Trichoderma strophariensis E. Tarafder & F. H. Tian, 2024

Tarafder, Entaj, Wenjun, Zhang, Karunarathna, Samantha C., Elgorban, Abdallah M., Huilian, Man, Nan, Wu, Zeng, Xiangyu, Yong, Wang & Tian, Feng-Hua, 2024, Unveiling two new species of Trichoderma (Hypocreales, Hypocreaceae) that cause green mold disease on Stropharia rugosoannulata from Guizhou Province, China, MycoKeys 110, pp. 361-383 : 361-383

Trichoderma strophariensis E. Tarafder & F. H. Tian sp. nov.

Figs 3 , 4

Diagnosis.

Trichoderma strophariensis differs from T. britannicum by smaller stromata (0.9–2.2 × 0.8–2 mm) with dark green surface, margin free; surface finely rugose or tubercular, brownish between black ascomata; ostiolar dots absent, inconspicuous or convex to distinctly papillate measuring (27 –) 35–64 (– 90) mm diam. Additionally, it is easily distinguished from T. viridistromatis by its relatively larger ascospores (8.4–16.9 × 5.5–8.1 µm) and conidia (8.5–25.5 × 5.7–17.9 μm). Phylogenetically, T. strophariensis forms a distinct clade and is closely related to T. viridistromatis , T. britannicum , and T. aerugineum with 100 % ML and 0.90 BYPP statistical support (Fig. 1 ).

Holotype.

HGUP 24-0001 .

Etymology.

The specific epithet ‘ strophariensis ’ refers to the occurrence of the new taxon in cultivated mushrooms Stropharia rugosoannulata .

Description.

Stromata, when fresh 1–14 mm in diameter, 1–11 mm thick (n = 10), solitary to sometimes aggregated, discoid or undulate, with brownish margin and pale red, depressed center when young, becoming reddish with rugose surface when mature. Attached to the host by hyphae, easily detached; sides often attenuated downward, surrounded at the base by white cottony mycelium when young. Surface finely rugose, tubercular, brownish between black ascomata; Ostiolar dots are convex to umbilicate, greenish, overall colors light green, darker green when dry, surface and spores green when mature. Ostiole 14–21 μm wide at apex, 41–59 μm high (n = 30). Ascomata (139 –) 175–295 (– 347) × (113 –) 151–248 (– 290) μm (n = 20), flask-shaped or sub-globose, crowded. Peridium 18–28 μm thick at the base and sides (n = 40), light brown. Asci (67 –) 110–146 (– 207) × (3.7 –) 5.8–7.7 (– 9.4) μm, stipe (3 –) 7–11 (– 18) μm long (n = 50), containing 16 - ascospores, apex slightly thickened, hyaline, cylindrical. Ascospores (8.4 –) 9.2–11.6 (– 16.9) × (5.5 –) 6.6–7.8 (– 8.1) μm, l / w (1.2 –) 1.4–1.6 (– 2.1) (n = 90), green, verruculose; sub-globose, oblong, elongated, thick-walled.

Culture characteristics.

Optimal growth at 25 ° C on all media, poor and limited growth at 30 ° C, no growth at 35 ° C.

On MEA and PDA growth is slow, colony creamy white, finely farinose by scant effuse conidiation; on PDA reverse brownish, surface turning greenish-brown. On MEA at 25 ° C after five days colony radius 5–7 mm; colony circular, dense, thick, first whitish, becoming zonate after a few weeks, turning olive-green to brown with yellow greenish, farinose center; conidiation effuse, on short odd verticillium like conidiophores. On SNA colony radius at 25 ° C after 2 weeks 6–9 mm; colony dense, hyaline, turning greenish or olivaceous from conidia. Conidiation following growth, effuse, on aerial hyphae and short odd verticillium-like conidiophores, spreading from the plug. Conidiophores simple, 1–4 level are branched and tapered at the tips, bearing few asymmetric side branches, terminated by solitary phialides of 2–3 divergent phialides. Phialides (10.5 –) 37–44 (– 55) × (1.5 –) 2.5–11 (– 12.5) μm (n = 50), mostly gregarious, cylindrical, less commonly subfusiform, often thickest near the base. Conidia (8.5 –) 12.5–16.4 (– 25.5) × (5.7 –) 6.5–10.7 (– 17.9) μm (n = 70), one-celled, variable shape and size, typically oblong and pale olive green when fully mature, sub-globose, oval or ellipsoid and hyaline when immature, straight or slightly curved, sides sometimes pinched, smooth; base often truncate, thick-walled.

Habitat.

On mushroom cultivated field, associated with Stropharia rugosoannulata .

Distribution.

China, Guizhou Province, Guiyang City, and Liupanshui City; Guizhou City in Anshun Province.

Material examined.

China • Guizhou, Liupanshui City, Shuicheng District , 23°55'39.36"N, 120°11'30.64"E, on soil surfaces of Stropharia rugosoannulata cultivated field, 16 - November- 2023, E. Tarafder and F. H. Tian ( HGUP 24-0001 , holotype); ex-type living cultures GUCC TB 1117 , GUCC TB 1118 and GUCC TB 1119 .

GenBank accession numbers.

GUCC TB 1117 ( ITS: PP 920011; rpb 2: PP 954941; tef 1 - α: PP 954947); GUCC TB 1118 ( ITS: PP 920012; rpb 2: PP 954942; tef 1 - α: PP 954948); GUCC TB 1119 ( ITS: PP 920013; rpb 2: PP 954943; tef 1 - α: PP 954949).

Notes.

Morphologically, our new isolates are most similar to T. danicum in the size of stromata (5–20 mm) but can be distinguished by its generally smaller ascospores and conidia (Table 3 ); the presence of deeper color of stromata and ascospores, less pigment on media, and faster growth rate on PDA and SNA. However, our new isolates differ from T. britannicum by smaller stromata (0.9–2.2 mm) with dark green surfaces ( Jaklitsch, 2009). In addition, it differs from other new species ( T. viridistromatis ) in producing cylindrical, less commonly subfusiform phialides (10.5–55 × 1.5–12.5 μm) and larger conidia (8.5–25.5 × 5.7–17.9 μm), typically oblong, subglobose, oval, sometimes ellipsoid and pale olive green after maturity. Phylogenetically, our isolate ( HGUP 24-0001) forms an independent clade and clustering with Trichoderma britannicum , T. aerugineum , T. danicum , T. viridistromatis , and T. spinulosum within the Spinulosum lineage with 100 % ML and 1.00 BYPP statistical support (Fig. 2 ). It exhibits 4 % sequence differences (28 / 610 nucleotides, four gaps) in the ITS region, 4 % differences (48 / 1080 nucleotides, no gaps) in the rpb 2 gene, and 5 % differences (64 / 1244 nucleotides, twenty-five gaps) in tef 1 - α gene when compared with T. britannicum . Additionally, the differences between our isolate with T. viridistromatis are 4 % (29 / 610 nucleotides, four gaps) in the ITS region, 4 % (46 / 1080 nucleotides, no gaps) differences in the rpb 2 gene, and 5 % (65 / 1244 nucleotides, twenty-five gaps) differences in the tef 1 - α gene. In contrast, the differences in our isolate with T. danicum are more than 4 % (25 / 610 nucleotides, eight gaps) in the ITS region, 9 % (99 / 1080 nucleotides, no gaps) in rpb 2 gene, and 8 % (105 / 1244 nucleotides, three gaps) in tef 1 - α gene (Table 2 ). Therefore, based on both morphological and phylogenetic distinctions, T. strophariensis is introduced as a new species from cultivated mushrooms.

Figures

Figure 1. Field symptoms of green mold disease on Stropharia rugosoannulata and pathogenicity tests of isolates GUCC TB 1117 and GUCC TB 1120 with spore suspension a healthy fruiting bodies of S. rugosoannulata b field symptoms of green mold disease on S. rugosoannulata c large stroma of the pathogen T. strophariensis (GUCC TB 1117) d control, no disease after seven days of inoculation with distilled water e – g pathogenicity tests after spraying with 0.5 mL spore suspension (1 × 10 6 conidia mL – 1) e, f hyphal blocks and pathogen stroma (F = yellow arrow) appear on the surface of the soil after five days of inoculation g whole rotten fruiting bodies after seven days of inoculation h, i rotten fruiting bodies of S. rugosoannulata in the field with T. viridistromatis (GUCC TB 1120) j Aggregated stroma of the pathogen T. viridistromatis (GUCC TB 1120) with typical green symptoms k, l yellow arrows showing pathogen hyphal blocks and stroma appear on the surface of the soil after five days of inoculation. Scale bars: 20 mm (a – e); 10 mm (f); 20 mm (g); 10 mm (h); 20 mm (I, j); 10 mm (k, l).

Figure 2. A phylogram was constructed using ML analysis, utilizing a combined ITS, rpb 2, and tef 1 - α sequences dataset. The green-spored T. longibrachiatum (CBS 81668) was used as the outgroup taxon following Zeng et al. (2022). The tree with the highest score according to RAxML, with a final probability value of - InL = 37957.575772, is displayed. Maximum Likelihood (ML) values equal to or greater than 70 % and Bayesian Inference (BI) values equal to or greater than 0.90 are given above the nodes (ML values on the left side of ‘ / ’ in regular font and BI values on right side of ‘ / ’ in italics). Type strain sequences are indicated in red bold, while newly generated sequences are shown in black bold. Strain numbers for the sequences are shown in the tree following the taxon name. ‘ T’ denotes ex-holotype strains.

Figure 3. Morphology of Trichoderma strophariensis (HGUP 24-0001, GUCC 24-0002) a, b disease in the field habitat c fresh stromata on natural habitat d dry stromata e ostiolar dots on stromata surface f cortical and subcortical tissues in section g ascomatal tissue in section h asci with ascospores i ascospores. Scale bars: 10 mm (a, b); 20 mm (c); 100 mm (d – f); 50 μm (g); 20 μm (h, i).

Figure 4. a cultures on MEA (five days) b cultures on PDA (five days) c cultures on SNA (4 days) d conidiophores e phialides f conidia. Scale bars: 10 μm (d, e); 5 μm (f).