Characterization of acetolactate synthase genes and resistance mechanisms of multiple herbicide resistant Lolium multiflorum

Combining imidazolinone-tolerant wheat with imazamox presents an effective solution to combat weed resistance. However, Lolium multiflorum, a troublesome resistant weed infesting wheat fields, may have developed resistance to imazamox, and the potential resistance mechanisms are intriguing. In this study, we explored the susceptibility of L. multiflorum to imazamox and investigated the resistance mechanisms, including the contribution of the target enzyme acetolactate synthase (ALS) to resistance and the presence of non-target-site resistance (NTSR). Eight L. multiflorum populations suspected of being resistant to imazamox were collected, and six populations exhibited resistance, ranging from 2.45-fold to 16.32-fold. The LmALS1 gene from susceptible population D3 plants and multiple copies of the LmALS gene (LmALS1, LmALS2, LmALS2α, LmALS3, LmALS3α, LmALS3β) from resistant populations D5 and D8 plants were separately amplified. Two mutations (Pro/Gln197 to Thr, Trp574 to Leu) were found in LmALS1 in the resistant populations. Compared to D3, LmALS1 was overexpressed in D5 but not in D8. The presence of LmALS1 mutants (LmALS1-Thr197 and LmALS1- Leu574), along with LmALS2, LmALS3, and their subunits, contribute to the resistance phenotype by increasing bonding energies, weakening hydrogen bonds, or decreasing protein binding pocket volumes and surface area. Additionally, D5 and D8 populations exhibited multiple resistance (>40-fold) to three other ALS inhibitors: pyroxsulam, flucarbazone-sodium, and mesosulfuron-methyl. Pre-treatment with malathion and 4-chloro-7-nitrobenzoxadiazole (cytochrome P450 monooxygenase and glutathione S-transferase inhibitors respectively) reversed the resistance of the D8 population and partially reversed the resistance of the D5 population to imazamox. This study characterizes ALS genes and extends our knowledge into the ALS resistance mechanisms involved in L. multiflorum. It also deepens our understanding of the complex diversification resistance mechanisms, thereby facilitating advances in weed resistance management strategies in wheat fields.