Chemical clues to infection: Metabolome differentiation underlies host colonization of potential biocontrol agents from the entomopathogenic genus Cordyceps

Cordyceps species are widely recognized as entomopathogens, with some developed as biocontrol agents. These fungi produce bioactive metabolites contributing to their ecology and pathogenicity, yet their specific role during host infection remains poorly understood. To gain insights into how these fungi control their insect hosts, we investigated the metabolome and virulence traits of two potential biocontrol agents from the genus Cordyceps . Virulence assays on beet armyworms ( Spodoptera exigua , Lepidoptera) revealed varying levels of pathogenicity, with C. javanica BCC 82944 exhibiting a higher virulence than C. blackwelliae BCC 37653, which revealed intermediate pathogenicity. Using state-of-the-art metabolomics, combined with 3D electron diffraction (3D ED) crystallography and comprehensive 1D/2D NMR spectroscopy, we identified diverse metabolites, including the cyclodepsipeptides beauverolides. Cordyceps javanica exhibited remarkable beauverolide diversity, featuring various amino acid rearrangements and fatty acid chain lengths, including three previously undescribed derivatives ( 1 – 3 ). While the main products of C. blackwelliae were diketopiperazines, feature-based molecular networking (FBMN) analysis uncovered the production of unprecedented beauverolides. To explore the functional relevance of these unique natural products, we analyzed the original insect cadavers from which each fungus was isolated. Our results revealed the presence of beauverolides and beauvericins in the host tissue, providing for the first time direct evidence of their involvement in fungal colonization during infection. Notably, not all beauverolides induced insect mortality in vitro , suggesting differentiated biological functions dependent on their amino acid organization. These findings indicate that distinct secondary metabolites may contribute to specific steps of the infection process. Moreover, the detection of species-specific metabolite profiles in the insect corpses suggests that Cordyceps species have developed chemically divergent infection strategies, possibly shaped by host specificity and ecological niche adaptation.