Effects of Supplementation with Different Concentrations of Selenium on Fruiting Bodies and Substrate Microbial Community Dynamics in Pleurotus ostreatus Cultivation

To investigate the effects of substrate selenium on mushroom growth, nutritional quality, and associated microbial community structure, the thermo-tolerant Pleurotus ostreatus cultivar “Daye 39” was used as the experimental material. Sodium selenite was applied at gradient concentrations (0–4000 mg·kg−1), and the total selenium, organic selenium, crude protein, and soluble polysaccharide contents of the fruiting bodies were determined across successive flushes. High-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer (ITS) regions was performed on the substrate, and selenium-tolerant bacterial strains were isolated and identified. Our results showed that substrate selenium concentrations ranging from 80 to 400 mg·kg−1 significantly increased fruiting body yield by 8.60% to 13.87%, whereas concentrations equal to or exceeding 600 mg·kg−1 resulted in progressive yield declines. Both total and organic selenium contents in the fruiting bodies increased markedly after the third flush, with the organic selenium proportion stabilizing at ≥65%, predominantly in the forms of selenomethionine (81.6%). The crude protein content increased from 25.52 g·100 g−1 in the first flush to 42.99 g·100 g−1 in the eighth flush. Selenium caused the relative abundance of Pseudomonadota increasing sharply from 34.35% in the control to 74.76% in the Se2–Se7 treatments, whereas Bacillota decreased from 38.96% to 15.16%. The fungal community was consistently dominated by P. ostreatus at all timepoints throughout the experiment. Two highly efficient selenium-transforming bacterial strains, Cedecea neteri S1 and Pseudomonas composti S15, both belonging to the phylum Pseudomonadota, were successfully isolated from the high-selenium substrate and attained organic selenium proportions of 98.02% and 99.23% in their cells, respectively. The present study elucidates the dual role of selenium in simultaneously increasing the nutritional value of edible mushrooms and reshaping the substrate microbial ecology. Our findings suggest that the recommended substrate selenium concentration of 160 mg·kg−1 is appropriate to optimize selenium-enriched P. ostreatus cultivation techniques and to explore functional microbial resources relevant to selenium biofortification.