Cryphonectria kunmingensis Lu L., K.D. Hyde & Tibpromma S., sp. nov. 

MycoBank number: MB 900438; Index Fungorum number: IF 900438; Facesoffungi number: FoF 10843; Fig. 1

Etymology – based on the location where the fungus was collected.

Saprobic on dead stem of unidentified plant. Sexual morph: Ascostromata (excluding neck) 300–660×240–420 μm (x=526 μm, n=342), single, semiimmersed, orange, oval to irregular. Perithecia immersed, single or aggregated, subglobose or irregular, multi-loculate, with long necks, ostiolar canal sometimes immersed in ascostromatic tissues, or come out from the substrate, orange. Neck composed of umber stromatic cells of textura porrecta. Peridium consists of two distinct layers, cells of textura angularis, outer layer dense, thick, yellow to pale brown, inner layer 4–5-layered, hyaline. Hamathecium 6–9 μm wide (x=7 μm, n=20) consists of a cellular, septate, hyaline, paraphyses attached at base. Asci 55–70×8–10 μm (x=64.5 × 8.9 μm, n=20), 8-spored, unitunicate, cylindrical, hyaline, with distinct J- refractive ring, no stalk. Ascospores 9.5–15×3–5 μm (x=13×4.2 μm, n=20), overlapping uniseriate to biseriate, ellipsoid, 1–2-septate when mature, constricted at septa, hyaline, smooth-walled, without a mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics – colonies on PDA reaching 60 mm diam after 4 weeks in 20 °C, circular, flat, lobate, pale yellow, flocculent, a lot of aerial mycelia front view pale yellow, reverse view orange center turning pale yellow towards the margin, produced pigments in culture.

Material examined – CHINA, Yunnan Province, Kunming, Chang chong Mountain, on dead stem of unidentified plant, 20 June 2021. Li Lu, CCS 4, (HKAS 121976, holotype), ex-type culture, KUMCC 21–0216.

GenBank numbers – ITS: ON359918, ON359919; LSU: ON340718, ON340719; β-tubulin: ON599004, ON5999005; rpb2: ON599000, ON599001; tef1-α: ON599002, ON599003

Notes – In the combined ITS, LSU, rpb2, β-tubulin and tef1-α gene analysis, our collection (HKAS 121976) formed a distinct subclade (Fig. 2) with MP/BI=98%/1.00 bootstrap support. ITS sequence of our strain is 93.6% similar to Cryphonectria nitschkei (GQ290656) while LSU is 99.6% similar to C. nitschkei (AF408341). The blast results of tef1-α is 90% similar to C. macrospora (AY308952), rpb2 is 96.5% similar to Chromendothia citrina (DQ862015) and β-tubulin is 92% similar to Cryphonectriaceae sp. (JQ862910). Morphologically, our collection is similar to general characters of Cryphonectria (Jiang et al. 2020), however, it differs from other Cryphonectria species in having 2-septate ascospores. In addition, our ascospores (9.5–15) are longer compared to the morphologically similar species C. nitschkei (8.5–12.5) (Myburg et al. 2004). Therefore, we introduce our collection as a new species, Cryphonectria kunmingensis. Four species have been collected from China
viz. C. japonica, C. neoparasitica, C. quercus and C. quercicola and this is the fifth species.

Figure 1 – Cryphonectria kunmingensis (HKAS 121976, holotype). a–c Ascostromata on the host surface. d Cross section of ascostromata. e Peridium. f Paraphyses. g–j Asci (stained in Congo Red reagent). k J−Refractive ring (stained in Melzer’s reagent). l Germinated ascospore. m–q Ascospores (stained in Congo Red reagent). r Upper and reverse views of colonies on PDA. Scale bars: d=300 µm, e=30 µm, f=25 µm, g–l=20 µm, m–q=5 µm

Figure 2 – Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, rpb2, β-tubulin and tef1-α sequence data. 45 taxa were included in the combined analyses, which comprised 3280 characters (LSU=869 bp, ITS=569 bp, rpb2=1,113 bp, β-tubulin=475 bp, tef1-α=254 bp) after alignment. The best scoring RAxML tree with a final likelihood value of − 7795.712285 is presented. The matrix had 510 distinct alignment patterns, with 48.97% of undetermined characters or gaps. Estimated base frequencies were as follows: A=0.227853, C=0.275760, G=0.262261, T=0.234126; substitution rates: AC=1.967589, AG=4.751144, AT=3.030078, CG=1.335415, CT=15.093804, GT=1.0; gamma distribution shape parameter α=0.02. Bootstrap support for maximum likelihood (ML) equal to or greater than 50% and clade credibility values greater than 0.90 (the rounding of values to 2 decimal proportions) from Bayesian inference analysis are labelled at each node. Ex-type strains are in black bold, while the new isolate is indicated in blue bold. The tree is rooted to Amphilogia gyrosae (CMW 10469) and Endothia gyrosae (CMW 2091)