Multi-gene metabolic detoxification conferring cyhalofop-butyl resistance in Echinochloa crus-galli (Barnyard grass)

With the long-term and widespread application of cyhalofop-butyl in Chinese rice cultivation systems, resistant barnyard grass (Echinochloa crus-galli) populations have been continuously detected and reported, indicating that herbicide resistance has become a serious threat to the unit area yield of rice. In this study, we aimed to characterize the resistance level of a suspected cyhalofop-butyl-resistant barnyard grass population, elucidate its resistance mechanisms, and identify potential resistance genes. Whole-plant bioassays confirmed cyhalofop-butyl resistance, showing a resistance index of 7 and a GR₅₀ of 240 g a.i. ha^-1. Sequencing of the EcACCase gene coupled with RT-qPCR analysis revealed no mutations at known resistance-conferring sites in cyhalofop-butyl-resistant plants and EcACCase expression levels did not differ between cyhalofop-butyl-resistant and susceptible plants. Consequently, target-site resistance was excluded as the resistance mechanism. To probe potential metabolic resistance, a multifaceted strategy integrating transcriptomics, NBD-Cl/malathion pretreatment, RT-qPCR, and molecular docking simulations was utilized. Synergist assays indicated that NBD-Cl and malathion pretreatment reduced the GR₅₀ value of the resistant population to 179 and 75 g a.i. ha^-1, respectively, indicating the presence of GST- and P450-mediated metabolic resistance. Ultimately, this study identified several candidate genes possibly linked to cyhalofop-butyl resistance, encompassing two CYP450 genes, one GST gene, two GT genes, and one ABC transporter gene, among which CYP72A616, GT2, and GT4 represent the first report of their association with cyhalofop-butyl resistance. These findings expand the catalog of candidate genes for non-target-site resistance to cyhalofop-butyl, providing a theoretical basis for understanding resistance mechanisms and developing management strategies.