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1 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, P.O. Box
85167, 3508 AD, Utrecht, The Netherlands
2 Department of Microbiology and Plant Pathology, Forestry and Agricultural
Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South
Africa
3 Estación Fitopatológica "Do Areeiro" Subida a la
Robleda S/N E-36153 Pontevedra, Spain
4 Departemento de Fitopatologia, Universidade Federal de Viçosa,
36.570 Viçosa, MG, Brazil
*
Correspondence: Pedro W. Crous,
crous{at}cbs.knaw.nl
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Taxonomic novelties: Colletogloeopsis stellenboschiana Crous sp. nov., Mycosphaerella davisoniellae Crous sp. nov. (anamorph Davisoniella eucalypti H.J. Swart), Mycosphaerella eucalyptorum Crous & M.J. Wingf. sp. nov. Mycosphaerella gamsii Crous sp. nov., Mycosphaerella perpendicularis Crous & M.J. Wingf. sp. nov., Mycosphaerella pluritubularis Crous & J.P. Mansilla sp. nov., Mycosphaerella pseudafricana Crous & T. Coutinho sp. nov., Mycosphaerella pseudocryptica Crous sp. nov. (anamorph Colletogloeopsis sp.), Mycosphaerella pseudoendophytica Crous & G. Hunter sp. nov. (anamorph Pseudocercosporella sp.), Mycosphaerella pseudosuberosa Crous & M.J. Wingf. sp. nov. (anamorph Trimmatostroma sp.), Mycosphaerella quasicercospora Crous & T. Coutinho sp. nov., Mycosphaerella scytalidii Crous & M.J. Wingf. sp. nov. (anamorph Stenella sp., synanamorph, Scytalidium-like.), Mycosphaerella secundaria Crous & A.C. Alfenas sp. nov., Mycosphaerella stramenti Crous & A.C. Alfenas sp. nov., Mycosphaerella stramenticola Crous & A.C. Alfenas sp. nov., Mycosphaerella sumatrensis Crous & M.J. Wingf. sp. nov., Mycosphaerella verrucosiafricana Crous & M.J. Wingf. sp. nov., Septoria eucalyptorum Crous sp. nov., Septoria provencialis Crous sp. nov., Stenella pseudoparkii Crous & M.J. Wingf. sp. nov. (teleomorph Mycosphaerella sp.), Stenella xenoparkii Crous & M.J. Wingf., sp. nov. (teleomorph Mycosphaerella sp.).
Keywords Ascomycetes / Colletogloeopsis / Davisoniella / DNA sequence comparisons / Mycosphaerella / Pseudocercospora / Pseudocercosporella / Scytalidium / Septoria / Stenella / systematics / Trimmatostroma
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Mycosphaerella leaf blotch has been associated with severe defoliation, shoot die-back, and even tree death. This damage has mostly been attributed to M. cryptica (Cooke) Hansf. and M. nubilosa (Cooke) Hansf. (Carnegie et al. 1994, Crous & Wingfield 1996, Wingfield et al. 1996, Cheah 1977, Dungey et al. 1997). In recent years, it has become apparent that there are many more species of Mycosphaerella Johanson occurring on eucalypts than previously realised. While some of these fungi cause serious disease problems, others cause minor leaf spots, rarely resulting in severe disease (Crous 1998, Crous et al. 2004b). Little is known regarding some of these less important species but some could become more important in genetically uniform plantations of susceptible clonal hybrids or where trees are exposed to conditions of stress.
The genus Mycosphaerella Johanson includes more than 2000 species names (Corlett 1991), and several thousand anamorphs that lack known teleomorphs (Crous & Braun 2003). Of these, 55 species from eucalypts were treated by Crous (1998) and several additional species have been described more recently (Carnegie & Keane 1998, Braun & Dick 2002, Maxwell et al. 2003, Crous et al. 2004b, Hunter et al. 2004). Species of Mycosphaerella are usually assumed to be host-specific, and presently there are little data available that can be used to refute this supposition. Although some taxa have been found to infect other, secondary hosts (Crous et al. 2004c, Groenewald et al. 2005), most seem to have narrow host ranges. Interestingly, where species have been reported to have wider host ranges within a plant family, e.g. as reported for Ramularia Unger anamorphs by Braun (1998), DNA-based techniques have clearly shown that in most cases these morphologically similar taxa are phylogenetically quite distinct (Crous & Groenewald, unpubl. data). Further confusion could result from species colonising atypical host tissue in an attempt to jump to an ideal host when this becomes available. Crous & Groenewald (2005) have referred to this unusual behavioural pattern as the "pogo stick hypothesis". In Mycosphaerella it has been observed to be true for teleomorph as well as anamorph states. When isolates of these fungi colonising atypical substrates are collected without proving their pathogenicity, incorrect conclusions pertaining to host range could arise.
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The present study presents the second in a series characterising the Mycosphaerella species occurring on eucalypts. A major aim of this study was to use comparisons of DNA sequence data to clarify as many as possible of the formerly published host and distribution records (Crous 1998). Furthermore, while previous descriptions focused on species associated with leaf spots, this study also includes species from eucalypt leaf litter.
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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DNA phylogeny
The protocol of Lee & Taylor
(1990) was used to isolate
genomic DNA from fungal mycelium, grown on MEA in Petri dishes. The primers
ITS1 and ITS4 (White et al.
1990) were used to amplify part of the nuclear rRNA operon
spanning the 3' end of the 18S rRNA gene, the first internal transcribed
spacer (ITS1), the 5.8S rRNA gene, the second ITS region and the 5' end of the
28S rRNA gene. The PCR reaction mixture and conditions were the same as those
used by Crous et al.
(2004b).
The ITS nucleotide sequences generated in this study were added to other sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov) and the alignment was assembled using Sequence Alignment Editor v. 2.0a11 (Rambaut 2002) with manual adjustments for visual improvement where necessary. Due to the size and the complexity of the original alignment, the sequences were split over four smaller alignments, each containing genetically similar sequences. The four datasets were each treated identically. Phylogenetic analyses of sequence data were done using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Phylogenetic analysis of the aligned ITS sequence data consisted of neighbour-joining analysis with the uncorrected ("p"), the Kimura 2-parameter and the HKY85 substitution model in PAUP. Alignment gaps were treated as missing data and all characters were unordered and of equal weight. When they were encountered, ties were broken randomly. Sequence data were deposited in GenBank (Table 1) and the alignments in TreeBASE.
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Taxonomy
Wherever possible, 30 measurements (x 1000 magnification) were made
of structures mounted in lactic acid, with the extremes of spore measurements
given in parentheses. Colony colours (surface and reverse) were assessed after
1 mo on MEA, oatmeal agar (OA) and potato-dextrose agar (PDA)
(Gams et al. 1998) at
25 °C in the dark, using the colour charts of Rayner
(1970). All cultures obtained
in this study are maintained in the culture collection of the Centraalbureau
voor Schimmelcultures (CBS) in Utrecht, the Netherlands
(Table 1). Nomenclatural
novelties and descriptions were deposited in MycoBank
<www.mycoBank.org>.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The second alignment (Fig. 2) contains 90 taxa (including the two outgroups) and 535 characters including alignment gaps. Of these characters, 246 are parsimony-informative, 51 are variable and parsimony-uninformative, and 238 are constant. Neighbour-joining analysis using the three substitution models yielded trees with identical topologies and similar bootstrap values. Parsimony analysis yielded 481 most parsimonious trees (TL = 862 steps; CI = 0.613; RI = 0.927; RC = 0.568). The topology of the distance trees differed from the trees obtained using parsimony only in the placement of the Mycosphaerella sp. CPC 11171 clade (data not shown). The second alignment and derived tree mainly includes the M. marksii Carnegie & Keane complex (100 % bootstrap support), the M. heimii Crous complex (60 % bootstrap support), the M. walkeri R.F. Park & Keane (100 % bootstrap support) and the M. parva R.F. Park & Keane complex (100 % bootstrap support). Five new species of Mycosphaerella are indicated in the tree.
The third alignment (Fig. 3) contains 71 taxa (including the two outgroups) and 529 characters including alignment gaps. Of these characters, 237 are parsimony-informative, 71 are variable and parsimony-uninformative, and 221 are constant. Neighbour-joining analysis using the three substitution models yielded trees with identical topologies and similar bootstrap values. Parsimony analysis yielded 4319 most parsimonious trees (TL = 853 steps; CI = 0.626; RI = 0.856; RC = 0.536). The topology of the distance trees differed from the trees obtained using parsimony mainly at the deeper nodes (data not shown). The third alignment and derived tree mainly includes species of Pseudocercospora Speg., Cercospora Fresen., Septoria Sacc. and Stenella Syd. New species indicated in the tree include three in Mycosphaerella, two in Septoria, and two in Stenella.
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Taxonomy
Several collections represented Mycosphaerella spp.
morphologically and phylogenetically distinct from ex-type strains of the
morphological species to which they had originally been assigned. These fungi
are described as new taxa as follows:
Colletogloeopsis stellenboschiana Crous, sp. nov. MycoBank MB500833. Fig. 5.
Etymology: Refers to Stellenbosch, where the fungus was collected.
Coniothyrio ovato similis sed conidiis minoribus, (6.5–)7–9(–10) x (33.5(–4) µm, distincta.
Leaf spots amphigenous, circular to subcircular, 0.5–3 mm diam, pale brown, with a raised border and red-purple margin. Conidiomata amphigenous, pycnidial, medium brown, globose, 80–120 µm diam; wall of 3–4 layers of brown textura angularis. Conidiogenous cells discrete, ampulliform to subcylindrical, pale to medium brown, finely verruculose, proliferating 1–3 times percurrently near the apex, 3–6 x 3–4 µm. Conidia holoblastic, solitary, aseptate, ellipsoidal, with subobtuse apex and subtruncate base with minute marginal frill, medium brown, finely verruculose, widest below the middle, (6.5–)7–9(–10) x (3–)3.5(–4) µm.
Holotype: South Africa, Western Cape Province, Stellenbosch Mountain, on leaves of Eucalyptus sp., 4 Dec. 2004, P.W. Crous, CBS H-19688, holotype, culture ex-type CBS 116428 = CPC 10886.
Cultures: Colonies after 3 wk on MEA 15–40 mm diam; on PDA erumpent, spreading, producing copious amounts of slime, olivaceous-black at the centre, aerial mycelium olivaceous-grey, with a vinaceous-grey outer zone and wide olivaceous-black margin that is smooth but uneven; reverse olivaceous-black; on OA surface smoke-grey with a wide, grey-olivaceous border, forming a characteristic yellow pigment; on MEA grey-white on surface, with sectors of smoke-grey; margin thin, submerged, smoke-grey; reverse olivaceous-black; aerial mycelium sparse to moderate, grey-white; colonies fertile.
Host: Eucalyptus sp.
Distribution: South Africa.
Notes: Numerous species of Coniothyrium Corda and several species of Colletogloeopsis cause spots on eucalypt leaves. Colletogloeopsis stellenboschiana is easily distinguished from the taxa occurring on eucalypt leaves (Crous 1998), and from representatives of the "Coniothyrium ovatum" species complex specifically, based on its conidial morphology. Phylogenetically it is closely related to members of the the Colletogloeopsis complex that cause stem cankers on eucalypt trees (Cortinas et al. 2006 – this volume)
Mycosphaerella davisoniellae Crous, sp. nov. MycoBank MB500834. Fig. 6.
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Asci subcylindrici, subsessiles, 50–70 x 9–12 µm. Ascosporae bi-vel triseriatae, tenuitunicatae, rectae, obovoideae, apicem versus latissimae, in medio uniseptatae, vix vel haud constrictae ad septum, 10–14 x 3–4 µm.
Leaf spots amphigenous, subcircular to irregular, 1–7 mm diam, discrete to confluent, medium brown, surrounded by raised, red-purple margin. Ascomata hypophyllous, embedded in a raised, black, subepidermal stroma, ostiolate, becoming erumpent up to 120 µm diam. Asci subcylindrical, subsessile, straight or slightly incurved, 8-spored, 50–70 x 9–12 µm. Ascospores bi- to triseriate, overlapping, hyaline, thin-walled, straight, obovoid with rounded ends, widest near the apex, medianly 1-septate, not to slightly constricted at the septum, tapering toward both ends, but more prominently toward the base, 10–14 x 3–4 µm. Conidiomata of Davisoniella embedded in the same black subepidermal stroma that contains ascomata, subepidermal, ostiolate, up to 450 µm diam; wall of 2–3 layers of brown textura angularis. Conidiogenous cells subcylindrical to ampulliform or doliiform, 5–15 x 3–4 µm, medium brown, verruculose, proliferating several times percurrently near the apex. Conidia solitary, brown, aseptate, verruculose, thick-walled, oval with an obtuse apex and a truncate to subtruncate base with a prominent basal frill, which can extend up to 2 µm from the brown basal rim of the conidium, (8–)10–12(–14) x 4.5–)5–6(–6.5) µm (av. 11 x 5.5 µm). Synanamorph: Conidiomata intermingled between that of D. eucalypti and ascomata of M. davisoniellae. Conidiogenous cells phialidic, hyaline, subcylindrical to ampulliform, with visible periclinal thickening, 8–15 x 2.5–3.5 µm. Conidia hyaline, curved, subcylindrical, widest in the middle, apex bluntly rounded, obtuse, base truncate, 17–30 x 2–1.5 µm.
In vivo: No cultures available.
Specimen examined: Australia, Darling Ranges W.A., Mundlimup Block, on leaves of Eucalyptus marginata, 24 Nov. 1981, F. Tay, DAR 58999, holotype of D. eucalypti and M. davisoniellae.
Notes: Swart (1988) reported that this fungus is associated with abundant leaf spots on saplings and the foliage of recently felled trees. Conidiomata of D. eucalypti were described as unilocular and subepidermal, occurring in a stroma which could result in some of them appearing as multilocular. Swart (1988) considered the fungus to be the stromatic counterpart of Coniothyrium. Davisoniella eucalypti is clearly related to species in the Colletogloeopsis complex that occurs on eucalypts, having characteristic aseptate, brown, verruculose conidia that arise from percurrently proliferating conidiogenous cells. Davisoniella is unique by virtue of its stroma, that gives rise to the uni- or multilocular conidiomata. Conidia of D. eucalypti exude in slimy masses. In many cases, the exudates included aseptate, hyaline, curved, subcylindrical conidia of a synanamorph. The latter anamorph was produced from unilocular conidiomata that formed in the same stromata that gave rise to D. eucalypti. Surprisingly, many of the stromata investigated also contained ascomata of a Mycosphaerella species, which most likely also belong to the same fungus. The latter state is described here as M. davisoniellae.
Mycosphaerella eucalyptorum Crous & M.J. Wingf., sp. nov. MycoBank MB500835. Fig. 7.
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Mycosphaerellae parkii similis, sed ascosporis maioribus, 12–17 x 3.5–4.5 µm, modo B germinantibus, distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 2–20 mm diam, medium brown, with raised, brown borders, and thin, red-purple margins. Ascomata pseudothecial, amphigenous but predominantly epiphyllous, single, black, erumpent, globose, up to 120 µm diam; apical ostiole 10–15 µm diam, with prominent periphyses lining the ostiolar channel; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid, straight or slightly incurved, 8-spored, 35–50 x 8–12 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in middle of apical cell, not constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (12–)14–15(–17) x (3.5–)4(–4.5) µm in vivo; some ascospores with slightly asymmetrical apical cells, as commonly observed in M. marksii.
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Ascospore germination on MEA after 24 h: Type B. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, not distorting, becoming slightly constricted upon germination, becoming up to 4 µm diam.
Cultures: Colonies on MEA after 3 wk 25–30 mm diam; on MEA flat, spreading, folding, with sparse aerial mycelium, olivaceous-grey, margins smooth, regular, reverse iron-grey; on PDA slightly erumpent, centre olivaceous-grey; outer zone pale olivaceous-grey; reverse iron-grey; on OA with sparse to moderate pale olivaceous-grey aerial mycelium and patches of olivaceous-grey.
Host: Eucalyptus sp.
Distribution: Indonesia.
Notes: Conidia of a Stenella anamorph were found on some lesions. This link is, however, unconfirmed, and isolates did not produce anamorph structures in culture. Mycosphaerella eucalyptorum is phylogenetically closely related to a Mycosphaerella sp. from Colombia that forms Stenella pseudoparkii in culture. Ascospores of M. eucalyptorum (12–17 x 3.5–4.5 µm) germinate with a Type B germination pattern as observed in M. gracilis (10–20 x 2–3 µm) (Fig. 8) and M. marksii (11–22.5 x 2–3.5). It is easily distinguished from these taxa, however, based on its ascospore morphology and growth characteristics in culture (Crous 1998).
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Etymology: Named after the collector, well-known mycologist and friend, Prof. dr Walter Gams.
Mycosphaerellae stramenticolae similis, sed ascosporis minoribus, (8–)9–10 x (2–)3 µm, modo C germinantibus, distinguenda.
Leaf spots amphigenous, irregular, 1–20 mm diam, medium brown, with a raised, dark brown border. Ascomata pseudothecial, amphigenous, but predominantly hypophyllous, single, black, subepidermal, becoming erumpent, globose, up to 90 µm diam; apical ostiole 5–10 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight or slightly incurved, 8-spored, 25–35 x 7–9 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10 x (2–)3 µm in vivo.
Holotype: India, Palampur, on leaves of Eucalyptus sp., Mar. 2004, W. Gams & M. Arzanlou, CBS H-19690, holotype, culture ex-type CBS 118495 = CPC 11138–11140. 5/6-6
Ascospore germination on MEA after 24 h: Type C. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, but also variable in direction; becoming constricted upon germination, up to 5 µm diam.
Cultures: Colonies on MEA 28–35 mm diam after 3 wk; on MEA spreading, folding, flat, with moderate smoke-grey aerial mycelium in the centre; outer region olivaceous-grey; margins smooth, regular; reverse iron-grey; on PDA with moderate aerial mycelium, pale olivaceous-grey, outer region olivaceous-grey with drops of slime; reverse iron-grey; on OA with moderate aerial mycelium, pale olivaceous-grey, with patches of olivaceous-grey.
Host: Eucalyptus sp.
Distribution: India.
Notes: Mycosphaerella gamsii is phylogenetically closely related to M. stramenticola, but is distinguishable in having a Type C ascospore germination pattern, as is found in species such as M. heimii, M. gregaria, M. molleriana, M. nubilosa and M. walkeri. Mycosphaerella gamsii has ascospores that are 8–10 x 2–3 µm, thus shorter than those of the species listed above, and it also lacks an anamorph in culture.
Mycosphaerella perpendicularis Crous & M.J. Wingf., sp. nov. MycoBank MB500837. Fig. 10.
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Mycosphaerellae heimioide similis, sed ascosporis longioribus, (8–)9–10(–12) x (2.5–)3 µm, modo M germinantibus distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 5–15 mm diam, medium brown, frequently with a orange-red discoloration in the central part; border raised, dark brown. Ascomata pseudothecial, epiphyllous, single, black, subepidermal, globose, up to 90 µm diam; apical ostiole 10–15 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, slightly incurved, 8-spored, 25–35 x 7–8 µm. Ascospores multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–12) x (2.5–)3 µm in vivo.
Holotype: Colombia, Suiza, on leaves of Eucalyptus eurograndis, Jan. 2004, M.J. Wingfield, CBS H-19691, holotype, culture ex-type CBS 118367 = CPC 10983–10985.
Ascospore germination on MEA after 24 h: Type M. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes 90° to the long axis of the spore, and distorting upon germination, becoming up to 5 µm wide.
Cultures: Colonies on MEA reaching 28–37 mm diam after 3 wk; colonies folding, spreading, flat, with sparse aerial mycelium, which is olivaceous-grey on the agar surface, and with smoke-grey aerial mycelium; margins are smooth, regular; reverse iron-grey at the centre, olivaceous-grey in the outer zone; on OA with moderate aerial mycelium, olivaceous-grey at centre, greenish black in outer zone; on PDA olivaceous-grey with some drops of slime, iron-grey in reverse.
Host: Eucalyptus eurograndis.
Distribution: Colombia.
Notes: Germinating ascospores of M. perpendicularis have a characteristic Type M germination pattern, similar to that of M. heimioides. Mycosphaerella perpendicularis can easily be distinguished from M. heimioides, however, by virtue of the fact that the ascospores distort at germination. In addition, the germ tubes of M. heimioides never quite reach 90° to the long axis of the spore, whereas those of M. perpendicularis are at right angles.
Mycosphaerella pluritubularis Crous & J.P. Mansilla, sp. nov. MycoBank MB500838. Figs 11, 12.
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Mycosphaerellae nubilosae similis, set ascosporis brevioribus, (8–)9–10(–11) x 3(–4) µm, saepe plures quam 2 tubos germinationis proferentibus, distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 5–15 mm diam, pale to medium brown, surrounded by a thin, raised, dark brown border. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 100 µm diam; apical ostiole 10–15 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to subcylindrical, straight to slightly incurved, 8-spored, 30–45 x 7–10 µm. Ascospores multiseriate, overlapping, hyaline, prominently guttulate, thin-walled, straight, obovoid with subobtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) x 3(–4) µm in vivo.
Holotype: Spain, on leaves of E. globulus, Nov. 2004, J.P. Mansilla, CBS H-19692 holotype, culture ex-type CBS 118508 = CPC 11697).
Ascospore germination on MEA after 24 h: Type F. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, and distorting prominently upon germination, becoming up to 11 µm diam; frequently germinating with more than two germ tubes.
Cultures: Colonies after 3 wk 17–22 mm diam on MEA; on PDA colonies forming copious amounts of slime; surface olivaceous-black with patches of olivaceous-grey and pale olivaceous-grey; aerial mycelium sparse; margins feathery, uneven; reverse iron-grey; on OA surface smoke-grey with patches of olivaceous-grey; on MEA with sparse aerial mycelium, colonies erumpent, iron-grey, margins feathery, irregular; reverse olivaceous-black; colonies sterile.
Host: E. globulus.
Distribution: Spain.
Notes: Mycosphaerella pluritubularis is characterised by its distinct ascospore germination pattern (Type F), but where ascospores form more than two germ tubes, thus distinguishing it from other species like M. nubilosa that have more typical type F germination patterns.
Mycosphaerella pseudafricana Crous & T. Coutinho, sp. nov. MycoBank MB500839. Fig. 13.
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Mycosphaerellae africanae similis, sed ascosporis maioribus, 8–11 x 2.5–3 µm, distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 2–7 mm diam, medium brown, surrounded by a thin, raised, concolorous border. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 120 µm diam; apical ostiole 10–15 µm diam; wall of 2–3 cell layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, slightly incurved, 8-spored, 35–45 x 7–9 µm. Ascospores tri- to multiseriate, overlapping, hyaline to pale brown, guttulate, thin-walled, straight to slightly curved, smooth to finely roughened, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) x (2.5–)3 µm in vivo. Spermatogonia similar to the ascomata in morphology. Spermatia hyaline, smooth, rod-shaped with bluntly rounded ends, 3–4 x 1–1.5 µm.
Holotype: Zambia, on leaves of E. globulus, Aug. 1995, T. Coutinho, PREM 54973 holotype, culture ex-type CBS 114782 = CPC 1230; 1229–1231.
Ascospore germination on MEA after 24 h: Type G. Ascospores darkening and becoming verruculose on MEA; germinating from both ends as observed in M. africana, with germ tubes irregular to the long axis of the spore, and distorting prominently upon germination, becoming up to 8 µm wide.
Cultures: Colonies reaching 12–17 mm diam after 3 wk on MEA; colonies erumpent, irregular, surface iron-grey with olivaceous-grey, sparse aerial mycelium in central part; margins catenate, smooth; reverse greenish black; on PDA colonies erumpent, olivaceous-black with sparse olivaceous-grey aerial mycelium in the central part, margins smooth, catenate; reverse greenish black; on OA olivaceous-grey with smooth, catenate margins and green-olivaceous central part.
Host: E. globulus.
Distribution: Zambia.
Notes: Ascospores of M. pseudafricana (8–11 x 2.5–3 µm) germinate with a Type G pattern similar to that observed in M. africana (7–11 x 2–3 µm). Ascospores of M. pseudafricana are more verrucose than those of M. africana, but both taxa have very similar ascospore dimensions and germination patterns. They do differ, in the symptoms with which they are associated. Lesions of M. pseudafricana are generally larger, and they lack the red-purple margin found in M. africana. The easiest means to distinguish these taxa from each other is to compare their growth in culture: colonies of M. africana are black, produce a brown pigment in MEA, and form clusters of chlamydospores, whereas cultures of M. pseudafricana also produce clusters of chlamydospores on MEA, but are iron-grey, and lack the diffuse brown pigment observed in colonies of M. africana.
Mycosphaerella pseudocryptica Crous, sp. nov. MycoBank MB500840. Figs 14, 15.
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Etymology: Morphologically similar to M. cryptica.
Mycosphaerellae crypticae similis, sed ascosporis minoribus, (11–)12–14(–15) x (3–)3.5(–4) µm, saepe utrinque germinantibus, distinguenda.
Leaf spots amphigenous, irregular to subcircular, 0.5–2 mm diam, pale brown, with a raised, red-brown margin. Ascomata pseudothecial, hypophyllous, arranged in dense clusters in pale brown areas next to the leaf spots associated with conidiomata of the anamorph, black, immersed, globose, up to 70 µm diam; apical ostiole 10–15 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, straight or slightly incurved, 8-spored, 35–45 x 9–11 µm. Ascospores multiseriate, overlapping, hyaline, granular, thin-walled, straight, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (11–)12–14(–15) x (3–)3.5(–4) µm, in vivo; frequently encased in an irregular mucous sheath. Mycelium internal, consisting of branched, septate, medium brown, smooth, 3–4 µm wide hyphae. Conidiomata intermixed among ascomata or separate, predominantly on the lower leaf surface, pycnidial, substromatal, up to 120 µm diam; wall of 3–4 layers of brown textura angularis. Conidiophores 0–1-septate, but mostly reduced to conidiogenous cells. Conidiogenous cells discrete, ampulliform to subcylindrical, medium brown, smooth to finely verruculose, proliferating 1–3 times percurrently near apex, but also intercalary and sympodially, 5–15 x 3–5 µm. Conidia holoblastic, solitary, aseptate, fusoid with obtuse to subobtuse apices and truncate bases, medium brown, finely verruculose, (10–)12–14(–17) x (3.5–)4(–6) µm; inconspicuous basal marginal frill present.
Holotype: New Zealand, Wellington Botanical Garden, on leaves of Eucalyptus sp., Mar. 2004, J.A. Stalpers, CBS H-19693, holotype, culture ex-type CBS 118504 = CPC 11267; 11267–11269 (teleomorph), CPC 11264–11266 (anamorph).
Ascospore germination on MEA after 24 h: Type A. Ascospores smooth, becoming olivaceous on MEA, germinating predominantly from both ends, with germ tubes at some angle to the long axis of the spore, and with a constriction at the ascospore septum; ascospores becoming up to 7 µm wide.
Cultures: Colonies slow growing, 3–8 mm diam after 3 wk on MEA; on MEA colonies erumpent, aerial mycelium sparse to absent, margins smooth, surface white-grey to smoke-grey, or with a reddish tinge in patches; reverse fuscous-black; on PDA erumpent, white to smoke-grey with patches of vinaceous-grey; reverse vinaceous-grey, with a diffuse red pigment visible in the agar, up to 2 cm from colony margins; on OA pale grey-olivaceous with a pale vinaceous grey pigment diffusing into the agar.
Host: Eucalyptus sp.
Distribution: New Zealand.
Notes: Ascospores of M. pseudocryptica germinate with a Type A pattern (as observed in M. cryptica), except that they tend to germinate from both ends. It is possible, therefore, that collections of M. pseudocryptica have in the past been confused with those of M. cryptica. Isolates also form a Colletogloeopsis anamorph in culture, which is similar to M. cryptica. Ascospores of M. pseudocryptica are 11–15 x 3–4 µm, and conidia 10–17 x 3.5–6 µm, while ascospores of M. cryptica are 9–17.5 x 2–5.5 µm, and conidia are 8.5–18 x 4–6 µm. Phylogenetically M. pseudocryptica is closely related to the M. molleriana complex (Fig. 1), and distinct from M. cryptica.
Mycosphaerella pseudoendophytica Crous & G. Hunter, sp. nov. MycoBank MB500841. Figs 16, 17. Anamorph: Pseudocercosporella sp.
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Mycosphaerellae endophyticae similis, sed ascosporis modo C germinantibus distinguenda.
Leaf spots amphigenous, irregular to subcircular or angular, 2–5 mm diam, brown, with a raised, dark brown margin. Ascomata pseudothecial, amphigenous, black, subepidermal, erumpent to superficial, globose, up to 120 µm diam; apical ostiole 5–10 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight or slightly incurved, 8-spored, 30–40 x 8–10 µm. Ascospores multiseriate, overlapping, hyaline, sparsely guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with obtuse ends, medianly 1-septate, widest in the middle of the apical cell, not to slightly constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)9–10(–11) x (2–)2.5–3 µm, in vivo. Mycelium internal, consisting of branched, septate, pale to medium brown, smooth, 3–4 µm wide hyphae. Conidiomata in vitro sporodochial, hyaline. Conidiogenous cells aggregated, unbranched or branched, hyaline, smooth, tapering to flat-tipped apical and lateral loci, proliferating sympodially, 8–15 x 2–3.5 µm. Conidia holoblastic, solitary, but frequently undergoing microcyclic conidiation, giving rise to one or several additional conidia, smooth, hyaline, obclavate, apex subobtuse, base long obconically subtruncate to truncate, irregularly curved, 0–3-septate, 12–40 x 1.5–2 µm; hila inconspicuous.
Holotype: South Africa, KwaZulu-Natal, Enon, Richmond, on leaves of E. nitens, 3 May 2000, G. Hunter, CBS H-19694, holotype, culture ex-type CBS 113288 = CMW 9098.
Ascospore germination on MEA after 24 h: Type C. Ascospores smooth, not darkening on MEA, germinating from both ends, with germ tubes parallel to the long axis of the spore, and with a constriction at the ascospore septum; ascospores becoming up to 3.5 µm wide.
Cultures: Similar to those of M. endophytica (Crous 1998).
Host: E. nitens.
Distribution: South Africa.
Notes: Mycosphaerella pseudoendophytica has been known to us for some time, but its formal description required a molecular comparison with ex-type strains of M. endophytica (which it resembles in anamorph morphology), and M. ellipsoidea (which it resembles in ascospore germination pattern). As can be seen here, M. pseudoendophytica (Fig. 4) is clearly a distinct species, sharing features of both of these taxa.
Mycosphaerella pseudosuberosa Crous & M.J. Wingf., sp. nov. MycoBank MB500842. Fig. 18. Anamorph: Trimmatostroma sp.
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Mycosphaerellae suberosae similis, sed ascosporis minoribus, (11–)12–14(–15) x (3–)3.5(–4) µm, distinguenda.
Leaf spots amphigenous, associated with brown, corky spots on leaf petioles. Ascomata pseudothecial, single to aggregated, black, immersed becoming erumpent, globose, up to 120 µm diam; apical ostiole 10–20 µm diam; wall of 3–6 layers of brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight or slightly incurved, 8-spored, 35–45 x 12–16 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thick-walled, straight to slightly curved, fusoid-ellipsoidal with obtuse ends, medianly 1-septate, widest at the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (11–)12–14(–15) x (3–)3.5(–4) µm in vivo; frequently surrounded by an irregular mucous sheath.
Holotype: Uruguay, on leaves and petioles of Eucalyptus sp., Apr. 2005, M.J. Wingfield, CBS H-19695, holotype, culture ex-type CBS 118911 = CPC 12085.
Ascospore germination on MEA after 24 h: Type H. Ascospores darkening and becoming verruculose on MEA, germinating from both ends, with germ tubes primarily parallel to the long axis of the spore, and distorting prominently upon germination, becoming up to 11 µm wide.
Cultures: Colonies extremely slow growing, erumpent, uneven, black; aerial mycelium absent; colonies powdery, producing a Trimmatostroma anamorph.
Host: Eucalyptus sp.
Distribution: Uruguay.
Notes: Mycosphaerella pseudosuberosa is morphologically similar, and phylogenetically closely related to M. suberosa. It can be distinguished by its ascospores that are slightly narrower (3–4 µm vs. 3–6 µm), having a mucous sheath, and germinating via two germ tubes (predominantly) that originate from the ends of the spore. Germinating spores exude mucus, and become pale brown and verruculose, which differs from the numerous germ tubes and dark brown ascospores observed in M. suberosa. Furthermore, cultures of M. suberosa are hard and resistant to being cut, while those of M. pseudosuberosa are powdery, producing a Trimmatostroma anamorph in culture. From the phylogenetic data available, it appears that there may be more species within the M. suberosa complex awaiting description (Fig. 1).
Mycosphaerella quasicercospora Crous & T. Coutinho, sp. nov. MycoBank MB500843. Fig. 19.
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Mycosphaerellae nubilosae similis, sed ascosporis brevioribus, 10–14 x 3–4 µm, distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 2–10 mm diam, pale brown, surrounded by a thin, raised, dark brown border; spots becoming confluent with age. Ascomata pseudothecial, hypophyllous, single, black, immersed becoming erumpent, globose, up to 100 µm diam; wall of 2–3 cell layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoid, straight to slightly incurved, 8-spored, 35–50 x 10–12 µm.
Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, obovoid with subobtuse ends, unequally 1-septate, widest close to the apex of the apical cell, not constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (10–)12–13(–14) x (3–)3.5(–4) µm in vivo; apical cell 4–6 µm long, basal cell 6–8 µm long.
Holotype: Tanzania, on leaves of E. maidenii, May 1995, T. Coutinho, PREM 54971, holotype, culture ex-type CBS 111161 = CPC 1098.
Ascospore germination on MEA after 24 h: Type F. Similar to M. nubilosa.
Cultures: Colonies after 3 wk on MEA reaching 6–15 mm diam; on MEA erumpent with sparse aerial mycelium, pale olivaceous-grey; margins smooth, regular; reverse ochraceous with patches of pale olivaceous-grey; on PDA erumpent, centres white to pale olivaceous-grey, outer zone olivaceous-grey, margins irregular, feathery; reverse smoke-grey in the central part, olivaceous-grey in the outer region; colonies sterile.
Host: E. maidenii.
Distribution: Tanzania.
Notes: Ascospores of M. quasicercospora (10–14 x 3–4 µm) germinate with a Type F germination pattern, similar to that observed in M. nubilosa (11–16 x 3–4.5 µm), but are somewhat shorter, and also cluster phylogenetically apart (Fig. 3). Of particular interest is the fact that it aligns with sequences of Cercospora apii, for which no teleomorph is known. Cultures are sterile, and a re-examination of the original specimen also failed to reveal the presence of a Cercospora state. This is presently the only Mycosphaerella teleomorph clustering with Cercospora apii other than C. acaciae-mangii (Crous et al. 2004b) (Fig. 3).
Mycosphaerella scytalidii Crous & M.J. Wingf., sp. nov. MycoBank MB500844. Fig. 20.
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Synanamorph: Scytalidium-like.
Etymology: Referring to the Scytalidium-like synanamorph.
Mycosphaerellae parkii similis, sed ascosporis minoribus, 8–10 x (2.5–)3 µm, modi I germinantibus, distinguenda.
Leaf spots amphigenous, irregular to sub-circular, 1–8 mm diam, grey to medium brown, with a raised, dark brown border. Ascomata pseudothecial, amphigenous, single, black, immersed becoming erumpent, globose, up to 90 µm diam; apical ostiole 5–10 µm diam; wall of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoid, straight or slightly incurved, 8-spored, 25–30 x 7–9 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in the middle of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, 8–10 x (2.5–)3 µm in vivo. Mycelium internal and external, consisting of septate, branched, verruculose hyphae, 2–3 µm wide. Caespituli fasciculate, amphigenous on the leaves, brown, up to 50 µm wide and 60 µm high. Conidiophores aggregated in loose fascicles arising from the upper cells of a brown stroma up to 50 µm wide and 30 µm high, or situated on the top of the ascomata; conidiophores medium brown, finely verruculose, 1–4-septate, subcylindrical, straight to geniculate–sinuous, unbranched, 20–40 x 2–4 µm. Conidiogenous cells terminal, unbranched, medium brown, smooth to verruculose, tapering to the flat-tipped apical loci, proliferating sympodially, 7–15 x 2–3 µm, with thickened, darkened, refractive scars. Conidia solitary, or in simple chains, medium brown, verruculose, subcylindrical to ellipsoidal, apex obtuse, base subtruncate, 1–2-septate, frequently constricted at the septa, 7–15 x 3–3.5 µm; hila thickened, darkened, refractive. Aerial mycelium disarticulating into hyaline, smooth arthroconidia that are Scytalidium-like, 12–35 x 3–5 µm.
Holotype: Colombia, Angela Maria, on leaves of Eucalyptus urophylla, Jan. 2004, M.J. Wingfield, CBS H-19696 holotype, culture ex-type CBS 118493 = CPC 10998.
Ascospore germination on MEA after 24 h: Type I. Ascospores not darkening on MEA, and germinating from both ends, with germ tubes parallel to the long axis of the spore, lateral branches present, and spore distorting upon germination, becoming up to 5 µm wide.
Cultures: Colonies on MEA reaching 18–30 mm diam after 3 wk; colonies erumpent, folding, margin smooth, irregular, aerial mycelium moderate, pale olivaceous-grey; reverse iron-grey; on PDA with moderate aerial mycelium, olivaceous-grey with patches of pale olivaceous-grey; reverse olivaceous-black; on OA pale olivaceous-grey with patches of olivaceous-grey and iron-grey.
Host: Eucalyptus urophylla.
Distribution: Colombia.
Notes: Several other as yet undescribed species occur on Eucalyptus leaves in Colombia, and some, such as M. longibasalis Crous & M.J. Wingf. (Crous 1998) (Fig. 21), is still not known from culture. Isolate CPC 10986 clusters with CPC 11002, and in culture they are distinct from CPC 11004. We were, however, unable to trace these isolates back to ascomata due to several species being present on the same leaf spots. Thus, further collections will be required before these taxa can be named.
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Etymology: Referring to the ecology of this fungus as a secondary coloniser on lesions of M. suberosa.
Mycosphaerellae parkii similis, sed ascosporis minoribus, 8–10 x 2.5–3 µm, distinguenda.
Occurring as a secondary colonist on leaf spots caused by M. suberosa, or M. perpendicularis. Ascomata pseudothecial, amphigenous, single, inconspicuous, sparsely distributed, black, subepidermal, rarely erumpent, globose, up to 90 µm diam. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid, straight or slightly incurved, 8-spored, 20–30 x 7–9 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight, ellipsoidal with subobtuse ends, medianly 1-septate, widest close to the apex of the apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, 8–10 x 2.5–3 µm in vivo.
Holotype: Brazil, Bahia, Teixeira de Freitas, on leaves of Eucalyptus sp., 8 Jun. 2004, A.C. Alfenas, CBS H-19697, holotype, culture ex-type CBS 118507 = CPC 11551–11553.
Ascospore germination on MEA after 24 h: Type D. Similar to M. parkii.
Cultures: Colonies on MEA after 3 wk reaching 25–35 mm diam; on MEA olivaceous-grey, flat, spreading, folding, with sparse aerial mycelium and smooth, even margins; reverse iron-grey; on PDA iron-grey with olivaceous-grey aerial mycelium in central part, and drops of slime throughout; reverse iron-grey; on OA flat, spreading, olivaceous-grey.
Host: Eucalyptus spp.
Distribution: Brazil, Colombia.
Notes: When this species was initially collected in 1992 (CPC 504), it was noted that it occurred in lesions ascribed to M. suberosa, presumably as a secondary pathogen. We have now been able to recollect this fungus where it had colonised lesions caused by M. suberosa, as well as those of Cryptosporiopsis eucalypti Sankaran & B. Sutton on eucalypts in Brazil. In the same phylogenetic clade accommodating M. secundaria from Brazil, isolates collected in Colombia were also found which were apparently associated with lesions caused by M. perpendicularis (Fig. 2). Mycosphaerella secundaria has thus far only been collected in association with other species of Mycosphaerella that we believe are the primary pathogens. Mycosphaerella perpendicularis (ascospores 8–10 x 2.5–3 µm) was originally treated as M. parkii (ascospores 8–15 x 2–3.5 µm) (Crous 1998).
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Mycosphaerella stramenti Crous & A.C. Alfenas, sp. nov. MycoBank MB500846. Fig. 23.
Etymology: Refers to the occurrence of this fungus on leaf litter.
Mycosphaerellae parkii similis, sed ascosporis minoribus, (8–)10–12(–13) x 3(–3.5) µm, modo I germinantibus, distinguenda.
Leaf spots absent, ascomata associated with leaf litter. Ascomata pseudothecial, amphigenous, but predominantly hypophyllous, single, black, immersed becoming erumpent, globose, up to 120 µm diam. Asci aparaphysate, fasciculate, bitunicate, subsessile, narrowly ellipsoid to subcylindrical, straight or slightly incurved, 8-spored, 25–40 x 7–8 µm. Ascospores tri- to multiseriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, fusoid–ellipsoidal with subobtuse ends, medianly 1-septate, widest in middle of apical cell, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8–)10–12(–13)
