Double Mutations Drive Multiple Resistances to Herbicides in Greek Rigid Ryegrass (<i>Lolium rigidum</i> Gaudin)

Based on the complaints of malt barley growers about the insufficient control of rigid ryegrass (<i>Lolium rigidum</i> Gaudin) after applying the ACCase inhibitor pinoxaden, a survey was conducted during the early spring growing season of 2019/20; 20 barley fields located in Thessaloniki and 20 fields in Serres were marked with poor weed control levels. Before the barley harvest, representative weed seeds were collected from all 40 fields. After performing seed germination tests, fourteen populations (six from Thessaloniki and eight from Serres) with the highest seed germination ability were selected for further study. The whole-plant dose–response assays conducted in 2019–2020 indicated that most of the populations were multi-resistant to ACCase and ALS inhibitors. The estimated GR₅₀ values (the herbicide dose required to reduce the fresh weight of treated plants by 50%) for pinoxaden and mesosulfuron-methyl + iodosulfuron-methyl-sodium ranged from 1.15 to 52.41 g ai ha⁻¹ and 4.75 to 31.25 g ai ha⁻¹, respectively. Furthermore, the sequencing of <i>acccase</i> gene fragments from plants that survived pinoxaden application revealed that 11 out of 14 plant populations had a double <i>accase</i> point mutation at Ile1781 and Ile2041 codons. In addition, the sequencing of <i>als</i> gene fragments from the plants that survived mesosulfuron-methyl + iodosulfuron-methyl-sodium application revealed that 11 out of 14 plant populations had a point mutation at the Pro197 codon and 2 of them had a second <i>als</i> mutation at the Trp574 codon. These findings indicate that <i>L. rigidum</i> populations are multi-resistant to ACCase and ALS inhibitors, with individuals exhibiting either double <i>accase</i> or double <i>als</i> mutations.