First Report of Postharvest Rot of Morchella sextelata Caused by Trichoderma polysporum in China

Morchella sextelata is an economically important, commercially cultivated morel species in China, with an annual cultivation area of over 216,000 mu and an annual fresh fruiting-body production of approximately 204,589.752 tons (China Edible Fungi Association, 2024). Fresh fruiting bodies are highly perishable and susceptible to fungal infection during storage, but postharvest diseases of this species remain understudied. In 2025, an unknown postharvest fungal disease was observed on fresh M. sextelata fruiting bodies stored in Guiyang City, Guizhou Province (26°38′49″ N, 106°37′48″ E). The disease was characterized by tissue rot and white mycelial overgrowth and caused economic losses of up to 25%. In February 2025, 58 healthy M. sextelata fruiting bodies were collected from the cultivation base, surface-disinfected with 75% ethanol for 30 s, rinsed with sterile distilled water, and incubated in a humid chamber at 90 ± 5% RH and 18 °C; after 5 days, 80% of fruiting bodies exhibited typical symptoms, including tissue softening and white, floccose mycelia at the pileus–stipe junctions. Diseased tissue pieces (5 × 5 mm) containing the healthy–diseased tissue interfaces were surface-sterilized with 75% ethanol for 30 s and 0.1% NaClO for 1 min, rinsed three times with sterile distilled water, and plated on PDA at 25 °C in the dark. Ten single-spore isolates with similar cultural characteristics were obtained; three representative isolates (GPYJ3, GPYJ4, GPYJ5) that best reflected the range of colony phenotypes observed among the ten isolates and yielded high-quality DNA for sequencing were selected for further morphological and molecular analysis, whereas the remaining seven isolates displayed essentially identical cultural characteristics and were therefore not further characterized to avoid redundancy. After 7 days at 25 °C on PDA, colonies were white, villous, loose floccose, flat-spreading, with white obverse and faint pale yellow reverse; at 15 days, dense white granular conidial masses formed, and reverse pigment deepened to pale yellowish-brown. On SNA, colonies were sparsely villous, thin translucent, radially spreading with irregular margins, and scattered white punctate conidia. On CMD, colonies were loose floccose with fluffy aerial mycelia, uniform white, and no obvious conidial masses. Microscopically, conidiophores were septate, colorless, laterally branched; phialides ampulliform, mostly densely verticillate in 1–4 per whorl and clustered. Conidia were globose, hyaline, aseptate, 4.72 ± 0.58 × 3.39 ± 0.42 μm (n=100), consistent with Trichoderma polysporum (Druzhinina et al. 2004). Genomic DNA was extracted from fresh mycelia. ITS, TEF1, and RPB2 genes were amplified using primers ITS5/ITS4 (White et al. 1990), 983F/2218R (Carbone and Kohn, 1999), and fRPB2-5f/RPB2-7cR (Liu et al. 1999), respectively. The 20 μL PCR system contained 1 μL each forward/reverse primer, 10 μL 2× Rapid Taq Master Mix, 1 μL template DNA, and 7 μL sterile ddH₂O. Amplification conditions: 94 °C for 5 min; 30 cycles of 94 °C (1 min), annealing (55 °C for ITS, 58 °C for TEF1 and RPB2, 1 min), 72 °C (1 min); final extension at 72 °C for 5 min. PCR products were sequenced by Qingke Biotech (Guizhou). BLAST analysis showed 98.32%-99.86% similarity to T. polysporum. Maximum likelihood phylogeny based on combined ITS-TEF1-RPB2 sequences clustered the three isolates with T. polysporum 8147 and 8232 (100% support). Sequences were deposited in GenBank (ITS: PZ477575, PZ477576, PZ477577; TEF1: PZ502546, PZ502547, PZ502548; RPB2: PZ502549, PZ502550, PZ502551). Koch’s postulates were fulfilled and verified in the present study via spore suspension inoculation. Pathogenicity assays were performed using three representative T. polysporum isolates. Uniform and healthy M. sextelata fruiting bodies were surface-inoculated with spore suspensions of each isolate, with nine biological replicates established per isolate. Fruiting bodies inoculated with sterile spore-free distilled water served as blank controls. All inoculated M. sextelata fruiting bodies exhibited typical disease symptoms at 5 days post-inoculation under incubation conditions of 18 °C and 90 ± 5% relative humidity. T. polysporum was consistently re-isolated from symptomatic diseased tissues of inoculated fruiting bodies, satisfying the key criteria of Koch’s postulates. No disease symptoms were observed on any control fruiting bodies during the entire experimental period. The whole inoculation experiment was independently repeated three times to confirm the stability and reproducibility of the pathogenicity results. T. polysporum has been reported on other edible fungi (Kim et al. 2012). To our knowledge, this is the first report of T. polysporum causing postharvest disease on M. sextelata worldwide, providing critical information for postharvest disease management of morels.