Trichobelonium miscanthi 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

Trichobelonium miscanthi Itagaki & Hosoya sp. nov.

Figs 12 , 13 , 14I

Etymology.

Named after the genus of its host, Miscanthus .

Diagnosis.

Characterized by 5-septate ascospores and sparse subiculum

Holotype.

TNS-F-17835, Sugadaira Montane Research Center, Ueda City, Nagano Pref., 17 September 2005, on decaying culm of Miscanthus sinensis , ex-holotype culture NBRC 115566.

Description.

Apothecia developed from scuta. Scuta superficial, scattered to gregarious, flat discoid, 145-180 µm diam., dark brown (C80M100Y80K60), textura epidermoidea, gradually becoming textura porrecta and connecting to subiculum. Apothecia sessile, globose to pulvinate when immature, discoid to saucer-shape when mature, flat to slightly convex when fresh, 0.2 mm high, with brown (C30-60M60Y80-100K60) receptacle; disc 0.5-1.5 mm diam., entire to slightly undulate, without hairs at margin, waxy, white to pale yellow (Y10-30) when fresh, shrunk to 0.3-1 mm diam., pulverulent, cream (Y20-40K10) when dried. Ectal excipulum 25-35 µm thick at base, approximately 25 µm thick at the upper flank to margin, textura globulosa and angularis, composed of 2-4 layers of brown thick-walled cells, not gelatinized, without crystals or exudates; cortical cells in middle to lower flank pyriform to clavate, with protruded cells, 16-21(-23) × 5-7 µm, containing with refractive vacuoles at margin when mounted fresh in water; anchoring hyphae connecting the cortical calls of the flank and subiculum, radially extending from apothecium, 2.5-3 µm width, thin-walled, brown. Medullary excipulum 40-75 µm thick, textura intricata to prismatica, hyaline, composed of loosely interwoven thin-walled hyphae which is frequently dichotomously branching. Asci (77-)79-85(-90) × 12.5-15(-17.5) µm, cylindrical-clavate to saccate, 8-spored, arising from croziers, containing hyaline oil globules in cytoplasm that disappear when mature, with a thick-walled conical apex; apex MLZ+ with or without 3% KOH pretreatment. Ascospores (32-)37-47(-57.5) × 4.5-5.5 µm, long fusiform, with acute extremes, curved to sigmoid, occasionally constricted, thin-walled, often 5-septate, hyaline, containing large or abundant minute guttles. Paraphyses (70-)83-105(-115) × 2.5-3(-4) µm, simple, occasionally branching at base, cylindrical, often becoming slightly wider toward the apex, 2-3-septate, thin-walled, hyaline, (2-)3-septate, containing long refractive vacuoles when mounted fresh in water. Subiculum thinly developed the surface of substrates in patches, sparse to especially abundant around the mature apothecia, shiny brown; subicular hyphae straight to curved, usually swelling in a globose, 3-5 µm diam. with 0.5-1 µm thick-walls, septate every 15-30 µm, perpendicularly branched. Colony of NBRC 115566 on PDA, flat to slightly convex with aerial hyphae, cottony, grayish orange (C0-20M60Y60K10) from the surface, appearing cinnamon (C20-40M80Y100K30) from reverse side, with apricot (M20-40Y60) soluble pigment uniformly diffuse in agar; aerial mycelium dense, white to pale yellow (Y10-20); crystals aggregating plate-like or small clusters, acicular, moderately abundant on colony surface and surrounding agar, 0.1-0.5 mm across, pale yellow. Conidiophores macronematous to mononematous, arising from subicular hyphae, straight, pale to dark brown, thick-walled, smooth, 2-3 µm width; phialides ampulliform, up to 15 µm long, 2.5-4 µm width, integrated, arranged penicillately, pale brown, thick-walled, with cylindrical to wide funnel-shape collarettes of 3.5-5 × 2-3 µm; conidia aseptate, spherical to subspherical, 2-2.5 µm diam., hyaline, thin-walled.

Additional specimens examined.

TNS-F-30037, Hachimantai City, Iwate Pref., 12 October 2009 on decaying culm of Miscanthus sinensis , culture NBRC 115652; TNS-F-81751, Kiritappu Wetland, Hamanaka City, Akkeshi County, Hokkaido, 29 August 2019, on decaying culm of Phragmites australis ; TNS-F-86581, Higashi Ward, Sapporo City, Hokkaido, 13 August 2021, on decaying culm of Ph. australis ; TNS-F-86672 (culture NBRC 115667) and 86695, Yuzawa Town, Minami-uonuma County, Niigata Pref., 17 and 31 October 2021 (respectively), on decaying culm of M. sinensis ; TNS-F-86700, Daigenta Lake, Yuzawa Town, Minami-uonuma County, Niigata Pref., 31 October 2021, on decaying culm of M. sinensis , culture NBRC 115668; TNS-F-86715, Toukamachi City, Niigata Pref., 31 October 2021, on decaying culm of M. sinensis .

Notes.

Trichobelonium miscanthi occurs with Neobelonopsis cinnabarina as they share the same host, Mollisia sinensis , and fruiting season (autumn). Brown phialides (Fig. 12E ) and spherical conidia, regarded as asexual stage of T. miscanthi , were observed to accompany subiculum, but we could not induce conidial reproduction under culture.

From the reverse of the two months old colony of T. miscanthi on CMA, clumps of dark cells strongly attached to the bottom of the Petri dish (Fig. 12U ) were observed. The clumps are composed of swollen cells with melanized ring. The swollen cell is usually obovoid to pyriform, sometimes lobed or hyphoid, 10-15 × 6-10 µm, arising vertically from hyphae, thick-walled, and containing abundant guttles (Figs 12V-X , 14I ). The brown ring structure has an outer diameter of 8-10 µm and inner diameter of 2-3 µm, and is formed at the cell and Petri dish interface. Very similar hyphal structures were reported by Aebi (1972) in the culture of T. kneiffii , but its function is unknown. The clumps of dark cells of Phialocephala bamuru P.T.W. Wong & C. Dong, known as plant pathogen, are interpreted as appressoria with infected pegs ( Wong et al. 2015). Although this structure may be appressorium, direct observation of the mycelium of T. miscanthi on the host epidermis and inoculation experiments are needed to clarify whether the clamps of T. miscanthi function as an appressorium during the infection process.

Trichobelonium miscanthi resembles T. albobarbatum in remarkable oil globules in immature asci, anchoring hyphae, and saccate form of asci. Although T. miscanthi lacks crystals in the excipulum, the culture produced abundant acicular crystals on PDA (Fig. 12R, S, T ).

Figures

Figure 12. Trichobelonium miscanthi (TNS-F- 17835, holotype) A fresh apothecia on the decaying culm of Miscanthus sinensis B dried apothecia with stromata (arrows) C brown connective hyphae extending from the base of fresh apothecium D texture of stroma (in LA) E conidiophore (in LA) F vertical section of the apothecium (in LA) G fresh outermost cells of ectal excipulum with refractive vacuoles (in water) H ascospores (in CB / LA) I fresh ascospores with droplets (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 long refractive vacuoles in fresh paraphysis (in water) M branching paraphysis (in CB / LA) N immature ascus containing oil globes in the cytoplasm (in CB / LA) O ascus with mature ascospores (in CB / LA) P immature ascus (in water) Q ascus with ascospores (in water) R one month old colony on PDA S pale yellow crystals formed on the edge of the colony T acicular crystals (in water) U hyphal mass attached to the bottom of the Petri dish V-X swollen cells with melanized appressorium-like structure. Scale bars: 1 mm (S); 0.5 mm (A, B); 0.25 mm (C); 50 μm (F, U); 20 μm (G, T); 10 μm (D, E, H-Q, V-X).

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.