The nortriterpenoid helvolic acid (HA) has potent antibiotic activities and can be produced by different fungi, yet HA function remains elusive. Here, we report the chemical biology of HA production in the insect pathogen Metarhizium robertsii. After deletion of the core oxidosqualene cyclase gene in Metarhizium, insect survival rates were significantly increased compared to those of insects treated with the wild type and the gene-rescued strain during topical infections but not during injection assays to bypass insect cuticles. Further gnotobiotic infection of axenic Drosophila adults confirmed the HA contribution to fungal infection by inhibiting bacterial competitors in an inoculum-dependent manner. Loss of HA production substantially impaired fungal spore germination and membrane penetration abilities relative to the WT and gene-complemented strains during challenge with different Gram-positive bacteria. Quantitative microbiome analysis revealed that HA production could assist the fungus to suppress the Drosophila cuticular microbiomes by exerting a bacteriostatic rather than bactericidal effect. Our data unveil the chemical ecology of HA and highlight the fact that fungal pathogens have to cope with the host cuticular microbiomes prior to successful infection of hosts. IMPORTANCE Emerging evidence has shown that the plant and animal surface microbiomes can defend hosts against fungal parasite infections. The strategies employed by fungal pathogens to combat the antagonistic inhibition of insect surface bacteria are still elusive. In this study, we found that the potent antibiotic helvolic acid (HA) produced by the insect pathogen Metarhizium robertsii contributes to natural fungal infection of insect hosts. Antibiotic and gnotobiotic infection assays confirmed that HA could facilitate fungal infection of insects by suppression of the host cuticular microbiomes through its bacteriostatic instead of bactericidal activities. The data from this study provide insights into the novel chemical biology of fungal secondary metabolisms.

Endophytic fungi of medicinal plants are symbiotic with the host and play an important role in determining metabolites. To understand the relationship between the accumulation of Sophora alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi, here we collected samples from S. alopecuroides at four developmental stages (adult, flowering, podding, and mature) and different organs (roots, stems, leaves, and seeds) at the mature stage. We then used high-performance liquid chromatography-mass spectrometry and high-throughput sequencing on the internal transcribed spacer region to identify the medicinal compounds and endophytic fungal communities in each sample. The endophytic fungal community characteristics and accumulation of medicinally bioactive compounds of S. alopecuroides varied with the host's developmental stages and organs, with the highest total alkaloids content of 111.9 mg/g at the mature stage. Membership analysis and network connection analysis showed a total of 15 core endophytic fungi in different developmental stages and 16 core endophytic fungi in different organs at the mature stage. The unclassified Ascomycota, Aspergillus, and Alternaria were significantly and positively correlated with the medicinal compounds of S. alopecuroides at the mature stage (r > 0.6 or r < -0.6; P < 0.05). In this study, we identified key endophytic fungal resources that affect the content of medicinally bioactive compounds in S. alopecuroides. This discovery could lay the foundation for enhancing the yield of medicinally bioactive compounds in S. alopecuroides and the development and application of functional endophytic fungi.IMPORTANCESophora alopecuroides is a traditional Chinese herbal medicine. The major medicinal chemicals are considered to be quinolizidine alkaloids. Quinolizidine alkaloids have been widely used for the treatment of tumors, dysentery, and enteritis. Previous studies have found that endophytic fungi in S. alopecuroides can promote the accumulation of host quinolizidine alkaloids. However, the relationship between the accumulation of S. alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi remains unclear. In this study, we screened the key endophytic fungal resources affecting the content of medicinally bioactive compounds and laid the foundation for subsequent research on the mechanism by which endophytic fungi promote the accumulation of medicinally bioactive compounds in S. alopecuroides.