Mutation of Trp-574-Leu ALS gene confers resistance of radish biotypes to iodosulfuron and imazethapyr herbicides

ABSTRACT. Acetolactate synthase inhibitors are the main group of herbicides used in winter crops in Southern Brazil where their intensive use has selected for herbicide-resistant biotypes of radish. The resistance affects the efficacy of herbicides, and identifying the resistance mechanism involved is important for defining management strategies. The aim of this study was to elucidate the resistance mechanism of radish biotypes by quantifying the enzyme activity, ALS gene sequencing and evaluating the response of biotypes to iodosulfuron and imazethapyr herbicide application after treatment with a cytochrome P450 monooxygenase inhibitor. The susceptible (B1) and resistant (B4 and B13) biotypes were from wheat fields in the Northwest of Rio Grande do Sul State. The results demonstrated that the enzyme affinity for the substrate (KM) was not affected in biotypes B4 and B13 but that the Vmax of the resistant biotypes was higher than that of biotype B1. The resistant biotypes showed no differential metabolic response to iodosulfuron and imazethapyr herbicides when inhibited by malathion and piperonyl butoxide. However, gene sequencing of ALS showed a mutation at position 574, with an amino acid substitution of tryptophan for leucine (Trp-574-Leu) in resistant biotypes.

摘要。乙酰乳酸合酶（Acetolactate synthase, ALS）抑制剂是巴西南部冬作物田中应用的主要除草剂类群，由于长期高强度施用，该类除草剂已筛选出具有除草剂抗性的萝卜生物型。此类抗性会降低除草剂的防控效果，明确其抗性机制对制定田间管理策略至关重要。本研究旨在通过酶活性定量检测、ALS基因测序，以及评估经细胞色素P450单加氧酶抑制剂处理后，萝卜生物型对碘甲磺隆（iodosulfuron）与咪唑乙烟酸（imazethapyr）的药剂响应，解析该类抗性萝卜生物型的抗性机制。供试的敏感生物型（B1）与抗性生物型（B4、B13）均采自巴西南里奥格兰德州西北部的小麦田。结果表明，抗性生物型B4与B13的酶对底物的亲和性（KM）未受显著影响，但二者的最大反应速率（Vmax）显著高于敏感生物型B1。经马拉硫磷（malathion）与胡椒基丁醚（piperonyl butoxide）这两种细胞色素P450单加氧酶抑制剂处理后，抗性生物型对碘甲磺隆与咪唑乙烟酸并未表现出差异化的代谢响应。然而，ALS基因测序结果显示，抗性生物型的ALS基因在第574位位点发生突变，氨基酸发生色氨酸替换亮氨酸的突变（Trp-574-Leu）。