Cryptovalsa ampelina (Nitschke) Fuckel, Jb. nassau. Ver. Naturk. 23–24: 212 (1870) [1869–70] Basionym –  Valsa ampelina Nitschke, Pyrenomycetes Germa-nici 1, p. 156, 1867.
Index Fungorum number: IF 241474, Facesoffungi number: FoF 01800, Figs. 1 and 2

Saprobic on bark. Sexual morph Stromata poorly developed, immersed in bark, with occasionally protruding perithecial necks, single or in groups, irregularly scattered. Ascomata 510 – 580 μm high, 340 – 440 μm diam. (x̄ = 530 × 391 μm, n = 8), solitary to gregarious, immersed, dark brown to black, arranged in a single layer, singly arising, in rows globose to subglobose, often compressed, ostiolate, with cylindrical necks, raising above the epidermis and forming black, blister-like areas, periphysate. Peridium 35 – 45 μm wide, composed of two layers; outwardly comprising several layers of thick-walled, dark brown to black cells of textura angularis, inwardly comprising 3 – 5 layers of thin-walled, hyaline cells of textura angularis to textura prismatica. Hamathecium comprising dense, 2 – 4 μm wide, hyaline, aseptate, anastomosing paraphyses. Asci (98–)118 – 133(–146) × (7–)7 – 11(–14) μm (x̄ = 119 × 9 μm, n = 30), polysporous, unitunicate, cylindric- clavate, long pedicellate, apically rounded to truncate with indistinct, amyloid apical annulus. Ascospores (7–)7.5 – 9(–10) × (1–)2 – 2.5(–3) μm, (x̄ = 8.3 × 2.4 μm n = 60), crowded, pale yellowish to pale brown at maturity, allantoid–reniform, 1-celled, smooth-walled, with small guttules. Asexual morph Coelomycetous, forming on MEA. Conidiomata 150 – 260 mm diam., pycnidial, superficial, solitary or aggregated, dark brown to black, globose to subglobose, covering by yellow to light brown interwoven, thick-walled, hyphae. Conidiophores 10 – 22 × 1.5 – 2 μm (x̄ = 18 × 2 μm, n = 10), septate, bicellately to verticillately branched, arranged in dense palisades, cylindrical, hyaline, smooth, arising from the base. Conidiogenous cells 8 – 14 × 1 – 2 μm (x̄ = 11 × 1.5 μm, n = 20), holoblastic, sympodial to synchronous, straight or curved, subcylindrical, hyaline, apically distorted on conidial secession. Conidia 16.5 – 20 × 1 – 1.5 μm (x̄ = 18.6 × 1.3 μm, n = 55), hyaline, cylindrical to filiform, unicellular, slightly curved, apically rounded, with truncate base.

Culture characteristics – Ascospores germinating on MEA within 24 h, germ tubes produced at both ends cell, colonies on MEA reaching 4 mm diam. after 7 days in darkness condition at 25 °C, medium dense, raised, circular with fimbriate edge, fluffy to fairy fluffy, white from above, light yellowish from below, forming asexual morph, with black, stromatic after 15 days.

Material examined – ITALY, Fiumana di Predappio, Province of Forlì-Cesena [FC], on dead branch of Vitis vinifera L. (Vitaceae), 5 January 2015, E. Camporesi, (MFLU 16–0007, KUN-HKAS 93731, reference specimen designated here), living culture, MFLUCC15–0139, KUMCC 16-0003).

Notes – Cryptovalsa ampelina is a pathogen of grapevines (Vitis vinifera L.) and is abundant on pruned canes and necrotic wood of living plants (Mostert et al. 2004; Luque et al. 2006; Trouillas et al. 2010; Pitt et al. 2013a). The species was reported as a pathogen from South Africa, Australia, North East of Spain, California and Eastern United States (Mostert et al. 2004; Vasilyeva and Stephenson 2005; Luque et al. 2006; Trouillas et al. 2010; Pitt et al. 2013a). Cryptovalsa ampelina causes internal wood discolouration, similar to that caused by Eutypa lata (Pers.) Tul. & C. Tul (Ferreira 1987; Mostert et al. 2004). However, the species can be distinguished from E. lata in having polysporous asci and pigmented allantoid ascospores (Luque et al. 2006).

The asexual morph of Cryptovalsa ampelina has been reported in the coelomycetous genus Libertella, which is characterized by sporodochium-like conidiomata, hyaline, branched conidiophores, with hyaline, subcylindrical, conidiogenous cells which proliferate sympodially and hyaline, filiform, slightly curved to hamate, unicellular conidia, with a truncate, flattened base (Mostert et al. 2004; Luque et al. 2006). In this study, the asexual morph formed in culture on MEA after 20 days. The characters of our taxon are similar to previous studies, although our taxon differs due to its slightly smaller conidia.

Based on phylogenetic analysis of ITS gene dataset (Fig. 73), Cryptovalsa ampelina clearly separates from Eutypa lata and clusters with Quaternaria quaternata (GNF13, EL60C). However, C. ampelina can be distinguished from Q. quaternata by its polysporous asci. Our strain (MFLU 15-0139) forms a well-supported clade (100 % ML) with other strains of C. ampelina (KHJ 20 and A 001) in the family Diatrypaceae (Fig. 1). Our isolate is similar to the protolgue described by Nitschke (1867) as well as Trouillas et al. (2010). Nevertheless, it differs from the type protoloque in having larger asci (from Saccardo (1882), 75 –90 × 8 – 9 versus (98–)118 – 133(–146) × (7–)7 – 11(–14), this study) and slightly smaller ascospores (from Saccardo (1882), 9 – 10 × 2.5 versus, (7–)7.5 – 9(–10) × (1–)2 – 2.5(–3), this study). Therefore, we propose our new collection as a reference specimen Fig. 3.

Fig. 1 Phylogram generated from maximum likelihood analysis based on ITS sequence data of the family Diatrypaceae. The new isolates are in red and ex-type strains are in bold. The tree is rooted with Xylaria hypoxylon.

Fig. 2 Cryptovalsa ampelina (MFLU 16–0007, reference specimen) a Appearance of stromata on host surface b Longitudinal section through stromata showing globose ascomata embedded in stromatal tissues c Ostiole with periphysate ostiolar neck d Peridium e Paraphyses f, g Asci h Immature ascus i Ascus with apical apparatus inconspicuously bluing in Melzer’s reagent j – l Ascospores m Germinating ascospore n, o Culture in MEA, note n is from above and o is from below. Scale bars: a = 200 μm, b = 500 μm, c = 30 μm, d = 50 μm, e – g = 20 μm, h, i,m = 10 μm, j – l = 5 μm.

Fig. 3 Culture of Cryptovalsa ampelina in PDA (MFLU 15–0139) a, b Conidiomata on the culture c Hyphae on conidiomatal surface d Section of conidiomata e Conidiophore with young conidia f Conidiogenous cells with conidia g Conidiophores h – j Conidia. (Note: f, i, j with cotton blue) Scale bars: b = 200 μm, d = 20 μm, c, e – j = 5 μm.