稻田千金子对氰氟草酯的抗性机理研究

Leptochloa chinensis (L.) Nees. is a pernicious weed next to Echinochloa crusgalli (L.) Beauv.. Cyhalofop-butyl is one of the main herbicides on L. chinensis (L.) Nees., which was used more than 10 years in some rice grown regions in Jiangxi province. We studied in early days to find that the resistant ratio of L. chinensis (L.) Nees. to cyhalofop-butyl were 33.64～40.86 in paddy fields by direct sowing in successive years in Jiangxi province, and low in paddy fields by alternate cultivated means of direct sowing, throwing rice seedlings and so on. The enhanced metabolism, the mutation of target gene site and excessive expression of target enzyme of herbicide were considered as the main reasons of the resistance, but the resistance mechanism of L. chinensis (L.) Nees. to cyhalofop-butyl was not clear yet. In order to determine the popularization of the resistance, L. chinensis (L.) Nees. seeds were collected from the main rice grown regions in Jiangxi province and the resistance level was determined using whole-plant bioassay and seed bioassay. In order to ascertain the resistant mechanism of L. chinensis (L.) Nees. to cyhalofop-butyl, several types of metabolic enzyme activities and target enzyme activity, and target enzyme gene were determined using molecular biology methods and enzymology methods. The occurrence reasons of the resistance were understood from the source, which provided a basis for laying down integrated control strategy on L. chinensis. There was the important sense in the theory and practice.

千金子是直播稻田仅次于稗草的恶性杂草，氰氟草酯是防治千金子的主要除草剂之一，在江西一些稻区使用年限达到 10 年以上。我们前期研究发现，江西连年直播稻田的千金子对氰氟草酯的抗性高达33.64～40.86倍，直播、抛秧等栽培方式交替的稻田也表现出低抗水平。除草剂的代谢作用增强和作用位点的改变或靶标酶的过量表达是杂草产生抗性的重要原因，但千金子对氰氟草酯的抗性机理尚不清楚。为探究江西省主要稻区抗性千金子的发生情况，拟从不同稻区采集千金子种子，通过整株生物测定法和种子生物测定法测定千金子对氰氟草酯的抗性水平；为探明千金子对氰氟草酯的抗药性机理，采用分子生物学和酶学等方法对抗性千金子的几类代谢酶和靶标酶活性及靶标酶基因进行分析，从源头上了解抗性发生的原因，为制定千金子综合防除策略提供依据，在理论与实践上均具有重要意义。

氰氟草酯是防治直播稻田千金子的主要除草剂之一。根据计划任务书，主要开展了如下工作：一是千金子对水稻生长和产量的影响及其经济阈值研究；二是江西稻田千金子对氰氟草酯的抗药性测定；三是氰氟草酯对不同抗性水平的千金子生物型解毒酶和保护酶活性的影响；四是氰氟草酯对高抗型(R)和敏感型(S)千金子靶标酶ACCase活性的影响；五是高抗生物型体内氰氟草酯靶标酶ACCase基因的突变分析；六是氰氟草酯靶标酶ACCase基因的表达量与千金子抗药性的关系。.获得的主要结果如下：.1、千金子对水稻的抑制效应与其密度呈正相关，早稻、中稻和晚稻间存在差异。与无千金子对照比较，在千金子密度为21株/m2时，早稻、中稻和晚稻产量分别降低39.22%、44.36%和37.80%。通过对经济阈值分析，使用氰氟草酯和 嗪草酮进行化学除草时，早稻、中稻和晚稻的经济危害允许水平分别为2.26%～2.72%、1.56%～1.88%和1.77%～2.12%，经济阈值分别为0.06～0.09、0.59～0.72和0.25～0.32株/m2。.2、2014-2015年不同地区稻田千金子对氰氟草酯的抗性测定结果表现为：进贤温圳生物型＞朱港农场生物型＞恒湖农场生物型＞宁都青塘生物型＞赣州农科所生物型＞奉新赤岸生物型＞吉安农科所生物型＞南昌广福生物型。其中进贤温圳生物型属低抗水平，朱港农场和恒湖农场生物型处于敏感性下降阶段，宁都青塘、赣州农科所、奉新赤岸、吉安农科所和南昌广福5地生物型仍处于敏感性阶段。.3、对抗性型与敏感型千金子体内解毒酶和保护酶活性测定结果表明，药剂处理后4种酶均表现为先升后降，药后4 h不同地区抗性生物型体内解毒酶GSTs活性显著高于敏感生物型，体内保护酶SOD活性无显著影响，而POD和CAT活性显著高于敏感生物型，地区间抗性型/敏感型比值表现为进贤温圳生物型＞朱港农场生物型＞恒湖农场生物型、赣州宁都生物型、赣州农科所生物型和宜春奉新生物型＞吉安农科所生物型＞南昌广福生物型。.4、氰氟草酯对稻田高抗型（R）和敏感型（S）千金子靶标酶ACCase的抑制活性表明，进贤温圳、朱港农场和恒湖农场抗型千金子靶标酶ACCase的活性分别为敏感型的7.21、5.04和4.87倍，赣州宁都抗性型千金子靶标酶ACCase的活性为敏感型的2.64倍。.5、通过克隆进贤温圳、朱港农场、恒湖农场和赣州宁都4个千金子生物型