First Report of Didymium bahiense on Cultivated Schizophyllum commune in China

From the paper

Abstract

Schizophyllum commune Fr., known as split-gill mushroom, is an edible, medicinal mushroom widely distributed in the world. Commercial cultivation of S. commune is rapidly increasing in China since the 1980s. In October 2022, a slime mold disease was found in S. commune mushroom houses in Wanan County, Ji’an City, Jiangxi Province, China. Disease incidence ranged from 5 to 12% of cultivation bags in houses, resulting in estimated 10 to 30% yield losses. The substrate and mushroom pilei in infected cultivation bags were initially covered by brown, vein-like plasmodia. Plasmodia aggregated and produced numerous pale gray sporangia when the relative humidity dropped. Sporangia were collected and examined microscopically. Sporangia were stalked, depressed globose, usually two or three fused, white to gray, 0.3 to 0.5 mm in diameter and 1.2 to 1.6 mm high; peridium membranous, covered with white, stellate lime crystals; stalk slender, tapering upward, orange ochraceous above, dark brown below; pseudocolumella a thickened basal plate, calcareous, looking like a stalked columella; capillitium delicate, branching, brown with pale tips; spores 8 to 10 μm in diameter, brown, densely warted, and with clusters of dark warts. To isolate the causal agent, five sporangia were gently crushed in a 1.5 ml Eppendorf tube, and 500 μl of sterilized distilled water was added to make a spore suspension. The tube was incubated at 25°C in the dark and spores were successfully germinated after 13 h of inoculation. Then, 50 μl of the suspension was cultured on 1% water agar media, where plasmodia were seen after 17 days. Plasmodia were repeatedly purified by transferring the leading edge to new 1% water agar media with antibiotics (penicillin and streptomycin at final concentrations of 100 μg/ml) and fed sterilized oat powder. One of the mature plasmodia could develop into sporangia after exposure to diffused light for 48 h. The sporangia had identical characteristics as described above, and were deposited in Fungal Herbarium of Jiangxi Agricultural University (HFJAU14001). Genomic DNA was extracted from the sporangia following Schnittler et al. (2020). The 18S rRNA and EF1A genes were amplified and sequenced with primers S1/SU19R and PB1F/PB1R, respectively (Novozhilov et al. 2014; Schnittler et al. 2017). The gene sequences were deposited in GenBank (OR214927 and OR237844, respectively). The 18S rRNA gene BLASTn had 98.27% similarity with that of Didymium bahiense (KP323372), and the EF1A gene had 90.85% similarity with Didymium melanospermum (OP616495). A two-gene phylogenetic analysis showed that HFJAU14001 was well clustered with D. bahiense. Based on morphological and molecular data, the causal agent was identified as D. bahiense Gottsb. To confirm pathogenicity, five cultivation bags of S. commune before primordium formation were inoculated with 15 mm agar discs covered with plasmodia from HFJAU14001. Control bags were inoculated with sterilized agar discs. All bags were cultured in a mushroom house at 24 to 26°C and 90 to 95% relative humidity. All treated bags had the same symptoms; controls had no symptoms. The gray sporangia on substrates and S. commune fruiting bodies were confirmed to be D. bahiense based on morphological and molecular characteristics. Many Myxogastria species have been reported to cause slime mold disease on cultivated mushrooms, such as Physarella oblonga, Stemonaria longa, and Stemonitis splendens on shiitake (Lee et al. 2014; Li et al. 2017; Zhang et al. 2018), and Physarum galbeum on Grifola frondosa (Dai et al. 2023). Plasmodia feed on fungal hyphae and can secrete lytic enzymes that cause wilting or rotting of mushroom fruiting bodies. S. commune usually has high resistance to adverse environmental and biotic conditions, and few diseases have been reported during cultivation. To our knowledge, this is the first report of D. bahiense causing slime mold disease on cultivated S. commune, which may harm S. commune production in China.