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

Background The Chinese caterpillar fungus, Ophiocordyceps sinensis, is an entomopathogenic fungus widely used medicinallyand as a health supplement, especially in Asia. However, fruiting body abortion occurs during its artificial cultivation. Failure in stroma development of Chinese Cordyceps severely impairs its yield and quality. The interactions between the fungal pathogen (O. sinensis), its host insects, and soil areessential elements for the occurrence and persistence of Chinese Cordyceps.Results Multi-omics analyses revealed that abnormal stroma development affected the microbial community structure and metabolite production in endophytic larvae and external mycosphere soil. Compared with normal samples, abnormal development significantly increasedfungal diversity in both endophytic larvae and external mycosphere soil, whereas bacterial diversity was significantly reduced in endophytic larvae. Meanwhile, the composition and structure of the internal fungal and bacterial communities differed significantly, particularly characterized by the high abundance enrichment of Penicilliumfungi and marked depletion of Bacillusbacteria inside and around the abnormal samples. In the cross-kingdom microbial network of abnormal samples, edges number, average degree, and network density were all lower, indicating reduced network stability. Metabolomic analysis further demonstrated that N1-hydroxy-roquefortine C and glandicoline A, both downstream derivatives of roquefortine C, were signifincantly enriched in the internal and external abnormal samples, respectively, along with the upregulation of multiple antibiotic biosynthesis pathways. Partial Least Squares Structural Equation Modellingshowed that Penicillium toxicity could induce microbial dysbiosis, and that the disruption of microbial homeostasis was the main reason for the abortion of Chinese Cordyceps fruiting bodies.Conclusion Our results revealedthat increased fungal diversity and high abundance of Penicilliumwere detrimental to the normal fruiting body formation. Both microbial community structure and cross-kingdom networks were significantly altered in abnormally developed samples. Penicillin toxicity likely induced microbial dysbiosis in the internal and external microenvironments of O. sinensis, which in turn led to fruiting body abortion. Additionally, Bacillusplay acritical role in the suppression of Penicillium, thud providing a potential avenue for the screening of biocontrol agents in subsequent studies. This study first elucidates the key role of Penicillium-mediated toxicity and microbial imbalance in stroma development disorders of Chinese Cordyceps.