Core microbes in Cordyceps militaris sclerotia and their nitrogen metabolism-related ecological functions

infects insects and forms sclerotia within the insect remains, establishing insect-microbe complexes. Here, . sclerotia samples from a single location in China over a 5-year period were subjected to high-throughput DNA sequencing, and the core microbes (which were stably enriched in the sclerotia over the 5 years) were identified. Next, seven bacterial strains were isolated from the sclerotia, their biochemical characteristics were assessed, and they were co-cultured with to study their effects on metabolite production and biomass. Furthermore, the effects of NH , NO , and peptone media on were compared. The results showed that , , , , , , , , and were the core microbes. Although co-culture of with the seven bacterial strains isolated from the sclerotia did not directly increase the cordycepin level, they all had NO reduction ability, and four had urea decomposition ability. Meanwhile, in NH medium had an increased cordycepin level compared to in the other two media. From this, we inferred that bacteria in the sclerotia can convert NO to NH , and then cordycepin is produced using NH , which was confirmed by RNA-seq and real-time fluorescence quantitative PCR. Thus, bacteria in the sclerotia may indirectly affect the metabolite production by regulating nitrogen metabolism. In summary, there are stable core microbes in the sclerotia, and they may directly and indirectly affect the growth and metabolite production of . The model species is rich in therapeutic compounds. It has recently been demonstrated that symbiotic microbes in sclerotia affect growth, development, and secondary metabolite production. In this study, core microbes were identified based on sclerotia samples obtained from the same site over 5 years. Additionally, bacterial strains isolated from sclerotia were found to affect metabolite production and nitrogen utilization, based on functional tests. Moreover, based on the bacterial nitrogen metabolism capacity in the sclerotia and its influence on metabolite production, we deduced that bacteria in the sclerotia can indirectly affect metabolite production by regulating nitrogen metabolism. This is the first report on how bacteria in the sclerotia affect metabolite production from the perspective of the nitrogen cycle. The results increase our understanding of microbial functions in sclerotia.