Supplementary material (Penicillium-mediated toxicity and microbial imbalance induce fruiting body abortion in artificial cultivation of Ophiocordyceps sinensis)

The Chinese caterpillar fungus, Ophiocordyceps sinensis, is a valuable medicinal entomopathogenic fungus, but artificial cultivation is hindered by fruiting body abortion, severely impacting yield and quality. The interactions between the fungus, its host insects, and soil are critical for its development. Multi-omics analyses were conducted to compare normally and abnormally developing samples, examining microbial communities and metabolites in endophytic larvae and the surrounding mycosphere soil. Abnormal stroma development significantly altered microbial ecology. It increased fungal diversity in both larvae and soil, while reducing bacterial diversity within larvae. The microbial composition shifted markedly, characterized by a high enrichment of Penicillium fungi and a depletion of Bacillus bacteria in and around abnormal samples. Cross-kingdom microbial network analysis in these samples showed fewer connections and lower stability. Metabolomic profiling revealed significant enrichment of the antibiotics N1-hydroxy-roquefortine C and glandicoline A, both derivatives of roquefortine C, alongside the upregulation of multiple antibiotic biosynthesis pathways. Partial Least Squares Structural Equation Modelling indicated that Penicillium toxicity likely induces microbial dysbiosis, and this disruption of microbial homeostasis is the primary cause of fruiting body abortion. The findings suggest that Penicillium overgrowth is detrimental to normal stroma formation. Conversely, Bacillus may play a crucial role in suppressing Penicillium, offering a potential target for biocontrol strategies. This study is the first to elucidate the key role of Penicillium-mediated toxicity and microbial imbalance in the developmental disorders of Chinese Cordyceps.