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

Figs 5 , 6

Diagnosis.

Trichoderma viridistromatis differs from T. aerugineum by its smaller stromata (0.5–2 mm diam, to ca. 1 mm thick in T. aerugineum ) and bigger phialides measuring 7–23 × 2.4–4 μm in T. aerugineum . In addition, it is easily distinguished from T. strophariensis by its smaller ascospores (3.4–5.6 × 2.4–3.3 µm) and conidia (2.8–4 × 1.7–3.2 μm). Phylogenetically, T. viridistromatis forms a distinct clade and is closely related to T. strophariensis , T. britannicum , and T. aerugineum with 100 % ML and 0.90 BYPP statistical support (Fig. 1 ).

Holotype.

HGUP 24-0004 .

Etymology.

The epithet “ viridistromatis ” refers to an entirely green-colored stroma.

Description.

Stromata, when fresh 1–7 mm in diam., 0.5–2 mm thick (n = 10), mostly gregarious, aggregated, discoid or undulate, becoming pulvinate, compact; outline circular to oblong; margin attached or free, surface smooth when immature without ostiolar dots, with yellowish margin and pale red, depressed center when young, becoming reddish with rugose surface when mature. Outline circular, oblong or irregularly lobed. Surface smooth, tubercular or rugose, when young finely velvety. Ostiolar dots absent, ostiolar openings sometimes visible, (16 –) 20–30 (– 32) μm (n = 30) wide, inconspicuous, pale, more distinct and shinier after rehydration. Ostioles (18 –) 24–30 (– 45) μm long, plane with the surface, (8 –) 12–19 (– 23) μm wide at the apex (n = 30). Ascomata (69 –) 75–85 (– 96) × (36 –) 41–55 (– 60) μm (n = 30), numerous, 5–7 per mm stroma length, sub-globose or flask-shaped. Peridium (7 –) 11–19 (– 22) μm (n = 60) thick at the base and sides; hyaline to pale yellowish. Asci (63 –) 74–81 (– 85) × (3.2 –) 4.2–5 (– 5.5) μm, stipe (4 –) 5–11 (– 14) μm (n = 30) long, containing 16 - ascospores, apex not thickened, hyaline, cylindrical. Ascospores (3.4 –) 3.6–4.3 (– 5.6) × (2.4 –) 2.8–3.1 (– 3.3) μm, l / w 1–1.1 (– 1.2) (n = 34), hyaline, verruculose, single-celled, non-septate, sub-globose, oblong or slightly tapered downwards, thick-walled.

Culture characteristics.

Optimal growth at 25 ° C on all media, poor and limited growth at 30 ° C, no growth at 35 ° C. Although MEA exhibited good growth, precultures were made on it.

On MEA and PDA, growth is slow, colony is 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 (5.5 –) 7–10 (– 14) × (1.6 –) 2.5–2.9 (– 3.5) μm (n = 32), mostly gregarious, lageniform, less commonly subfusiform, not thickest near the base. Conidia (2.8 –) 3.1–3.7 (– 4) × (1.7 –) 2.2–2.7 (– 3.2) μm (n = 70), variable shape and size, typically oblong and pale yellowish green when fully mature, oval, ellipsoid and hyaline when immature, straight or slightly curved, sides sometimes pinched, smooth; base often truncate.

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 , 24°55'39.936"N, 121°11'30.264"E, on soil surfaces of Stropharia rugosoannulata cultivated field, 16 - November- 2023, E. Tarafder and F. H. Tian ( HGUP 24-0004 , holotype); ex-type living cultures GUCC TB 1120 , GUCC TB 1121 and GUCC TB 1122 .

GenBank accession numbers.

GUCC TB 1120 ( ITS: PP 922277; rpb 2: PP 954944; tef 1 - α: PP 954950); GUCC TB 1121 ( ITS: PP 926290; rpb 2: PP 954945; tef 1 - α: PP 954951); GUCC TB 1122 ( ITS: PP 922285; rpb 2: PP 954946; tef 1 - α: PP 954952)

Notes.

Morphologically, our newly described taxon Trichoderma viridistromatis shares common characteristics with T. aerugineum ( CBS 120541) and T. britannicum , a species previously isolated from dead stems and leaves of Calamagrostis epigejos . However, T. viridistromatis differs from T. aerugineum by having smaller stromata (0.5–2 mm in diameter, compared to ca. 1 mm thick in T. aerugineum ) and larger phialides (7–23 × 2.4–4 μm in T. aerugineum ) and ascospores (8–12 × 4–6 µm; Table 4 ) ( Chaverri and Samuels 2004). Additionally, it can be distinguished from T. strophariensis by its larger stromata (1–14 mm in diameter, 1–11 mm thick in T. strophariensis ) and significantly larger subglobose to elongated ascospores (8.4–16.9 × 5.5–8.1 µm). In comparison, T. britannicum has discoid, convex to turbinate stromata surrounded by light brown radial mycelium and much larger one-celled ascospores (10–16 × 4.5–6.2 μm; Table 4 ) ( Jaklitsch et al. 2014). The phylogenetic positions of the new taxon (Fig. 2 ) demonstrated that Trichoderma viridistromatis is closely related to T. strophariensis , T. britannicum , and T. aerugineum , with strong statistical support (Fig. 2 ). However, our isolate differs from T. britannicum with 3 % (17 / 610 nucleotides, with five gaps) in ITS region, 2 % (20 / 1080 nucleotides, no gaps) in rpb 2 gene, and 4 % (55 / 1244 nucleotides, twenty-one gaps) in tef 1 - α gene. Moreover, the difference in our collections with T. aerugineum is more than 2 % (10 / 610 nucleotides, ten gaps) in the ITS region, 7 % (79 / 1080 nucleotides, no gaps) in the rpb 2 gene, and 6 % (78 / 1244 nucleotides, eleven gaps) in tef 1 - α gene (Table 2 ). Additionally, the differences between our isolate with T. strophariensis are 4 % (29 / 610 nucleotides, four gaps) in ITS region, 4 % (46 / 1080 nucleotides, no gaps) differences in rpb 2 gene, and 5 % (65 / 1244 nucleotides, twenty-five gaps) differences in tef 1 - α gene also supported T. viridistromatis to be a distinct species compared to T. strophariensis and T. britannicum respectively.

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 5. Morphology of Trichoderma viridistromatis (HGUP 24-0004, GUCC 24-0005) a, b diseased in the field, c fresh stromata on natural substrate d cortical and subcortical tissues e ascomatal tissue in section f asci with ascospores g, h ascospores. Scale bars: 10 mm (a – c); 1,000 μm (d); 50 μm (e); 20 μm (f – h).

Figure 6. a cultures on MEA (five days) b cultures on PDA (five days) c cultures on SNA (four days) d conidiophores e phialides f conidia. Scale bars: 10 μm (d, e); 5 μm (f).