Nontarget site-based resistance to nicosulfuron and identification of candidate genes in Cucumis melo L. var. agrestis Naud. via RNA-Seq transcriptome analysis

Herbicide resistance is a worldwide concern for weed control. Cucumis melo L. var. agrestis Naud. (C. melo) is an annual trailing vine weed that is commonly controlled by nicosulfuron, acetolactate synthase (ALS)-inhibiting herbicides. However, long-term use of this herbicide has led to the emergence of resistance and several nicosulfuron resistant populations of C. melo have been found. Here we identified a resistant (R) C. melo population exhibiting 7.31-fold resistance to nicosulfuron compared with a reference sensitive (S) population. ALS gene sequencing of the target site revealed no amino acid substitution in R plants, and no difference in enzyme activity, as shown by ALS activity assays in vitro. ALS gene expression was not significantly different before and after the application of nicosulfuron. Pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion reduced nicosulfuron resistance in the R population. RNA-Seq transcriptome analysis was used to identify candidate genes that may confer metabolic resistance to nicosulfuron. We selected genes with annotations related to detoxification functions. A total of 20 candidate genes (7 P450 genes, 1 glutathione S-transferase (GST) gene, 2 ATP-binding cassette (ABC) transporters, and 10 glycosyltransferase (GT)) were identified; 12 of them (7 P450s, 1 GST, 2 ABC transporters, and 2 GTs) were demonstrated significantly differential expression between R and S by quantitative real-time RT-PCR (qRT–PCR). Our findings revealed that the resistance mechanism in C. melo was nontarget-site based. Our results also provide a valuable resource for studying the molecular mechanisms of weed resistance.