【作者】 刘冰；

【导师】 刘亚光；

【作者基本信息】 东北农业大学 ， 农药学， 2015， 硕士

【摘要】 耳叶水苋在稻田的危害日趋严重,苄嘧磺隆的防治效果越来越差。该草对ALS抑制剂类除草剂的交互抗性水平国内外未见报道。本论文测定不同耳叶水苋生物型对4种ALS抑制剂的交互抗性,并利用荧光定量PCR技术研究了耳叶水苋的抗性分子机制,研究结果如下:1、盆栽法测定结果表明,苄嘧磺隆对五种耳叶水苋生物型HZ001(浙江)、NB0143-05(浙江)、JX110(浙江)、JS039(江苏)、AH014(安徽)的鲜重抑制50%剂量(ED50值)分别为0.18、3.16、12.20、8.02和1.50gai/ha,生物型NB0143-05、JX110、JS039、AH014对苄嘧磺隆的抗性指数分别为44.63、67.90、17.59和8.37。五氟磺草胺、双草醚、咪唑乙烟酸对敏感生物型HZ001的ED50值分别为0.16、0.19和0.24,NB0143-05、JX110、JS039、AH014等四种生物型对五氟磺草胺的抗性指数分别为5.43、4.96、3.73和4.55,对双草醚的抗性指数分别为4.60、5.05、2.17和3.63,对咪唑乙烟酸的抗性水平分别为6.45、2.60、4.16和1.96。这表明,浙江、江苏和安徽的NB0143-05、JX110、JS039、AH014四种耳叶水苋生物型对苄嘧磺隆产生了中高水平抗性,且对三唑嘧啶类除草剂五氟磺草胺都产生了低水平交互抗性;对于嘧啶硫代苯甲酸酯类的双草醚,NB0143-05、JX110和AH014产生了低水平交互抗性;NB0143-05和JS039产生了低水平交互抗性。这是国内外耳叶水苋抗苄嘧磺隆生物型对其他ALS抑制剂交互抗性的首次报道。2、本研究获得浙江、江苏省稻田HZ001,NB0143-05,JS039和JX110四种耳叶水苋生物型ALS的全长序列,长度为2235bp,编码667个氨基酸。耳叶水苋三种抗性生物型的ALS基因第93位的亮氨酸(CTC)都被脯氨酸取代(CCC),但该突变位点不是ALS酶结合域氨基酸残基与除草剂的结合位点。未见其保守区突变。3、本文采用实时荧光定量PCR技术,以β-actin为内参基因,对耳叶水苋抗苄嘧磺隆生物型NB0143-05和敏感生物型HZ001在mRNA水平上进行分析。结果表明,耳叶水苋幼苗在3-4叶期进行苄嘧磺隆(0.1gai/ha)茎叶处理后第2、4、6d,生物型HZ001的ALS表达量分别是对照的1.23、2.74和2.41倍,生物型NB0143-05的ALS表达量分别是对照的1.83、4.45和2.72倍。这表明,耳叶水苋幼苗经苄嘧磺隆处理后ALS表达量逐渐增加,4d达到最大峰值,然后逐步降低。药后4天抗性生物型NB0143-05的ALS表达量是敏感生物型HZ001的1.86倍。ALS表达量增加可能是浙江省稻田耳叶水苋抗性生物型NB0143-05对苄嘧磺隆等除草剂产生抗性和交互抗性的重要原因,这是国内外耳叶水苋抗苄嘧磺隆生物型ALS基因表达量增加的首次报道。更多还原

【Abstract】 A.arenaria has increased serious harmly in paddy fields and control efficiency of bensulfuron-methyl(BSM) is becoming worse. Research on cross-resistance level of this weed to ALS inhibitor herbicide is not reported in the world. In this paper, it is determined the cross-resistance of different biotypes of A. arenaria to the four ALS inhibitors, and fluorescence quantitative PCR technique was used to study the molecular mechanism of A. arenaria, the results are as follows:1、The results of potted experiments showed that the ED50 value of BSM to five A. arenaria biotypes HZ001(Zhejiang), NB0143-05(Zhejiang), JX110(Zhejiang), JS039(Jiangsu) and AH014(Anhui) are respectively 0.18, 3.16, 12.20, 8.02 and 1.50gai/ha, the resistance index(RI) are respectively 44.63, 67.90, 17.59 and 8.37. The ED50 value of penoxsulam, bispyribac-sodium and imazethapyr to sensitive biotype HZ001 are respectively 0.16, 0.19 and 0.24, RI of the biotypes NB0143-05, JX110, JS039 and AH014 to penoxsulam are respectively 5.43, 4.96、3.73 and 4.55, RI to bispyribac-sodium are respectively 4.60, 5.05, 2.17 and 3.63, RI to imazethapyr are respectively 6.45, 2.60, 4.16 and 1.96. This suggests that NB0143-05, JX110, JS039 and AH014 develop high levels of resistance to BSM, and develop low resistance of interaction to penoxsulam( triazole pyrimidine herbicide). Three biotypes except JS039 develop low resistance of interaction to bispyribac-sodium( pyrimidine glucosinolates benzoic acid esters). NB0143-05 and JX110 develop low resistance of interaction to that. This is the first report that for cross-resistance level to other ALS inhibitor in the world.2、This study obtained the full length gene sequence of ALS of four Ammannia arenaria biotypes: HZ001, NB0143-05, JS039 and JX110 in paddy fields in Zhejiang and Jiangsu province. The results showed that the full DNA sequence length were 2235 bp, encoding 667 amino acids. The ALS gene 93 th leucine(CTCS) are replaced by the proline(CCC) in three Ammannia arenaria resistant biotypes. But the mutations are not binding site to amino acid and herbicide. Conservative region is not mutated.3、In this paper, it used real-time fluorescent quantitative PCR technology, β-actin as reference genes, to analyze expression difference of ALS gene by comparing resistant biotype (NB0143-05) to BSM and sensitive biotype(HZ001)at the m RNA level. The results show that Ammannia arenaria was sprayed the BSM at the dosage of 0.1 gai/ha in 3-4 leaf stage. At 2、4、6 after treatment(DAT), the expression of ALS gene of HZ001 were respectively 1.23、2.74 and 2.41 times higher than that of control, the expression of ALS gene of NB0143-05 were respectively 1.83、4.45 and 2.72 times higher. The results indicated that the expression of ALS gene increase gradually after spraying BSM, and reached maximum peak at 4 DAT, then steped down. At 4 DAT, resistant biotype was 1.86 times higher than that of sensitive biotype. The increasing expression of ALS gene may be key reason that NB0143-05 develop resistance and cross-resistance to BSM and other herbicides in paddy field of Zhejiang province. It reported for the first time that the expression of ALS gene increased after BSM treatment.更多还原