Table_1_Target-Site and Non-target-Site Resistance Mechanisms Confer Multiple and Cross- Resistance to ALS and ACCase Inhibiting Herbicides in Lolium rigidum From Spain.DOCX

Lolium rigidum is one the worst herbicide resistant (HR) weeds worldwide due to its proneness to evolve multiple and cross resistance to several sites of action (SoA). In winter cereals crops in Spain, resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has become widespread, with farmers having to rely on pre-emergence herbicides over the last two decades to maintain weed control. Recently, lack of control with very long-chain fatty acid synthesis (VLCFAS)-inhibiting herbicides has been reported in HR populations that are difficult to manage by chemical means. In this study, three Spanish populations of L. rigidum from winter cereals were confirmed as being resistant to ALS- and ACCase-inhibiting herbicides, with broad-ranging resistance toward the different chemistries tested. In addition, reduced sensitivity to photosystem II-, VLCFAS-, and phytoene desaturase-inhibiting herbicides were confirmed across the three populations. Resistance to ACCase-inhibiting herbicides was associated with point mutations in positions Trp-2027 and Asp-2078 of the enzyme conferring target site resistance (TSR), while none were detected in the ALS enzyme. Additionally, HR populations contained enhanced amounts of an ortholog of the glutathione transferase phi (F) class 1 (GSTF1) protein, a functional biomarker of non-target-site resistance (NTSR), as confirmed by enzyme-linked immunosorbent assays. Further evidence of NTSR was obtained in dose-response experiments with prosulfocarb applied post-emergence, following pre-treatment with the cytochrome P450 monooxygenase inhibitor malathion, which partially reversed resistance. This study confirms the evolution of multiple and cross resistance to ALS- and ACCase inhibiting herbicides in L. rigidum from Spain by mechanisms consistent with the presence of both TSR and NTSR. Moreover, the results suggest that NTSR, probably by means of enhanced metabolism involving more than one detoxifying enzyme family, confers cross resistance to other SoA. The study further demonstrates the urgent need to monitor and prevent the further evolution of herbicide resistance in L. rigidum in Mediterranean areas.

硬黑麦草（Lolium rigidum）是全球范围内危害最为严重的抗除草剂（herbicide resistant, HR）杂草之一，因其极易演化出针对多种作用位点（sites of action, SoA）的多重抗药性与交互抗药性。在西班牙的冬谷类作物田中，针对乙酰乳酸合酶（acetolactate synthase, ALS）与乙酰辅酶A羧化酶（acetyl-CoA carboxylase, ACCase）类除草剂的抗药性已广泛蔓延，近二十年来农户只能依赖芽前除草剂维持杂草防控效果。近期有研究报道，在难以通过化学手段防控的HR种群中，出现了超长链脂肪酸合成（very long-chain fatty acid synthesis, VLCFAS）类除草剂防控失效的情况。本研究中，3个采自西班牙冬谷类作物田的硬黑麦草种群被证实对ALS与ACCase类除草剂具有抗药性，且对受试的各类化学药剂均表现出广谱抗药性。此外，这3个种群均被证实对光系统II、VLCFAS以及八氢番茄红素脱氢酶类除草剂的敏感性显著降低。针对ACCase类除草剂的抗药性与该酶色氨酸2027位（Trp-2027）和天冬氨酸2078位（Asp-2078）的点突变相关，该突变可介导靶标位点抗药性（target site resistance, TSR），而ALS酶中未检测到此类突变。此外，HR种群中谷胱甘肽转移酶Phi类1（glutathione transferase phi (F) class 1, GSTF1）蛋白的同源物含量显著升高，该蛋白是非靶标位点抗药性（non-target-site resistance, NTSR）的功能性生物标志物，这一结论经酶联免疫吸附测定（enzyme-linked immunosorbent assays, ELISA）验证。非靶标位点抗药性的进一步佐证来自剂量反应实验：在使用细胞色素P450单加氧酶抑制剂马拉硫磷进行预处理后，喷施苗后除草剂prosulfocarb的抗药性被部分逆转。本研究证实，西班牙的硬黑麦草种群已演化出对ALS与ACCase类除草剂的多重与交互抗药性，其抗性机制兼具靶标位点抗药性与非靶标位点抗药性特征。此外，研究结果表明，非靶标位点抗药性（可能通过涉及多种解毒酶家族的增强代谢途径介导）可赋予对其他作用位点的交互抗药性。本研究进一步表明，在地中海区域的硬黑麦草种群中，亟需开展抗除草剂演化的监测与防控工作。