Non-target-site resistance to Clodinafop-Propargyl in Polypogon fugax involving PfCYP76P15

Polypogon fugax, a problematic weed in rapeseed fields across China, has evolved resistance to clodinafop-propargyl, posing a significant threat to agricultural production. In this study, a putative clodinafop-propargyl-resistant (R) population was collected from a rapeseed field in Qingshen County, Sichuan Province, China, with a long history of repeated clodinafop-propargyl application. Whole-plant dose-response bioassays confirmed that the R population had evolved moderate-level metabolic resistance to clodinafop-propargyl. Sequencing of the acetyl-CoA carboxylase (ACCase) gene revealed no known resistance-conferring mutations. Pretreatment with the cytochrome P450 (P450) inhibitor malathion and the glutathione S-transferase (GST) inhibitor 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) significantly reversed clodinafop-propargyl resistance in the R population. Quantitative real-time PCR (qRT-PCR) analysis identified the P450 gene PfCYP76P15 significantly upregulated in the R population. Heterologous expression of PfCYP76P15 in yeast enhanced tolerance to clodinafop-propargyl, and its overexpression in transgenic rice similarly increased resistance compared to control plants. Molecular docking analysis suggested that PfCYP76P15 may metabolize clodinafop acid. Consistently, high-performance liquid chromatography analysis showed that clodinafop acid content was significantly lower in PfCYP76P15-overexpressing (PfCYP76P15-OE) rice plants than in GFP-overexpressing (GFP-OE) controls following herbicide treatment. Collectively, these results indicate that resistance to clodinafop-propargyl in this P fugax population is conferred by non-target-site resistance, and that PfCYP76P15 is one important contributing gene.