, Queensland, Mareeba, S16°58’75.5” E145°20’60.8”, leaves of Mangifera indica, 10 Aug. 2009, P.W. Crous & R.G. Shivas, CBS H-20846, culture CPC 17352, 17351 = CBS 125467.

Trochophora R.T. Moore, Mycologia 47: 90. 1955. Fig. 19.

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Fig. 19.

Trochophora simplex (CBS 124744). A. Leaf spots on upper and lower leaf surface. B, C. Close-up of leaf spot with fruiting. D-G. Fascicles with conidiophores and conidiogenous cells. H, I. Conidia. Scale bars = 10 μm.

Foliicolous, but pathogenicity unproven. Colonies hypophyllous, medium to dark brown, consisting of numerous synnemata. Stroma absent, but with a superficial network of hyphae linking the various synnemata. Conidiophores synnematous, mostly unbranched and straight, or with 1-2 short branches, straight or curved, cylindrical, individual conidiophores tightly aggregated, but separating near the apex, pale to medium brown, smooth. Conidiogenous cells polyblastic, integrated, terminal, determinate to sympodial, with visible unthickened scar, clavate. Conidia solitary, terminal or lateral on conidiogenous cells, prominently curved to helicoid, pale to medium brown, smooth, transversely euseptate with a darkened, thickened band at the septa (adapted from Crous et al. 2009a).

Type species: Trochophora simplex (Petch) R.T. Moore, Mycologia, 47: 90. 1955.

Specimens examined: Japan, Shimane, on Daphniphyllum teijsmannii, 26 April 2008, C. Nakashima & I. Araki, MUMH 11134, culture MUCC 952. South Korea, Jeju, Halla arboretum, on D. macropodum, 29 Oct. 2005, H.D. Shin, CBS H-20847, culture CBS 124744.

Notes: Other pseudocercospora-like species found in this clade are P. colombiensis (foliar pathogen of Eucalyptus; Crous 1998), and P. thailandica (foliar pathogen of Acacia; Crous et al. 2004d), both also having mycosphaerella-like teleomorphs. Morphologically, these taxa appear typical members of Pseudocercospora s. str. so it would be difficult to identify these as different from Pseudocercospora without the aid of DNA sequence comparisons.

Clade 13: Passalora-like

Notes: This clade is represented by Passalora eucalypti, which was originally described as a leaf spot pathogen of Eucalyptus saligna in Brazil (Crous 1998, Crous & Braun 2003). Recently, a second species was found to belong to this clade, namely Passalora leptophlebiae, which was described from Eucalyptus leptophlebia leaves collected in Brazil (Crous et al. 2011a). Both species are charaterised by fasciculate conidiophores and catenate, pale brown conidia, with thickened, darkened and refractive scars and hila.