Cross- and Multiple-Resistant Perennial Ryegrass (Lolium perenne) to ALS or/and ACCase Inhibitors

Four putative resistant (R) to acetolactate synthase (ALS)- and/or acetyl-CoA carboxylase (ACCase)-inhibiting herbicides and one susceptible (S) perennial ryegrass (Lolium perenne L.) population were collected from winter cereal fields located in north-western Greece and subsequently were evaluated in whole-plant rate-response pot assays to investigate resistance status, patterns, level, and the underlying mechanism of resistance. All putative R populations were found cross-R to ALS inhibitors mesosulfuron-methyl + iodosulfuron-methyl-sodium and pyroxsulam, whereas two of them were also determined as multiple-R to ALS- and ACCase-inhibiting herbicides clodinafop-propargyl and pinoxaden. All R and S populations were effectively controlled with the ACCase inhibitor clethodim. Sequencing of selected ALS gene regions from R plants identified substitutions at Pro-197, Asp-376, and Trp-574 positions previously associated with resistance to ALS-inhibiting herbicides. Sequencing of selected regions of the ACCase gene identified the established resistance-associated substitutions Ile-1781-Leu and Ile-2041-Asn, together with additional amino acid substitutions at Leu-1701, Glu-1874, Asn-1878, and Gly-1946 whose functional contribution to herbicide resistance was not determined in the present study. No substitutions were detected at positions Val-1992, Glu-2039, Asp-2078, or Cys-2088. The presence of the Ile-1781-Leu and Ile-2041-Asn in the ACCase enzyme of the R4.1 plant and Pro-197-His/Pro-197-Leu and Asp-376-His in the ALS enzyme of the R1.1 plant suggests the existence of double mutations, while the respective presence of the Ile-1781-Leu and Pro-197-Leu mutant alleles in the R1.2 plant supports co-existence of multiple-resistance to ACCase and ALS inhibitors, respectively, which is recorded for the first time. In an additional pot experiment, all R and S populations were effectively controlled with preemergence prosulfocarb or chlorotoluron + diflufenican or early postemergence application of flufenacet + diflufenican, suggesting that chemical control options with herbicides having different modes of action still exist to manage multiple-R L. perenne populations.