High ‐abundance polyphyllin H in Paris qiliangiana : discovery of a novel CYP51 ‐targeting antifungal agent against Sclerotinia sclerotiorum

Abstract BACKGROUND Paris qiliangiana , a recently discovered member of the Paris genus, is rich in steroidal saponins known for diverse bioactivities, including antimicrobial effects. Plant pathogenic fungi like Sclerotinia sclerotiorum threaten global crop yields. Widespread pathogen resistance to conventional demethylation inhibitor (DMI) fungicides (triazole/imidazole scaffolds) creates an urgent demand for novel antifungal agents with distinct chemical skeletons and unique molecular mechanisms. This work identifies polyphyllin H as a natural steroidal saponin that acts as a novel cytochrome P450 sterol 14 α ‐demethylase (CYP51)‐targeted inhibitor, representing a dual innovation in both chemical scaffold and antifungal mode of action. RESULT A high‐abundance steroidal saponin, polyphyllin H, was isolated from Paris qiliangiana and exhibited potent antifungal activity against S. sclerotiorum , with a median effective concentration (EC 50 ) of 4.11 mg L −1 . Leaf bioassays demonstrated complete protection at 200 mg L −1 . Integrated mechanistic evidence from transcriptomics, CYP51 immunofluorescence, enzymatic activity assays, scanning electron microscopy and molecular docking confirmed that polyphyllin H suppresses CYP51‐mediated ergosterol biosynthesis and disrupts fungal membrane integrity – this CYP51 inhibitory pathway is mechanistically distinct from all commercial synthetic DMI fungicides with azole backbones. Acute oral toxicity tests in mice showed no observable toxic adverse effects at a single oral dose of 500 mg kg −1 . CONCLUSION Polyphyllin H delivers dual novelty in both chemical structure and molecular mechanism: it bears a unique pennogenin glycoside steroidal scaffold absent from existing CYP51 inhibitors, and exerts antifungal activity via a differentiated CYP51 binding mode to block ergosterol synthesis. This natural product provides a sustainable, eco‐friendly template for developing next‐generation fungicides to overcome DMI resistance in phytopathogens. © 2026 Society of Chemical Industry.