【作者】 程玉峰；

【导师】 高智谋； 高同春； 袁善奎；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2008， 硕士

【摘要】 本论文采用菌丝生长抑制法测定了2007年采自黑龙江和河北省的59株马铃薯晚疫病菌对甲霜灵和嘧菌酯的敏感性,建立了该菌对嘧菌酯的敏感基线,进行了马铃薯晚疫病菌对嘧菌酯的室内抗药性突变体的诱导和抗性风险评估,并开展了抗性机制的研究:1马铃薯晚疫病菌对甲霜灵和嘧菌酯的敏感性测定采用菌丝生长抑制法对采自黑龙江和河北省的59株马铃薯晚疫病菌进行了敏感性测定,结果表明河北省20株马铃薯晚疫对甲霜灵的抗性频率为100%,黑龙江的39株菌株敏感的占20.51%,抗性菌株占74.36%,抗性频率也达到相当高的水平。嘧菌酯对59株马铃薯晚疫的EC₅₀值介于0.0062μg/mL～0.147μg/mL之间,平均EC₅₀为0.0581（±0.0307）μg/mL,MIC均小于1μg/mL,不同菌株对嘧菌酯的敏感性频率分布呈连续性单峰曲线,接近正态分布,尚未出现敏感性明显下降的抗药群体,因此供试菌株的平均EC₅₀值可作为马铃薯晚疫病菌对嘧菌酯的敏感性基线用于将来开展田间抗性监测。2马铃薯晚疫病菌对嘧菌酯的室内抗药性风险评估在室内采用紫外诱变和药剂驯化的方法均获得了马铃薯晚疫病菌对嘧菌酯的抗性突变体,共获得4株抗嘧菌酯的突变体,其中两株抗性指数较低,两株（N2-4和N2-5）抗性指数较高分别为20.94和52.87倍。紫外诱变方法较药剂驯化方法更易于获得抗药性突变体,且其抗性水平高于药剂驯化方法。部分生物学性状研究显示,突变体的抗药性状稳定、产孢子囊能力、致病力与亲本敏感菌株相比没有显著差异。嘧菌酯与醚菌酯和烯肟菌酯之间存在正交互抗药性,与霜脲氰、烯酰吗啉、和代森猛锌之间均无交互抗药性。综合分析表明,马铃薯晚疫病菌对嘧菌酯具有较高的抗药性风险,建议生产上应将嘧菌酯与其它无交互抗性的药剂交替使用或混合使用,以避免或延缓抗药性的产生。3马铃薯晚疫病菌对嘧菌酯抗药性机制病原菌抗药性分子机制研究结果表明,与敏感菌株线粒体Cytb基因部分氨基酸序列比对,低抗菌株A32-5氨基酸序列中的98位由Phe突变为Leu（F99L）;N2-10氨基酸序列中的339位由Ile突变为Asn（I339N）;室内抗药性水平较高的突变体N-4和N-5氨基酸突变位点相同,均为氨基酸序列中的137位由Gly突变为Glu（G137E）;其中N2-5的869为碱基也发生了变化,由A变成了G,但氨基酸序列没有发生变化。根据相关甲氧基丙烯酸酯的抗性研究报道,抗性突变体发生突变的区域主要集中在Cytb序列上的氨基酸127～147和275～296位,不同的突变位点代表了不同抗性水平,在127～147位发生突变,表现高水平的抗药性,因此推测马铃薯晚疫病菌线粒体中Cytb基因的137位氨基酸的突变是靶标菌对嘧菌酯产生抗性的主要原因。4嘧菌酯与霜脲氰、烯酰吗啉和代森锰锌复配对马铃薯晚疫病菌的联合毒力嘧菌酯与3个无交互抗药性的杀菌剂按不同比例混配后的联合毒力测定表明,嘧菌酯与霜脲氰、烯酰吗啉按1:（1,2,3,4,5）混配对马铃薯晚疫病菌,均无增效作用;嘧菌酯与代森锰锌按（1:4、1:8、1:12、1:16、1:20）进行混配对马铃薯晚疫病菌的共毒系数均大于120,表明嘧菌酯与代森锰锌5个配比均有明显的增效作用,而1:16进行复配增效作用最为明显。更多还原

【Abstract】 The sensitivities of 59 single-lesion field isolates of potato Phytophthora infestans which were collected from Heilongjiang and Hebei provinces,to metalaxyl and azoxystrobin were determined in laboratory by mycelial growth inhibition method,and the baseline-sensitivity of P.infestans to azoxystrobin was established.The resistant mutants of P.infestans to azoxystrobin were obtained and the risk of resitance was assessed,as well as the mechanism of resistance was studied.1 Sensitivities of P.infestans isolates to metalaxyl and azoxystrobinThe results showed that all isolates from Hebei province were resistant to metalaxyl, and the metalaxyl-sensitive isolates accounted for 20.51%among 39 isolates which were collected from Heilongjiang province,while the high resistance isolates covered 74.36%, the resistant frequency also reached the relatively high level.The sensitivities to azoxystrobin of the 59 P.infestans isolates were tested by mycelial growth inhibition method.The baseline sensitivities to azoxystrobin,expressed as EC₅₀ values,ranged from 0.0062μg/mL to 0.1417μg/mL,with a mean of（±0.0307）μg/mL.The sensitivities of all.isolates distributed as a unimodel curve and no resistant subpopulation appeared.Thus,this baseline sensitivity was suitable for field resistance monitoring.2 Assessment of the resistance risk of P.infestans to azoxystrobinTotally four azoxystrobin-resistant mutants of P infestans were recovered in the laboratory by UV-irradiation and fungicide adaptation method.There were two mutants with relatively high resistant level（N2-4 and N2-5）,the resistant factors was 20.94 and 52.87 respectively The resistant mutants was easily obtained by UV irradiation method than fungicide adaptation method.Some biological characteristics of mutants such as stability,sporangia production and pathogenicity were not significantly different from those of their parental strains.The results also showed that there was a positive cross-resistance between kresoxim-methyl and enostrobilurin,but not with cymoxanil,dimethmorph and mancozeb.The laboratory studies revealed that azoxystrobin was a high risk fungicide. Therefore,the epidemic development of the resistant population of P.infestans would be possible in the field.It was suggested that the azoxystrobin should be alternated or mixtured use with other fungicides which with no cross-resistance to avoid or delay the development of resistance.3 Resistant mechanism of P.infestans to azoxystrobinThe single point mutation F98L and I339N were found in low resistance strains A32-5 and N2-10 respectively,and the other point mutations G137E was found in two relatively high resistant mutants N-4 and N-5,the 869 point of N2-5 has also changed for the basic group,substituted A by G,but the amino acid sequence has not changed.The documents reported that the strobilurin resistance mainly concentrate in the Cytb sequence,the amino acid mutation focus on 127-147 and 275-296,the different point mutation has represented the different resistant level,the mutation range in 127-147 denote high level resistance. This study suggested a mutation at position 137 was a major reason for the rapid development of high strobilurin resistance in the mutants.4 Synergistic effect of azoxystrobin with cymoxanil,dimethmorph and mancozeb to P.infestansThe synergistic effects of the mixtures of azoxystrobin with cymoxani,dimethmorph, mancozeb to P.infestans on the mycelial growth were studied in vitro,the resullts showed that azoxystrobin and cymoxanil,dimethmorph,azoxystrobinl:（1,2,3,4,5） and cymoxanil, dimethmorph to P infestans there were no synergistic effects,while the mixtures of azoxystrobin with mancozeb at the ratio of 1:4,1:8,1:12,1:16,1:20),their CTC values all exceeded 120,which indicated that there were obviously synergistic effect in these combination,and the best synergistic effect especially for the ratio of 1:16.更多还原