Eutypa flavovirens (Pers.) Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 57 (1863)

Index Fungorum number: IF 122506; MycoBank number: MB 122506; Facesoffungi number: FoF 00692; Fig. 1

Saprobic on dead land branch of Quercus sp. Sexual morph Stromata 0.7–1 mm diam., gregarious, aggregates are solitary to scattered, erumpent on the bark, black, outer thin carbonaceous layer, pustulate, with numerous ascomata in a single stroma. Ascomata 350–650 μm high, 300–450 μm diam., (x̅=360×490 μm, n=10) length with ostiole neck, immersed in the stroma, globose to broadly ovoid, narrowing towards the apex, very narrow at the base of papilla, ostiolate, papillate. Ostiole neck 100–130 × 250–275 µm (x̅= 90 × 260, n = 5), opening to outer surface, appearing as black spots, comprising outer dark brown and inner pale brown to yellowish parenchymatous cell layers, with periphyses oriented towards apical direction. Peridium 15–25 μm thick, with two layers, outer layer comprising 4–6 layers with thick-walled, dark brown to pale brown cells of textura angularis to textura prismatica cells, inner layer comprising 2–3 layers with thin-walled, hyaline cells of textura prismatica. Hamathecium 1–1.5 μm wide, filiform, longer than the asci, septate, unbranched, guttulate, hyaline paraphyses. Asci 80–120×8–10 μm (x̅=105×8.5 μm, n=20), 8-spored, unitunicate, thin-walled, clavate, with a J- apical ring, long, thin-walled pedicel, with cylindrical, thick-walled, swollen upper portion, apically flat. Ascospores 7–9.5×2–2.5 μm (x̅=8.5×1.5 μm, n=40), overlapping, 1–3-seriate, yellowish to brown, unicellular, ellipsoidal to cylindrical or elongate-allantoid, aseptate, straight to slightly curved, with or without guttules, smooth-walled. Asexual morph See Senanayake et al. (2015) and Niranjan et al. (2018).

Culture characteristics – Ascospores germinating on PDA within 24 h and germ tubes arising from one end of the ascospore. Colonies on PDA, reaching 4–5 cm diam. after 7 days at 25 °C, colonies medium dense, flat or effuse, slightly raised, cottony, white, margin rough.

Material examined – ITALY, Province of Bologna, Calistri—Porretta Terme, on the dead land branch of Quercus sp. (Fagaceae), 9 April 2019, E. Camporesi, IT 4287 (MFLU 19-0911, new record), living culture, MFLUCC 21-0069.

GenBank numbers – ITS = MZ456005, TUB2=MZ476771.

Notes – In the multigene phylogeny (ITS and TUB2) of this study, our isolate (MFLUCC 21-0069) and Eutypa flavovirens (E48C, CBS 272) clustered together with relatively high support (98% MLBS, 1.00 BYPP; Fig. 2). Eutypa flavovirens strains MFLUCC 12-0052, MFLUCC 13-0625 and PUFNI 310 were studied by Senanayake et al. (2015) and Niranjan et al. (2018) and they did not provide TUB2 DNA sequences to support. However, in our phylogenetic analyses, Eutypa flavovirens strains are grouped within Allodiatrype clade (Fig. 153) introduced by Konta et al. (2020). In our study Eutypa shown to be polyphyletic genus which are presently reside in A, B, C and D clades. According to Niranjan et al. (2018) several strains of Eutypa flavovirens have been identified with few differences in size of the asci and ascospores. Also, Senanayake et al. (2015) reported, the specimen collection in Thailand (MFLUCC 13-0625) may not be identical to species collected from Europe. Likewise, our isolate collected from Italy having ascomata, ostiole neck, peridium thickness are larger than previously studied and differs in having thinner paraphyses (Fig. 1). However, our isolate showed that the distinct yellowish green stromatic tissues the same as other Eutypa flavovirens isolates from Senanayake et al. (2015) and Niranjan et al. (2018). Based on the morpho-molecular analyses we conclude that our new collection is another record of Eutypa flavovirens and also a new host record on Quercus sp. (Fagaceae) in Italy.

Figure 1 – Eutypa flavovirens (MFLU 19-0717, new record). a, b Stromata on substrate. c Cross-section of stroma. d, e Vertical section through stromata showing ostiolar canals. f Peridium. g Paraphyses. h–k Asci. l–o Ascospores. p, q Culture on PDA from above and below. Scale bars: a=500 μm, b, c=200 μm, d=100 μm, e=50 μm, h–l=20 μm, f–g=10 μm, m–o=5 μm

Figure 2 – Phylogram generated from maximum likelihood analysis based on combined ITS and TUB2 sequence data representing Diatrypaceae in Xylariales. Related sequences are taken from Konta et al. (2020) and additions according to the BLAST searches in NCBI. Hundred and twenty-nine strains are included in the combined analyses which comprised 1180 characters (665 characters for ITS and 515 characters for TUB2) after alignment. Kretzschmaria deusta (CBS 826.72) and Xylaria hypoxylon (CBS 122620) in Xylariaceae (Xylariales) were used as the outgroup taxa. The best scoring RAxML tree with a final likelihood value of − 17362.853779 is presented. The matrix had 906 distinct alignment patterns, with 37.13% of undetermined characters or gaps. Estimated base frequencies were as follows: A=0.224946, C=0.263658, G=0.237000, T=0.274396; substitution rates: AC=1.093989, AG=3.037146, AT=1.238822, CG=0.814851, CT=4.048082, GT=1.000000; gamma distribution shape parameter α=0.183977. Bootstrap support values for ML equal to or greater than 75% are given above the nodes (left side). Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are given above the nodes (right side). Ex-type strains are in bold and newly generated sequences are in blue

Figure 2 – (continued)