The Insect Pathogenicity of Cordyceps militaris - a molecular, metabolic, ecological, and phylogenetic study

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Cordycepin, or 3'-deoxyadenosine, is a secondary metabolite secreted by the cosmopolitan entomopathogenic "caterpillar fungus" Cordyceps millitaris and closely related species. The metabolite is well known for its anti-inflammatory effects and thus has been used as both a traditional Chinese medicine for centuries and a focus of pharmacological research in recent years. However, studies on the ecological roles of cordycepin and other secondary metabolites in the infection of the insect larval host and the involvement of these on the sexual development which occurs following this process are in their infancy. Culture degeneration, a phenomenon observed in a broad range of ascomycete fungi, is the loss of ability to develop sexually and perform parts of secondary metabolism following subsequent subculturing in the vegetative state. This is a significant problematic issue in cordycepin production from laboratory and industrial-based cultures.Here, a Chinese strain of Cordyceps militaris, CM2 (MycoMedica) with high cordycepin production was successively sub-cultured on potato dextrose agar. Targeted analysis of cordycepin and associated metabolite pentostatin by liquid chromatography (LC)-coupled mass spectrometry (MS), combined with a gene expression analysis using RT-qPCR provided evidence of a link between phenotypes and gene expression of both sexual development and secondary metabolite biosynthesis. Cordycepin production was greatly reduced in degenerated, multiply-subcultured samples; although production was partially restored following long-term transfer of these to a new media type based on the host substrate (silk pupa powder agar). Significant decreases in the expression of a sexual development gene, as well as cordycepin and related biosynthesis genes were also detected. Untargeted LC-MS analysis was performed to compare degenerated and non-degenerated strains.