Pyrenophora verruculosa Madrid & Cantillo, sp. nov.
Index Fungorum number: IF 844464; Mycobank number: MB 844464; Facesoffungi number: FoF 10420; Fig. 1
Etymology – The name refers to the verruculose conidia produced by this species
Holotype – SGO 168420
Probably saprobic or pathogenic to an unidentified member of Poaceae. Sexual morph: Not observed. Asexual morph: Hyphomycetous. Vegetative hyphae septate, branched, light olivaceous to mid olivaceous brown, thin to thick-walled, smooth, 2–6 µm wide, anastomosing, occasionally showing deposits of a mucilaginous dark brown material. Conidiophores macronematous, mononematous, solitary, septate, simple, slightly flexuous to strongly geniculate, light olivaceous brown to dark brown, often paler at the apex, smooth to verruculose, with cell walls often thicker than those of the supporting vegetative hyphae, 300–1270×5–9 µm with subnodulose to nodulose intercalary swellings up to 11 µm wide. Conidiogenous
cells integrated, terminal and intercalary, mostly subcylindrical, mono- to polytretic, proliferating sympodially, 15–28 µm long. Conidia narrowly clavate, narrowly ellipsoidal to fusiform or subcylindrical, straight to slightly curved, light olivaceous brown to dark brown, verruculose, (26–)32–63(–74) × 12–21 µm, 3–5(mostly 4)-distoseptate, often constricted at the uppermost distoseptum, with a rounded apex and an obconically truncate or rounded base, basal cell sometimes delimited by a thick, dark septum. Hilum thick and dark. Microsclerotia abundant, mostly 45–240 µm wide.
Culture characteristics – Colonies on water agar with sterilized maize leaves dark brown, hairy, with abundant clumps of microsclerotia.
Material examined – Chile, El Loa Province, Atacama Desert, near Calama, isolated from unidentified dead Poaceae, 15 October 2015, H. Madrid & L. Linaje (SGO 168420, holotype).
GenBank numbers – ON722346 (ITS), ON722346 (LSU), ON736764 (gpdh).
Notes – Due to mobility restrictions during the SARSCoV-2 pandemic, the ex-type strain (HM 201), which had been preserved in sterile water, could not be properly maintained for several months. Recent attempts to reactivate the strain have been unsuccessful and the fungus probably died. However, the holotype was deposited at SGO and extype sequences of ITS, LSU and gpdh are available in GenBank. DNA sequence analyses revealed that Pyrenophora verruculosa is clearly distinct from all other members of Pyrenophora represented in GenBank. The closest hits in BLAST searches with the ITS sequence of strain HM 201 were Pyrenophora novozelandica (CBS 127934) (ex-type, GenBank MK539997, 96.55% similarity), P. fugax (CBS 509.77) (GenBank MK539985, 95.23% similarity), P. nisikadoi (CBS 190.29) (ex-type, as Bipolaris brizae, GenBank MH855213, 93.57% similarity), and P. nobleae (CBS 259.80) (GenBank MK539994, 91.33% similarity). BLAST searches with the gpdh sequence showed P. fugax (CBS 509.77) (GenBank AY004822, 95.70% similarity) and P. nobleae (CBS 966.87) (GenBank AY004824, 88.81% similarity) as the closest matches. With LSU, similar results were obtained, but with higher similarity percentages, as expected for this rather conserved locus. In the phylogenetic analysis, only ITS sequence data was used considering that LSU offers little resolution for closely related taxa in Pleosporales, and gpdh is available for a smaller number of species than ITS. In the ITS-based phylogenetic tree (Fig. 2), P. verruculosa, P. novozelandica and P. fugax formed a clade with 83% bootstrap support. These close relatives can easily be distinguished from P. verruculosa on the basis of conidial dimensions, i.e. smaller in P. novozelandica, 20.5–58×9.5–14 µm, and longer in P. fugax, 50–170 × 14–24 µm (Ellis 1961; Sivanesan 1987; Marin-Felix et al. 2019). Clumps of thick-walled, strongly pigmented cells superficially resembling the microsclerotia of P. verruculosa, have been reported in other Pyrenophora species, such as P. nisikadoi and P. pseudoerythrospila (Marin-Felix et al. 2019). These species, however, are phylogenetically clearly distinct from P. verruculosa (Fig. 2).
Figure 1 – Pyrenophora verruculosa (SGO 168420, holotype). a, c, d Conidia attached to conidiogenous cells. b Conidium passively released from a conidiogenous cell. e–h Conidia (verruculose ornamentation can be observed in e). i Hyphae with mucilaginous dark brown material. j, k Mycelia with microsclerotia. Scale bars: a–d=40 µm, e–i=20 µm, j–k=45 µm
Figure 2 – Maximum likelihood tree based on ITS sequences, showing the phylogenetic relationships of Pyrenophora species. Bootstrap support values>70% are shown near the internodes. The tree is rooted to Bipolaris maydis. GenBank accession numbers of ITS sequences are given, in parentheses, after each strain number. A authentic strain, T ex-type strain, E Tex-epitype strain, H Tholotype material. New species is in bold