AbstractThe WSSA Group 15 (HRAC Group K3) herbicide flufenacet is a key compound in weed resistance management, primarily used for PRE control of grass weeds in winter cereal–based crop rotations in Europe. Although resistance to compounds of its mechanism of action (inhibition of the synthesis of very-long-chain fatty acids) generally evolves slowly, reduced flufenacet efficacy due to enhanced glutathione transferase (GST) activity has been described in several blackgrass (Alopecurus myosuroides Huds.) populations. The present study aimed to better understand of the mechanism of flufenacet detoxification in A. myosuroides. Therefore, we characterized four A. myosuroides populations with different levels of flufenacet sensitivity. Flufenacet degradation was significantly slowed down in a sensitive population and a population with reduced flufenacet sensitivity by the use of the GST inhibitors tridiphane and ethacrynic acid at sublethal rates. Finally, an RNA sequencing (RNA-seq) study with the four A. myosuroides populations was conducted. In total, six differentially expressed GSTs and nine transcription factors as well as a keto-acyl-CoA reductase involved in the biosynthesis of very-long-chain fatty acids were identified as candidate genes among a set of 319 significantly more highly expressed gene-associated contigs. Among a set of 218 contigs with significantly lower expression levels, receptor kinase activity was the most frequent annotation. In summary, the likely GST-mediated reduction in sensitivity evolves in A. myosuroides at a slow rate and can partially be reversed by an interaction between flufenacet and the GST inhibitors tridiphane and ethacrynic acid. This provides further evidence for enhanced GST activity as a key mechanism in flufenacet resistance in A. myosuroides and supports the hypothesis that the six differentially expressed GSTs detected in the present RNA-seq study are potentially involved in flufenacet resistance.