日本看麦娘对精噁唑禾草灵和甲基二磺隆的多抗性机理研究

Japanese foxtail (Alopecurus japonicus Steud.), a kind of competitive annual weed in winter wheat fields, which is among the top ten weeds in China. The control of Japanese foxtail has been based on ACCase inhibitor herbicides, mostly fenoxaprop-P-ethyl. However, a high degree of resistance has been demonstrated in recent years. One of the alternatives is ALS inhibitors herbicides, such as mesosulfuron-methyl. But there was treatment failure existed and multi-resistance was found, so it has become an important issue to be solved in the production. Our preliminary results showed that the resistance ratios of Japanese foxtail to fenoxaprop-P-ethyl and mesosulfuron-methyl were 1914.6 and 104.3, respectively. Therefore, this research, by means of traditional physiological and biochemical methods, associate with PCR and AS-PCR, determine the difference between the resistant and sensitive biotype of Japanese foxtail in target and metabolic enzyme activities and target site mutation. Moreover, changes of gene expression level of target enzyme will also be evaluated by the Real-time PCR. The results of this study will assure the multi-resistance mechanism of Japanese foxtail to fenoxaprop-P-ethyl and mesosulfuron-methyl, which will be of essential guiding significance to control grass weeds such as Japanese foxtail.

日本看麦娘是小麦田恶性禾本科杂草，也是我国十大草害之一。主要采用以精噁唑禾草灵为主的ACCase抑制剂类除草剂进行防治，近几年已表现出高度抗药性，生产中主要以甲基二磺隆等ALS抑制剂类除草剂为替代产品，但部分地区出现防治失败的情况，表现出多抗性，已成为生产中亟待解决的重要问题。课题组前期研究结果表明，整株法测定日本看麦娘抗性生物型对精噁唑禾草灵的抗性倍数高达1914.6倍，对甲基二磺隆的抗药性倍数为104.3倍。本项目拟通过运用常规生理生化方法结合PCR、AS-PCR等现代分子生物学技术，研究日本看麦娘抗性、敏感生物型在靶标酶、代谢酶活性的差异以及靶位点基因的突变，并利用荧光定量PCR技术测定靶标酶基因表达量的变化。项目的完成可明确日本看麦娘对精噁唑禾草灵和甲基二磺隆的多抗性机理，从而对指导日本看麦娘等禾本科杂草的科学治理具有重要的现实意义。

日本看麦娘是小麦田恶性禾本科杂草，也是我国十大草害之一。主要采用以精噁唑禾草灵为主的ACCase抑制剂类除草剂进行防治，近几年已表现出高度抗药性，生产中主要以甲基二磺隆等ALS抑制剂类除草剂为替代产品，但部分地区出现防治失败的情况，表现出多抗性，已成为生产中亟待解决的重要问题。.本研究以采自安徽、江苏、山东、河南、湖北等5省的48个日本看麦娘生物型为试材，研究了不同日本看麦娘生物型对精噁唑禾草灵和甲基二磺隆的抗性水平，结果表明，AH-15、AH-16、AH-17和AH-23 等4个生物型对精噁唑禾草灵和甲基二磺隆均产生了高水平的抗性。.测定了日本看麦娘抗性、敏感生物型靶标酶ALS对甲基二磺隆的敏感性，结果表明，抗性生物型ALS受甲基二磺隆的抑制程度明显弱于敏感生物型，酶活力倍数为2.41倍。.对4个多抗性生物型、1个敏感生物型的质体型ACCase基因和ALS基因进行扩增、测序和比对。结果表明，AH-15、AH-16、AH-17质体型ACCase基因1781位氨基酸由异亮氨酸突变为亮氨酸，AH-23生物型ACCase基因2027位氨基酸由色氨酸（TGG）突变为精氨酸（CGT）；AH-15、AH-16生物型ALS基因574位氨基酸发生了突变，由色氨酸突变为亮氨酸，AH-17生物型ALS基因未发生突变，AH-23生物型ALS基因574位氨基酸由色氨酸（TGG）突变为精氨酸（CGG）。.测定了日本看麦娘抗性生物型和敏感生物型代谢酶P450和GSTs活性的差异。结果表明，抗性生物型AH-17对精噁唑禾草灵和甲基二磺隆的抗性可能与P450s活性的增强有关；与敏感生物型相比，抗性生物型体内GSTs活性变化无明显差异。.基于突变位点，利用酶切扩增序列多态性技术（CAPS）和衍生酶切扩增序列多态性技术（dCAPS）建立了日本看麦娘靶标位点突变的检测方法。.以β微管蛋白基因为内参，采用实时荧光PCR法比较了日本看麦娘抗性和敏感生物型质体ACCase和ALS基因在转录水平上的差异。结果表明，抗性和敏感日本看麦娘生物型在靶标酶基因转录水平上没有明显差异。.测定了不同日本看麦娘生物型对不同除草剂的抗性谱，对生产上科学合理地应用除草剂，延缓杂草抗药性的发生，为不同地区选择性地应用不同的替代药剂提供了重要的依据，对抗性日本看麦娘等禾本科杂草的化学治理具有重要的理论和现实意义。