First Report of <i>Lecanicillium aphanocladii</i> Causing Rot of <i>Morchella importuna</i> in China

Morchella importuna is a highly valued edible mushroom due to its rich nutritional and medicinal properties (Wen et al. 2019). In March 2023, a severe rot disease was detected at a mushroom farm in Nanchang, Jiangxi Province, China, with a disease incidence rate >30%. Infected fruiting bodies of M. importuna initially exhibited brown lesions, which were often covered with white mycelia, and eventually rotted. Tissue from lesion margins were placed onto potato dextrose agar (PDA) and incubated at 25℃ for three days. Ten isolates with similar morphological characteristics were obtained. Colonies on PDA were raised and white on the front side, purplish red on the reverse, and they produced diffusing purple pigments. Conidia were borne at the tips of conidiogenous cells, oval to subglobose, and 3.4 to 6.7 × 1.2 to 2.6 μm in size (n = 30). Based on morphological characteristics, the pathogen was tentatively identified as Lecanicillium sp. To further confirm the species, genomic DNA was extracted from two representative isolates (JFRL 03-5292 and JFRL 03-5293) using the CTAB method. The internal transcribed spacer (ITS) region, large subunit (LSU), small subunit (SSU), translation elongation factor 1-alpha (TEF1-ɑ), and RNA polymerase I beta subunit (RPB1) genes were amplified and sequenced using the primers ITS5/ITS4, LR7/LR0R, NS1/NS4, EF1-983F/EF1-2218R, and CRPB1A/RPB1Cr, respectively (Zhou et al. 2018). Nucleotide sequences were deposited in GenBank under accession numbers PV549406-PV549407 (ITS), PV549408-PV549409 (LSU), PV549404-PV549405 (SSU), PV567539-PV567540 (TEF1-ɑ), and PV550730-PV550731 (RPB1). A BLASTn homology search revealed 100% identity of these sequences with the LSU (KM283787, 1271/1271 nt), SSU (KM283763, 1008/1008 nt), TEF1-ɑ (KM2837811, 903/903 nt), and RPB1 (KM283833, 715/715 nt) sequences of Lecanicillium aphanocladii CBS 797.84. Maximum likelihood analyses were conducted on the combined ITS, LSU, SSU, TEF1-ɑ and RPB1 dataset using the IQtree web server (Trifinopoulos et al. 2016). In the resulting phylogenetic tree, isolates clustered with L. aphanocladii in a clade supported by 100% bootstrap values. Therefore, the fungus was conclusively identified as L. aphanocladii based on both morphological and molecular characteristics. To assess pathogenicity, six fruiting bodies of M. importuna (grown in a greenhouse) were selected. A conidial suspension (1 × 10⁷ conidia/ml) of isolate JFRL 03-5292 was sprayed onto six fruiting bodies, while another six fruiting bodies were treated with sterile water as controls. All treated fruiting bodies were incubated at 25℃ with 80%-90% humidity, and the experiment was repeated twice. After three days, inoculated fruiting bodies inoculated with L. aphanocladii exhibited symptoms similar to those previously observed, whereas controls remained asymptomatic. L. aphanocladii was consistently reisolated from symptomatic fruiting bodies and confirmed through morphological and molecular analyses, thereby fulfilling Koch’s postulates. L. aphanocladii is recognized as a significant pathogen causing rot (or cobweb disease) on various cultivated edible mushrooms in China, including M. sextelata, Tremella fuciformis, Sparassis latifolia, and Schizophyllum commune (Lin et al. 2024; Liu et al. 2018; Lv et al. 2022; Xia et al. 2024). To the best of our knowledge, this is the first report of L. aphanocladii causing rot on M. importuna in China, thereby expanding the known host range of this fungus. This pathogen poses a threat to M. importuna production in China, and its accurate identification is necessary to develop effective management strategies.