【作者】 张晓；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 农药学， 2008， 硕士

【摘要】 嘧菌酯（Azoxystrobin）是由先正达公司（原捷利康）开发的,也是第一个商品化的甲氧基丙烯酸酯类杀菌剂,具有杀菌活性高,防病谱广,与环境相容性好等突出优点。甲氧基丙烯酸酯类杀菌剂作用于真菌线粒体电子传递链的复合物Ⅲ氧化还原酶系,阻断电子传递,干扰能量合成,因其通过与细胞色素b（Cyt b）的Q_o部位结合而称之为Q_o抑制剂（Q_oI）。由于其独特的作用机制,与已有的所有杀菌剂无交互抗药性。但已有研究表明多种植物病原真菌对该类药剂产生抗药性自发突变频率较高,在高选择压下自然界易形成抗药性病原真菌群体。黄瓜霜霉病和番茄早疫病是中国黄瓜和番茄生产上的严重病害。但长期以来一直缺乏防治这两种病害的有效药剂,寻找新型高效杀菌剂来控制这两种病害的流行已成为化学防治工作者的目标。所以研究番茄早疫病菌和黄瓜霜霉病菌对嘧菌酯的敏感性及其在嘧菌酯处理下的生理生化变化和靶标基因表达水平上的变化具有重要的现实意义。离体培养条件下,嘧菌酯单独作用对番茄早疫病菌的菌丝生长和孢子萌发的抑制作用不明显。旁路氧化在番茄早疫病菌野生菌株的孢子萌发中提供大于27.03倍生理功能的补偿作用;在菌丝生长中提供4.83-9.95倍的补偿作用。嘧菌酯对番茄早疫病菌的菌丝生长、孢子萌发、孢子产生有抑制作用。12.5μg/mL的嘧菌酯处理1h对菌丝呼吸耗氧有良好的抑制作用,其抑制效果随药剂浓度提高而略有增加,但对菌丝生长的抑制率随药剂浓度的提高增加缓慢。随处理时间延长,药剂对菌丝耗氧的抑制作用下降,但仍然抑制菌丝生长。研究证明延长处理时间呼吸作用对药剂的敏感性下降的机理不是旁路氧化途径增强,也不是因为基质中药剂效力的下降而是存在其它机制。黄瓜霜霉病是由Pseudoperonospora cubensis引起的,能导致黄瓜产量的减少。在2006年从山东省寿光地区温室大棚中采集的106个黄瓜霜霉菌株中EC₅₀的范围是0.0063-0.0688μg/mL,平均EC₅₀为0.01955μg/mL,没有监测到抗性菌株。在2007年采集的97个黄瓜霜霉菌株中,有57株为抗性菌株,抗性菌株的EC₅₀为0.609 to＞51.2μg/mL,抗性倍数为21.15-＞2618.9。2007年从山东寿光地区采集的97个黄瓜霜霉菌株中,监测到57个抗性菌株。在57个抗性菌株中EC₅₀为0.609-10μg/mL有20株,EC₅₀为10-50μg/mL有23株,EC₅₀为＞51.2μg/mL有14株。抗性倍数为21.15-＞2618.9。从三个不同抗性水平范围中任意选取一个菌株,提取DNA,经过DNA单克隆及质粒提取,测序。实验结果发现抗药性菌株的Cytb基因发生了点突变（GGT-GCT）,导致143位由甘氨酸突变为丙氨酸。抗性水平不同的菌株突变位点却相同,而且在一年的时间内就产生了抗性菌株。更多还原

【Abstract】 Azoxystrobin,developed by Syngenta（Zeneca） company,was the first product of the strobilurin fungicides.It combines high level and broad-spectrum of fungicidal activity with excellent safety in the environment.Strobilurin fungicides inhibit mitochondrial respiration in fungi at a specific site on complexⅢand break energy synthesization of fungi.Strobilurins were called QoIs as they combined at Qo site of Cytb.This kind of fungicide was widely used because of their novel action mechanism and non-cross-resistance with any other presented fungicides.However,the frequency of spontaneous resistance mutation was high according to several research reports.It’s easy to form resistant-population under high fungicidal selection pressure.Cucumber downy mildew（Pseudoperonospora cubensis） and Tomato early blight （Alternaria solani） are one of the serious diseases on China cucumber and tomato production,respectively.Aimed to control the popularity of Cucumber downy mildew and Tomato early blight,fungicidal workers are searching new fungicides.So,it is essential to embark on bioactivity and toxicology at molecular biochemical level of azoxystrobin to Pseudoperonospora cubensis and Alternaria solani.The bioactivity of azoxystrobin to Alternaria solani which had never been exposured to QoIs or QiIs was determined and also its influence on the fungus’s biological properties was determined.We also made clear the synergistic effect of SHAM.The result was that azoxystrobin worked alone expressed less inhibition on mycelial growth and spore germination.Sensitivity of Alternaria solani to azoxystrobin could be decreased by the induction of alternative respiration.In the absence of a host,alternative respiration provided a＞27.03-fold rescue potential from azoxystrobin during the germination of conidia derived from five wild-type isolates ofAlternaria solani.And for mycelial growth, the rescue potential was 4.83～9.95-fold.SHAM had synergistic effect on the bioactivity of Azoxystrobin.But,azoxystrobin alone could strongly inhibit conidia production and delay the pigment formation.Oxygen consumption test of mycelia of Alternaria solani showed that azoxystrobin strongly inhibited mycelial respiration after 1 hour of the treatment at 12.5μg/mL and the effect became stronger when the concentration was increased.As treatment time went on, the respiration inhibition on the mycelia decreased,while the mycelium growth was still inhibited strongly.Results showed that the sensitivity decrease of mycelium respiration to azoxystrobin was not caused by induction of alternative respiration or degrade of the fungicide in medium.Cucumber downy mildew caused by Pseudoperonospora cubensis limits crop production in Shandong province of China.Since management of downy mildew is strongly dependent on fungicides,a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates of P.cubensis were obtained in 2006 and 2007,respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063-0.0688μg/mL with an average ECs0 value of 0.0196±0.0048μg/mL azoxystrobin which were considered sensitive isolates.However,57 field isolates of P.cubensis out of 97 collected in 2007 with EC₅₀ that ranged from 0.609 to＞51.2μg/mL were considered resistant to azoxystrobin.A total of 97 isolates of P.cubensis were obtained in 2007。There are 57 resistant isolates.The EC₅₀ values for resistant isolates ranged from 0.609 to＞51.2μg/mL,with the resistance factor 21.15-＞2618.9.Of all the 57 resistant isolates,the EC₅₀ values of 20 isolates,23 isolates and 14 isolates were 0.609-10μg/mL,10-50μg/mL and＞51.2μg/mL,respectively.One isolate was randomly selected from each of the three categories and one sensitive isolate was randomly selected for the molecular testing.A single point mutation（GGT to GCT） in the cytochrome b gene,resulting in substitution of glycine by alanine at position 143,was found in the three selected azoxystrobin-resistant isolates of downy mildew.This substitution in cytochrome b exhibited different resistance levels,with the resistance factor from 21.15 to greater than 2618.9.In addition,the different resistance levels seemed to appear within 1 year（within 2006 to 2007）.更多还原