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

Abstract

ABSTRACT Cordyceps militaris infects insects and forms sclerotia within the insect remains, establishing insect–microbe complexes. Here, C . militaris 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 C. militaris sclerotia, their biochemical characteristics were assessed, and they were co-cultured with C. militaris to study their effects on C. militaris metabolite production and biomass. Furthermore, the effects of NH 4 , NO 3 , and peptone media on C. militaris were compared. The results showed that Rhodococcus , Phyllobacterium , Pseudomonas , Achromobacter , Ensifer , Stenotrophomonas , Sphingobacterium , Variovorax , and Acinetobacter were the core microbes. Although co-culture of C. militaris with the seven bacterial strains isolated from the sclerotia did not directly increase the cordycepin level, they all had NO 3 reduction ability, and four had urea decomposition ability. Meanwhile, C. militaris in NH 4 medium had an increased cordycepin level compared to C. militaris in the other two media. From this, we inferred that bacteria in the sclerotia can convert NO 3 to NH 4 , and then cordycepin is produced using NH 4 , which was confirmed by RNA-seq and real-time fluorescence quantitative PCR. Thus, bacteria in the sclerotia may indirectly affect the C. militaris metabolite production by regulating nitrogen metabolism. In summary, there are stable core microbes in the C. militaris sclerotia, and they may directly and indirectly affect the growth and metabolite production of C. militaris . IMPORTANCE The model Cordyceps species Cordyceps militaris is rich in therapeutic compounds. It has recently been demonstrated that symbiotic microbes in sclerotia affect Cordyceps’ growth, development, and secondary metabolite production. In this study, core microbes were identified based on C. militaris sclerotia samples obtained from the same site over 5 years. Additionally, bacterial strains isolated from C. militaris 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 C. militaris metabolite production, we deduced that bacteria in the sclerotia can indirectly affect C. militaris metabolite production by regulating nitrogen metabolism. This is the first report on how bacteria in the sclerotia affect C. militaris metabolite production from the perspective of the nitrogen cycle. The results increase our understanding of microbial functions in C. militaris sclerotia.