Colletotrichum flexuosum Damm, sp. nov.

Index Fungorum number: IF 558527; MycoBank number: MB 558527; Facesoffungi number: FoF 10680; Fig. 1

Etymology – The species epithet is derived from the shape of the setae that are often flexuous.

Holotype – CBS H-21899

Associated with leaf spots of Xanthophyllum sylvestre. Asexual morph on synthetic nutrient-poor agar medium (SNA): Vegetative hyphae 1.5–8 µm diam., hyaline to pale brown, smooth-walled, septate, branched. Chlamydospores not observed. Conidiomata absent, conidiophores and setae formed directly on hyphae. Setae medium brown, smooth-walled, verrucous (warts 1–2 mm diam.) close to the tip, 80–140 µm long, 3–4-septate, base inflated, 6–10 µm diam., tip ± acute to round, often strongly bent. Conidiophores pale brown, smooth-walled, simple or septate, to 20 µm long. Conidiogenous cells pale brown, smooth-walled, cylindrical to subglobose, 9–20(–26) × 5–9 µm, opening 2–2.5 µm diam., collarette 1 µm long, periclinal thickening conspicuous. Conidia hyaline, smooth-walled, aseptate (few septate conidia observed), straight, cylindrical, with both ends rounded, (14.5–)16.5–24(–21) × (6.5–)7–8.5(–9.5) µm, mean ± S D = 18.9 ± 2.3 × 7.8 ± 0.8 µm, L/W ratio = 2.4 (n = 30). Appressoria single, pale brown, smooth-walled, with a navicular to clavate outline and a lobate to crenate margin, (9–)12.5–18(–24) × (4–)5.5–8.5(–10) µm, mean±SD =1 5.1±2.8 × 7.1±1.4 µm, L/W ratio=2.1 (n=30). Sexual morph: not observed.

Asexual morph on Anthriscus stem – Conidiomata, conidiophores and setae formed on medium brown, smooth-walled, roundish to angular cells, 5–11 µm diam. Setae medium to dark brown, smooth-walled, 100–180 µm long, 3–4-septate, straight to ± flexuous, base cylindrical or restricted, 4–8 µm diam., tip±round to acute. Conidiophores pale to medium brown, smooth-walled, septate, branched, to 90 µm long. Conidiogenous cells pale to medium brown, smooth-walled, cylindrical to clavate, 15–32 × 5–8 µm, opening 1.5–2 µm diam., collarette 0.5–1 µm long, periclinal thickening distinct. Conidia hyaline, smooth-walled, aseptate, cylindrical, with both ends rounded, sometimes a distinct membranous appendage remains at the base, (21–) 23.5–28.5(–33.5)×(5–)6–7.5(–8) µm, mean±SD=25.9± 2.6×6.9±0.8 µm, L/W ratio=3.8 (n=30). Sexual morph: not observed.

Culture characteristics – Colonies on SNA flat with entire margin, hyaline to honey, agar medium, filter paper and Anthriscus stem partly covered with grey acervuli and short floccose white aerial mycelium, reverse hyaline to honey, agar medium, filter paper and Anthriscus stem partly pale grey; growth 13.5–18 mm in 7 days at 20 °C under near UV light with 12 h photoperiod (21–28 mm in 10 days). Colonies on OA flat with entire margin; buff, grey olivaceous to olivaceous grey, partly covered with short floccose white aerial mycelium and dark grey to black spots, reverse olivaceous-grey, growth 20–21.5 mm in 7 days (30.5–32 mm in 10 days). Conidia in mass whitish.

Material examined – Vietnam, Ninh Bình Province, Cúc Phương National Park, rain forest, from leaf spots of Xanthophyllum sylvestre (Polygalaceae), 6 December 2012, Ulrike Damm (CBS H-21899, holotype); ex-holotype living culture, CBS 134419. Ninh Bình Province, Cúc Phương National Park, rain forest, from leaf spots of Xanthophyllum sylvestre (Polygalaceae), 6 December 2012, Ulrike Damm, living culture CBS 137338.

GenBank numbers – CBS 134419—MZ444580 (ITS), MZ444582 (tub2), MZ444584 (gapdh), MZ444586 (act), MZ444588 (chs-1), MZ444590 (his3). CBS 137338—MZ444581 (ITS), MZ444583 (tub2), MZ444585 (gapdh), MZ444587 (act), MZ444589 (chs-1), MZ444591 (his3).

Notes – Colletotrichum flexuosum was isolated from leaves of Xanthophyllum sylvestre, a tree species native to Laos, Thailand and Vietnam (http://www.plantsoftheworldonline.org). Few fungi were previously reported from Xanthophyllum, including an unidentified Colletotrichum species on X. octandrum in Australia (Simmonds 1966; Farr and Rossman 2022). No Colletotrichum species was previously described or reported from X. sylvestre. Based on blastn searches and sequence comparisons on NCBI GenBank, the closest neighbour of the strains from Xanthophyllum is C. vietnamense, belonging to the C. gigasporum complex. The ITS, tub2, gapdh, act, chs-1 and his3 sequences of this species were 80, 97, 91, 98, 98 and 96% identical (105, 23, 9, 4, 4and 15 nucleotides difference) with those of the ex-holotype strain of C. vietnamense (Liu et al. 2014). No strain was more than 82% identical with its ITS sequences. The closest match with the act sequences was Colletotrichum sp. gnqczg15 (KC293585, F. Huang, unpubl. study) with two nucleotides difference. In a phylogeny inferred from concatenated ITS, tub2, gapdh, act, chs-1 and his3 sequences of the C. gigasporum species complex (Fig. 2), the two strains formed a well-supported (BS 100%, BYPP 1) sister clade to C. vietnamense and a clade formed by two recently described species, C. subvariabile and C. variabile (Liu et al. 2022). Colletotrichum flexuosum can be identified with all loci included. The species from Xanthophyllum is morphologically different from all previously described species of the C. gigasporum complex. Its conidia are shorter than those of C. gigasporum (22–32×6–9 μm on PDA), C. magnisporum (av. 34.3×9.7 μm on SNA, av. 33.8×9.9 μm on Anthriscus stem), C. serranegrense (24–37×7–9 μm on MEA) and C. vietnamense (av. 31.2×9.6 μm on SNA, av. 32.3×9.5 μm on Anthriscus stem) and larger than those of C. jishouense (av. 10.8×3.7 μm on PDA). In contrast to C. arxii, C. jishouense, C. pseudomajus, C. radicis and C. vietnamense, neither curved nor clavate conidia were formed, while conidia with membranous appendages and distinctly flexuous setae as formed by C. flexuosum were previously not observed in the C. gigasporum complex (Rakotoniriana et al. 2013; Liu et al. 2014, 2022; Silva et al. 2018; Zhou et al. 2019). Conidiogenous cells with a distinct periclinal thickening are typical for species of the C. boninense complex; within the C. gigasporum complex this was previously only observed with C. zhaoqingense (Liu et al. 2022). However, in contrast to C. zhaoqingense, conidial bases of C. flexuosum sometimes end with a membranous appendage, while most species of the C. boninense and the C. dracaenophilum complexes develop a prominent scar (Damm et al. 2012, 2019; Liu et al. 2022).

Figure 1 – Colletotrichum flexuosum (CBS 134419, ex-type living culture) a–b. Conidiomata. c, h. Tips of setae. d, i. Bases of setae. e–g, j–k. Conidiophores. m–r. Appressoria. s–t. Conidia. a, c–g, s. from Anthriscus stem. b, h–r, t. from SNA. a–b. Dissecting microscope (DM). c–t. Differential interference contrast illumination (DIC). Scale bars: a=100 µm, e=10 µm. Scale bar of a applies to a–b. Scale bar of e applies to c–t

Figure 2 –   One of two most parsimonious trees obtained with PAUP v. 4.0b10 (Swofford 2003) from a heuristic search of the combined sequence alignment (gene boundaries of ITS: 1–550, tub2: 551–1240, gapdh: 1241–1510, act: 1511–1756, chs-1: 1757–2007, his3: 2008– 2385) of the Colletotrichum gigasporum species complex, rooted with C. gloeosporioides CBS 112999 (sequences from Damm et al. 2012; Rakotoniriana et al. 2013; Liu et al. 2014; Silva et al. 2018; Zhou et al. 2019; Liu et al. 2022). Bootstrap support values (BS) above 70% (bold) and Bayesian posterior probability (BYPP) values above 0.90 are shown at the nodes. Bootstrap support values have been calculated based on 10 000 replicates, and a Markov Chain Monte Carlo algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v. 3.2.6 (Ronquist et al. 2012). Numbers of ex-type strains are in bold