Metabolic nontarget-site mechanism confers dual resistance to cyhalofop-butyl and imazamox in Leptochloa chinensis from China

Chinese sprangletop ( Leptochloa chinensis (L.) Nees) has emerged as a dominant grass weed in rice fields, representing a serious risk to crop productivity because of its rapidly increasing resistance to herbicides. A putative resistant population, MASHS7 (R), was collected from an imidazolinone-tolerant rice field, where it had survived applications of cyhalofop-butyl (CYB) and imazamox (IMA) at the field-recommended rates. Dose–response assays indicated that compared with the susceptible (S) population, the R population developed resistance to CYB (26.29-fold) and IMA (5.50-fold). Additionally, both the cytochrome P450 (P450) inhibitor malathion and the glutathione S -transferase (GST) inhibitor 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) partially reversed the resistance of the R population to CYB and IMA. Target-site resistance mechanism analysis revealed that the R plants carried a Trp-to-Cys mutation at codon 1999 of ACCase , with no mutations in the ALS gene, and exhibited no overexpression of any target gene. Compared with the S plants, the R plants metabolized CYB, cyhalofop acid (CYA) and IMA faster. RNA sequencing analysis and quantitative real-time PCR (qPCR) validation confirmed that two P450s ( CYP709B2 and CYP81Q32b ) and one GST ( GST4 ) gene were constitutively upregulated in the R plants. Molecular docking results predicted favorable binding interactions of these enzymes with CYA and IMA.