Neobelonopsis multiguttata Itagaki & Hosoya, 2023

Itagaki, Hiyori & Hosoya, Tsuyoshi, 2023, A new genus Neobelonopsis and two new species of Trichobelonium (Helotiales, Ascomycota) discovered mainly from poaceous grasses native to Asia in Japan, MycoKeys 99, pp. 45-85 : 45

Neobelonopsis multiguttata Itagaki & Hosoya sp. nov.

Figs 8 , 13 , 14F

Etymology.

Named after the abundant number of guttules in the ascospores.

Diagnosis.

Resembles N. acutata , but distinguishable by more sparsely formed conidiophores, longer asci, and longer ascospores with rounded extremes (vs. more acute in N. acutata ).

Holotype.

TNS-F-86402, Sugadaira Research Station, Mountain Science Center, Ueda City, Nagano Pref., 5 June 2021, on decaying culms of Sasa kurilensis , ex-holotype culture NBRC 115371.

Description.

Apothecia developed from scuta. Scuta superficial, scattered to gregarious, flat discoid, approximately 0.2 mm diam., blackish brown (C80M100Y80-100K60), textura epidermoidea, consisting of thick-walled cells. Apothecia 0.2-0.4 mm high, with dark brown (C80M80Y80-100K60) receptacle; disc 0.5-1.6 mm diam., white to pale gray (K10) when fresh, shrunk to 0.4-1.3 mm diam., pale yellow (Y10) when dried. Ectal excipulum 37-50 µm thick at base, 25-35 µm thick at the upper flank to margin; cortical cells hemispherical to short clavate, 12-17 × 9-10(-12) µm at base, becoming slender and closely packed toward the upper flank to margin. Medullary excipulum 37-87 µm thick. Asci (63-)78-98(-105) × 5-8 µm, arising from croziers, with MLZ + apical pore. Ascospores (12-)17-26(-27.5) × 2.5-3.5 µm, long ellipsoid to fusiform with rounded extremes, (1-)3-septate, containing abundant guttules. Paraphyses (62-)74-90(-100) × 2.5-3 µm, simple, with long apical cell. Subiculum sparsely developed, covering the surface of substrates in patches, shiny brown; subicular hyphae straight to curved, usually constricted at septum, fascicular, 3-5 µm width with 0.5-1 µm thick-walls, septate every 15-25(-50) µm, branched at right angle, covered by gelatinous substance. Colony of NBRC 115371 on PDA flat to slightly winkled, entire to undulate, floccose to woolly, grayish brown (C20-30M40Y40K60) from the surface, forming indistinct section observed clearer from the reverse, without soluble pigment and crystals; aerial mycelium moderately abundant at the center, sparse at the edge, pale gray (K10-30) to white. Conidiophores semi-macronematous, short, arising vertically from aerial hyphae, pale to dark brown, smooth, thick-walled, constricted, occasionally loosely branched; phialides cylindrical to ampulliform, up to 16 µm long, 3 µm width at base, discrete, arranged terminal or intercalary, pale brown, thick-walled, with cylindrical funnel-shape collarettes of 4.5-6.5 × 2-3 µm; conidia aseptate, spherical to subspherical, abundantly aggregated in slimy heads, 1.5-1.7 µm diam., hyaline, thin-walled.

Additional specimens examined.

TNS-F-18023, Shirakamisanchi, Aomori Pref., 24 May 2006, on decaying culms of Sasa sp.; TNS-F-39229, Mt. Tsukuba, Tsukuba City, Ibaraki Pref., 22 April 2011, on decaying culms of Sasa sp.; TNS-F-54941, Omama Town, Midori City, Gunma Pref., 9 May 2018, on decaying culms of Sasa sp.; TNS-F-61278, Mt. Tsukuba, Tsukuba City, Ibaraki Pref., 16 April 2014, on fallen cupules of Fagus crenata ; TNS-F-61280, Hakone Town, Ashigara-shimo County, Kanagawa Pref., 23 May 2014, on fallen cupules of F. crenata Blume, culture NBRC 115655; TNS-F-81133, Sugadaira Research Station, Montane Science Center, Ueda City, Nagano Pref., June 2017, on decaying culms of Sasa kurilensis ; TNS-F-86224, Sekimoto Town, Kita-ibaraki City, Ibaraki Pref., 3 June 2020, on dead branches on living Stephanandra incisa , culture NBRC 115365; TNS-F-86426, Mt. Amari, Asahi Town, Nirasaki City, Yamanashi Pref., 14 June 2021, on decaying culms of Sasa sp.; TNS-F-86465, Nagataki Town, Noumi City, Ishikawa Pref., 18 June 2021, on decaying culms of Sasa palmata , culture NBRC 115661.

Notes.

Neobelonopsis multiguttata has a wide host range, such as Sasa spp., Fagus crenata , and Stephanandra incisa , and occurs on various substrates, such as culms, branches, and cupules. Neobelonopsis multiguttata was found in spring and its morphology overlaps with N. bicolor in the dimensions of apothecia and paraphyses. However, the ITS sequence similarity with N. acutata is only 93.8%. Further, the two species form phylogenetically distinct clades (Fig. 1 ). The conidiophores of N. multiguttata on CMA are discrete (Figs 8N-R , 14F ), rather than aggregated as in N. acutata (Fig. 3N, O ).

Based on a BLAST search for the ITS sequences of Neobelonopsis multiguttata in the GenBank database, the closest hit was Ascomycota sp. (MK842071), isolated from the needles and roots of pine trees in South Korea [Identities=531/531 (100%), no gaps]. The endophytic isolate was recognized as Mollisia sp. by Rim et al. (2021). This result suggests that N. multiguttata has an endophytic phase as part of its life cycle.

Figures

Figure 1. Maximum likelihood tree inferred from ITS-LSU-RPB 1 concatenate sequences used in Tanney and Seifert (2020), together with sequences from additional taxa of Neobelonopsis and two new species of Trichobelonium. Significant branch supported by SH-aLRT (> 80 %) / Ultrafast BS (> 95 %) / BPP (> 0.95) are indicated. Type species are in red. Collection numbers are shown at the beginning of the species name (type strains are in boldface). The tree is rooted with Cadophora dextrinospora and C. fastigiata.

Figure 3. Neobelonopsis acutata (TNS-F- 86648, holotype) A fresh apothecia on the decaying culm of Miscanthus sinensis B dried apothecia C stromata with sparse subiculum D one month old colony on PDA E vertical section of the apothecium (in LA) F refractive vacuoles in fresh marginal cells (in water) G asci with ascospores (in CB / LA) H paraphyses (in CB / LA) I paraphyses with a long refractive vacuole (in water) J blue-stained apical pore of ascus (in Melzer's solution after 3 % KOH pretreatment) K croziers at the base of ascus (in CB / LA) L ascospores (in CB / LA) M conidia (in water) N, O conidiophores (in water) P bulbile (in CB / LA) Q subicular hyphae (in CB / LA). Scale bars: 1 mm (A, B); 0.2 mm (C); 25 μm (E, Q); 10 μm (F-P).

Figure 8. Neobelonopsis multiguttata (TNS-F- 86402, holotype) A fresh apothecia on decaying culm of Sasa kurilensis B dried apothecia C stromata with sparse subiculum D vertical section of apothecium (in LA) E ascospores (in CB / LA) F asci with ascospores (in CB / LA) G paraphyses (in CB / LA) H blue-stained apical pore of ascus (in Melzer's solution after 3 % KOH pretreatment) I croziers at the base of asci (in CB / LA) J texture of stroma (in LA) K subicular hyphae (in lactic acid) L three months old colony on PDA M conidia (in CB / LA) N-R conidiophores, arrows in N indicated conidiogenous cells (in CB / LA). Scale bars: 0.5 mm (A-C); 50 μm (D, K); 20 μm (N), 10 μm (E-J, M, O-R).

Figure 13. Line-drawings of the Neobelonopsis and Trichobelonium species I vertical section of the apothecium showing the marginal structure of ectal excipulum, medullary excipulum, and hymenium II asci III paraphyses IV ascospores. Scale bars: 10 μm.

Figure 14. Asexual morph and hyphal structures of the Neobelonopsis and Trichobelonium species A conidiophores of N. acutata B monilioid hyphae of N. bicolor C conidiophores with conidia of N. cinnabarina D conidiophores with conidia of N. didymospora E conidiophores with conidia of N. microspora F conidiophores with conidia of N. multiguttata G conidiophores with conidia of N. obtusa H conidiophores with conidia of T. albobarbatum I hyphal mass with melanized appressorium-like structure of T. miscanthi. Scale bars: 10 μm.