Molecular phylogenetic analyses
The final concatenated alignment comprised of 130 ingroup taxa within the family Mycosphaerellaceae, with 2250 characters including alignment gaps (gene boundaries of LSU: 1–745, RPB2: 746–1554 and ITS: 1556–2250). The five characters artificially introduced as spacers between partitions were excluded from the phylogenetic analysis. Schizothyrium pomi (Mont.) Arx (isolate CBS 486.50) (Schizothyriaceae) was used as outgroup (Fig. 1).
Phylogenetic trees obtained from BI and MP analyses yielded trees with similar overall topologies at the generic level and are in agreement with previous studies, such as Videira et al. (2017). The results of MrModeltest recommended that Bayesian analysis should use dirichlet (1, 1, 1, 1) state frequencies and the GTR+I+G with an invgamma-shaped rate variation for all loci (LSU, RPB2 and ITS). The Bayesian analysis resulted in 4942 trees after 2,470,000 generations. The first 1234 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 3708 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (Fig. 1). The alignment contained a total of 1230 unique site patterns (217, 557 and 456 for LSU, RPB2 and ITS, respectively).
The Maximum Parsimony (MP) analyses generated a maximum of 1000 equally most parsimonious trees and the bootstrap support values (MP-BS) were mapped on the Bayesian tree as the second value in the tree nodes (Fig. 1). From the analyzed characters, 1074 were constant, 206 were variable and parsimony-uninformative and 970 were parsimony-informative. A parsimony consensus tree was calculated from the equally most parsimonious trees and the branches were mapped with a thicker line on the Bayesian tree (Length = 14,442, CI = 0.170, RI = 0.500, RC = 0.085, HI = 0.830).
Taxonomy
Based on phylogenetic analyses, the isolates collected in this study could be assigned to four different taxa in the Mycosphaerellaceae. The taxa are treated as follows.
Paracercosporidium microsorum (Sacc.) U. Braun, C. Nakash., Videira & Crous, Fig. 2
Description in planta:Leaf spots amphigenous, irregular to subcircular, dark brown to black, sometimes with yellow halo, 1–4 mm diam. Mycelium internal. Stromata small, substomatal to intraepidermal, brown, up to 45 μm diam. Caespituli amphigenous, pale brown, effuse. Conidiophores aggregated in loose fascicles, emerging from upper part of stromata, medium brown, smooth to slightly verruculose, 0–3-septate, unbranched, thick-walled, cylindrical, subcylindrical to geniculate-sinuous, irregular in width, frequently constricted due to sympodial proliferation, (15–)30–75 × 4–5.5 μm. Conidiogenous cells integrated, terminal or intercalary, polyblastic, proliferating sympodially, with conidiogenous loci rim-like, darkened and thickened, located at shoulders and at the apex, 1.5–2.5 μm diam. Conidia solitary, hyaline to pale brown, cylindrical to obclavate, obconically truncated and thickened at the base, rounded at the apex, 22–58 × 4–7 μm, 1–6-euseptate, hila thickened and darkened, 1.5–2.5 μm diam.
Cultural characteristics: Colonies on MEA, surface raised, folded, white in the centre, pink vinaceous towards the rim, with sparse aerial mycelium and lobate margin, reverse dark brown, reaching 11 mm after 20 days at 25°C.
Material examined: Iran: Mazandaran Province, Nowshahr, Mollakola, N 36°32'20", E 51°48'31", alt. 260 m, on leaves of Tilia platyphyllos Scop. (Malvaceae), 11 November 2019, M. Bakhshi, IRAN 18054F, IRAN 4116C (TEF1-α=XXX), IRAN 18055F, IRAN 4089C (TEF1-α=XXX), IRAN 4074C, IRAN 18056F, IRAN 4118C (TEF1-α=XXX).
Notes: To our knowledge, this study is the first molecular confirmation of Paracercosporidium microsorum for Asia and part of the TEF1-α locus was sequenced for the first time for this species.
Prathigadoides M. Bakhshi, Zare & U. Braun gen. nov.
MycoBank MB XXXXX.
Etymology. Composed of Prathigada, a genus name used for morphologically similar species, and the Latin suffix -oides (similar).
Type species.Prathigadoides gleditsiae-caspicae M. Bakhshi, Zare & U. Braun sp. nov.
Description: Plant pathogenic. Caespituli amphigenous. Conidiophores fasciculate, arising from a well-developed, substomatal to intraepidermal, semi-immersed, brown stroma, medium to dark brown, finely roughened, septate, unbranched, straight, slightly curved or geniculate-sinuous, subcylindrical, wall thickened. Conidiogenous cells integrated, terminal, finely roughened, polyblastic, proliferating sympodially, with conidiogenous loci thickened, darkened and protuberant. Conidia solitary, brown, subcylindrical to obclavate, usually distinctly rostrate, straight to gently curved, smooth to rough-walled, wall thickened, apex subobtuse to obtuse, base truncated to short obconically truncated, septate; hila thickened and darkened.
Prathigadoides gleditsiae-caspicae M. Bakhshi, Zare & U. Braun sp. nov. Fig. 3
MycoBank MB XXXX.
Etymology. Epithet derived from the host species of this fungus, Gleditsia caspica.
Holotype. Iran: Guilan Province, Talesh, Kishonben, on Gleditsia caspica L. (Fabaceae), 12 October 2012, M. Bakhshi (IRAN 16460F).
Ex-type strain: CBS 136121. GenBank: ACT = XXX; CAL = XXX; HIS = XXX; TEF1-α = XXX.
Description in planta: Leaf spots amphigenous, circular to subcircular, 1–4 mm diam., grey in center, with brown margin, occasionally with shot-holes in the centers. Mycelium internal. Stromata well-developed, substomatal to intraepidermal, semi-immersed, brown, 25–75 μm diam. Caespituli amphigenous. Conidiophores aggregated in loose to dense fascicles, erumpent through the cuticle or emerging through stomata, medium to dark brown, finely roughened, 0–3-septate, unbranched, straight, slightly curved or geniculate-sinuous, subcylindrical, wall thickened, regular in width, (25–)40–55(–80) × (3.5–)4–5 μm. Conidiogenous cells integrated, terminal, medium brown, finely roughened, polyblastic, proliferating sympodially, with conidiogenous loci thickened, darkened, protuberant and somewhat refractive, located at the apex and on shoulders formed by sympodial proliferation, 2–3.5 μm diam. Conidia solitary, brown, subcylindrical to obclavate, usually rostrate, straight to gently curved, smooth to rough-walled, wall thickened, apex subobtuse to obtuse, base truncated to short obconically truncated, (4–)10–16-septate, occasionally constricted at the septa, (42–)65–76(–110) × (4.5–)5.5–6 μm; hila thickened, darkened and refractive, 1–2 μm diam.
Cultural characteristics: Colonies on MEA, surface raised, folded, dark brown-grey, with sparse aerial mycelium and irregular margin, reverse dark brown, reaching 7 mm after 20 days at 25°C.
Additional material examined: Iran: Guilan Province, Gourab Zarmikh, N 37°18'02", E 49°12'39", on Gleditsia caspica, 7 August 2019, M. Bakhshi (IRAN 18053F, IRAN 4110C, IRAN 4111C); Guilan Province, Talesh, Kishonben, on Gleditsia caspica, October 2020, M. Bakhshi (P 829).
Notes: The cercosporoid fungus found in Iran on the endemic Gleditsia capsica, distributed in a small area near the southern coast of the Caspian See, proved to belong to a clade of its own distinct from all other clades within the Mycosphaerellaceae, which reflects a new undescribed genus. The type species of the new genus is morphologically close to Prathigada condensata (Ellis & Kellerm.) U. Braun [º Cercospora condensata Ellis & Kellerm, Passalora condensata (Ellis & Kellerm.) U. Braun] on the North American Gleditsia triacanthos L., but differs in having finely verruculose conidiophores with wider conidiogenous loci, 2–3.5 µm diam., and shorter conidia, (42–)65–76(–110) µm long, with numerous dense septa [(4–)10–16], and narrower hila, 1–2 µm diam. [versus, smooth conidiophores with narrower loci, 1.5–2.5 µm diam., and longer conidia, 40–150 µm long, with fewer, more spaced septa, 4–12, and larger hila, 2–2.5 µm diam. (Braun et al. 2013)]. Furthermore, the two hosts species, G. capsica and G. triacanthos, are geographically and phylogenetically clearly distinct. Gleditsia capsica belongs to the Asian G. japonica clade, which is distantly related to the G. triacanthos clade (Schnabel et al. 2003). In spite of the strong morphological similarity between the new species from Iran and species previously assigned to Prathigada, the latter genus is not available for the fungus on Gleditsia capsica. Based on phylogenetic examinations of the type species of Prathigada, P. cratevae (Syd. & P. Syd.) Subram., this genus was reduced to synonymy with Pseudocercospora (Braun et al. 2013). The conidiogenous loci and hila in the type species of Prathigada are conspicuous by being rigid and refractive, falling into the morphological variation of Pseudocercospora spp., but they are not truly thickened. The loci in Cercospora condensata are thickened and somewhat similar to those of Prathigadoides gleditsiae-caspicae, suggesting that the two species might be congeneric. However, the clarification of this question requires results of phylogenetic analyses of C. condensata. In any case, the North American cercosporoid species on G. triacanthos neither pertain to Cercospora nor to Passalora in the current sense and circumscription (Videira et al. 2017).
Pruniphilomyces circumscissus (Sacc.) Crous & Bulgakov, Fig. 4
Description in planta:Leaf spots amphigenous, circular to subcircular, 1–6 mm diam., pale brown to brown with distinct border. Mycelium internal. Stromata well-developed, substomatal to intraepidermal, semi-immersed, brown, 20–55 μm diam. Caespituli amphigenous. Conidiophores aggregated in loose to moderately dense fascicles, arising from stomata or erumpent through the cuticle, medium brown, smooth to slightly verruculose, 0–3-septate, unbranched, straight to geniculate-sinuous, subcylindrical, regular in width, (30–)38–49(–60) × 3–4.5 μm. Conidiogenous cells integrated, terminal, medium brown, polyblastic, proliferating sympodially, with conidiogenous loci thickened, darkened and refractive and located at the apex and shoulders, 1–2 μm diam. Conidia solitary, medium olivaceous brown, subcylindrical to obclavate, abruptly tapered towards the apex, straight to gently curved, apex subobtuse to obtuse, base obconically truncated, 1–7(–10)-septate, (18–)50–65(–105) × (3–)3.5–5 μm; hila thickened, darkened and refractive, 1–2 μm diam.
Cultural characteristics: Colonies on MEA, surface flat, folded, with moderate aerial mycelium and entire margin, dark olivaceous green, reverse olivaceous black, reaching 20 mm after 20 days at 25°C.
Material examined: Iran: Mazandaran Province, Kelardasht, Goharkela, N 36°28′59.04″, E 51°14′58.68″, alt. 1300 m, on Prunus avium L. (Rosaceae), 12 August 2018, M. Bakhshi (IRAN 18026F, IRAN 3478C), on Prunus avium, September 2020, M. Bakhshi (IRAN XXXF, P 943.a). Mazandaran Province, Marzan Abad, Foshkour, N 36°21′29.2″, E 51°11′43.04″, alt. 1770 m, on Prunus avium, 12 August 2018, M. Bakhshi (IRAN 3477C), on Prunus avium, September 2020, M. Bakhshi (IRAN XXXF, P 947). Iran: Ardabil Province, Moghan, on Prunus avium, October 2012, M. Bakhshi (IRAN 16459F, CBS 136122 = IRAN 2681C). Iran: West Azerbaijan Province, Kaleibar, on Prunus avium, November 2012, M. Arzanlou (IRAN 18022F, P 237). West Azerbaijan Province, Khoy, Firouragh, on Prunus cerasus L. November 2011, M. Arzanlou (IRAN 2656C). Iran: Guilan Province, Talesh, Lisar, on Prunus avium, May 2019, P. Emdadi (IRAN 18043F, IRAN 4099C). Talesh, Jowkandan, on Prunus avium, May 2019, M. Bakhshi (IRAN 18021F, P 830). GenBank numbers CBS 136122: ACT = XXX; CAL = XXX; HIS = XXX; TEF1-α = XXX.
Notes: To our knowledge, this study is the first molecular confirmation of Pruniphilomyces circumscissus for the Middle East. Furthermore, part of the ACT, TEF1-α, CAL and HIS loci is sequenced for the first time in this species.
Sirosporium celtidis (Biv.) M.B. Ellis, Fig. 5
Description in planta:Leaf spots amphigenous, subcircular to circular, olivaceous, reddish brown to dark blackish brown, velvety, 1–9 mm diam., without forming chlorosis or necrosis, which might be due to the semibiotrophic nature of the fungus. Caespituli hypophyllous, velvety. Stromata lacking. Mycelium partly immersed and partly external; consisting of branched, septate (septa 10–19 μm apart), pale brown to brown hyphae, 2.5–4 μm wide, smooth or somewhat verrucose, faintly rough-walled. Conidiophores arising from external hyphae, macronematous or semi-macronematous, solitary, not branched, dark brown, thick-walled, 0–1(–4)-septate, constricted at the septa, 0–2 times geniculate, 13–22(–30) × (4–)6–8 μm, often reduced to solitary conidiogenous cells. Conidiogenous cells terminal and lateral, proliferating sympodially, brown, smooth or verrucose, 5–10(–19) × (4–)6–8 μm, loci thickened and darkened, 2–5 μm diam. Conidia solitary, brown to dark brown, subcylindrical to obclavate, straight to curved or coiled, thick-walled, smooth, rugulose or verrucose, (2–)11–16(–31) transversely septate and occasionally with 1–3 longitudinal or oblique septa, often constricted at the septa, apex rounded, base rounded to truncated, (15–)62–85(–130) × (4–)6–7(–9) μm; hila rather inconspicuous, but thickened and somewhat darkened, 2–5 μm diam.
Cultural characteristics: Colonies on MEA, surface brown-grey, with sparse aerial mycelium and entire margin, colony surrounded by a red-brown halo, reverse dark brown, reaching 4 mm after 20 days at 25°C.
Material examined: Iran: Mazandaran Province, Jannat Roudbar, Dalkhani forest, N 36°49′10.49″, E 50°40′05.06″, alt. 660 m, on Celtis australis L. (Ulmaceae), 14 August 2018, M. Bakhshi & H. Taheri (IRAN 18030F, IRAN 3483C, IRAN 3484C). Mazandaran Province, Nowshahr, Mollakola, alt. 260 m, N 36°32'20", E 51°48'31", on leaves Celtis australis, 11 November 2019, M. Bakhshi (IRAN 18057F, IRAN 4117C, P 899.a). Iran: West Azerbaijan Province, Kaleibar, on Celtis australis, 10 November 2012, M. Arzanlou (IRAN 16465F, CBS 136019). GenBank numbers CBS 136019: ACT = XXX; CAL = XXX; HIS = XXX; TEF1-α = XXX.
Notes: To our knowledge, this study is the first molecular confirmation of Sirosporium celtidis for Asia. Furthermore, first sequences of the ACT, TEF1-α, CAL and HIS loci have also been generated for this species.