Didymella azollae E. Shams, F. Dehghanizadeh, A. Pordel & M. Javan-Nikkhah, sp. nov.

Index Fungorum number: IF558550; MycoBank number: MB558550; Facesoffungi number: FoF 09945; Figs. 1, 2

Etymology – Name refers to the host plant species, Azolla filiculoides.

Holotype – IRAN 18020 F

Endophytic from leave of Azolla filiculoides. Sexual morph Undetermined. Asexual morph Coelomycetous. Conidiomata 118–275 μm high, 80–200 μm diam., pycnidial, brown, solitary or confluent, globose, subglobose to pyriform, often with a short neck, glabrous or with some hyphal outgrows. Pycnidial wall pseudoparechymatous, comprising several layers of dark brown to lightly pigmented cells of textura angularis, outer layers 3–5 layers, 3–30 μm thick, pigmented, inner layers lightly pigmented. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic, hyaline, smooth, ampulliform to doliiform. Conidia 3–5×1.5–2 μm (x̅=4.2×2 μm, n=30), subglobose or ellipsoidal, hyaline, aseptate, smooth-walled.

Culture characteristics – Colonies on PDA, 40–48 mm diam. after 7 days, margin regular, densely covered by floccose aerial mycelia, greenish brown, reverse black (Fig. 2A). Colonies on OA, margin regular, floccose, aerial mycelia sparse, grayish brown, reverse black (Fig. 2B). Colonies on MEA, margin regular, floccose, buff, dense, reverse dark brown (Fig. 2C).

Material examined – IRAN, Guilan Province, endophytic from leave of Azolla filiculoides (Salviniaceae), 15 December 2016, E. Shams, (IRAN 18020F, holotype), ex-type living culture, IRAN 3058C.

GenBank numbers – ITS = MT514913, MT514914, MT514915, LSU = MT514910, MT514911, MT514912, TUB2=MT512516, MT512517, MT512518.

Notes – Didymella azollae was isolated from Azolla filiculoides in Guilan, Iran. The characters of the conidia and colony (Figs. 1 and 2) fit the generic concept of Didymella (Chen et al. 2015). The species identified in the present study is closely related to Didymella dactylidis (CBS 124513) and D. rhei (CBS 109177) (Fig. 3). Didymella azollae differs from D. dactylidis in type of conidia (Chen et al. 2017). In pairwise nucleotide comparisons of Didymella azollae with the type strain of D. dactylidis (CBS 124513), there is a nucleotide difference of 0.75% (6 bp) in LSU (of 799 nucleotides altogether) and 1.37% (5 bp) nucleotide difference in ITS (of 435 nucleotides altogether). However, in the proteincoding region of TUB2, the nucleotide difference was 6.7% (20 bp) across 286 nucleotides. Didymella rhei differs from D. azollae by conidia size (3.5–) 5–8 (–10.5)×1.5–3 μm). In pairwise nucleotide comparisons of Didymella azollae with the type strain of D. rhei (CBS 109177), there is a nucleotide difference of 0.85% (7 bp) in LSU (of 817 nucleotides altogether), 1.33% (6 bp) nucleotide difference in ITS (of 450 nucleotides altogether) and in the protein-coding region of TUB2, there is a large nucleotide difference of 6.7% (20 bp out of 286 nucleotides).

Figure 1 – Didymella azollae (IRAN 18020F, holotype). a Pycnidia on host. b Pycnidia. c Pycnidial wall. d Conidiogenous cells. e Conidia. Scale bars: b–e=10 μm

Figure 2 – Didymella azollae (IRAN 3058C, ex-type culture) cultures after 7 days at 25 °C on different media. a PDA. b OA. c MEA

Figure 3 – Maximum Likelihood tree inferred by MEGA v.7 from the combined ITS, LSU, and TUB gene regions of 92 isolates. Bootstrap support values from ML equal to or greater than 90% are provided
above or below the branches. Ex-type strains are in bold and novel species is shown in blue