【作者】 杜洪忠；

【导师】 朱杰华；

【作者基本信息】 河北农业大学 ， 植物病理学， 2007， 硕士

【摘要】 由于马铃薯晚疫病菌对常用杀菌剂抗药性的产生，明显降低了化学防治晚疫病的效果。本研究从马铃薯晚疫病菌对杀菌新药—阿米西达的敏感性测定、抗阿米西达菌株的诱导以及抗阿米西达分子标记筛选等方面进行研究，旨在初步评价马铃薯晚疫病菌对阿米西达的抗性风险，并得到能够用于鉴定阿米西达抗、感菌株的分子标记。为阿米西达的科学使用和马铃薯晚疫病菌对阿米西达产生抗药性后治理策略的制定提供理论依据。1．测定了采自不同地区、不同年份的30株马铃薯晚疫病菌菌株对阿米西达的敏感性。结果表明：不同地区、不同年份的马铃薯晚疫病菌菌株对阿米西达均表现敏感，其抑制马铃薯晚疫病菌菌丝生长的EC₅₀值介于0.0351～0.1492μg／mL。此外，马铃薯晚疫病菌对阿米西达和甲霜灵之间没有正负交互抗药性。2．建立了马铃薯晚疫病菌对阿米西达的敏感基线，其EC₅₀值为0.0604±0.0226μg／mL。3．选取5株对阿米西达敏感的马铃薯晚疫病菌菌株进行阿米西达驯化抗性诱导和人工诱变（阿米西达+紫外线），获得了1株对阿米西达具有稳定的低水平抗性菌株X-DC98-3，抗性水平是野生菌株的20倍。获得的其它对阿米西达具有低水平抗性的菌株，经过抗性稳定性验证发现，抗性程度均有不同程度的下降，抗性不稳定。4．利用AFLP分子标记对阿米西达诱导后的抗性菌株和诱导前的野生敏感菌株进行研究，从144对AFLP引物组合中筛选出8对引物组合能在诱导后的抗性菌株和诱导前的野生菌株之间扩增出差异性条带。其中：引物组合E4／M5、E4／M8、E7／M5、E15／M6、E16／M9均能在菌株X-DC98-3与DC98-3之间扩增出1～2条稳定的特异条带；引物组合E19／M4除能在菌株X-DC98-3中扩增出一条稳定250bp左右的特异条带外，在菌株X-DC98-3、X-DK98-1和DK98-1中均能扩增出170bp左右的特异条带；引物组合E15／M19在菌株X-DC98-3和UV-HD01-4中均能扩增出稳定280bp左右的特异条带；引物组合E20／M11在菌株X-DC98-3、A-HH00-2、A-HW00-5和UV-HD01-4中均能扩增出稳定200bp左右的特异条带。5．用2个SSR引物Pi4B和Pi4G对阿米西达诱导后的抗性菌株和诱导前的野生敏感菌株进行SSR基因型分析，所有抗性菌株与它们的野生敏感菌株间的SSR基因型均没有发生变化，说明马铃薯晚疫病菌的SSR基因型与其对阿米西达抗性可能没有相关性。更多还原

【Abstract】 Because of fungicide resistance of Phytophthora infestans, the effect of fungicides was decreasing on potato late blight. In this paper, the sensitivities of P. infestans to azoxystrobin were determined, the azoxystrobin-resistance mutants of P. infestans were induced, and molecular marker of P. infestans to azoxystrobin resistance was detected. The results derived from this study would preliminarily evaluate the risk of P. infestans to azoxystrobin resistance and gain the molecular marker which could be applied to identify the azoxystrobin-resistant and azoxystrobin-sensitive isolates of P. infestans. A theoretical basis was provided for the scientific application of azoxystrobin and the development of managing strategies when isolates of P. infestans to azoxystrobin resistance were occurred.1. The sensitivities to azoxystrobin of 30 P. infestans isolates collected from different geographical regions in different years were determined in vitro. The results showed that all the isolates were sensitive to azoxystrobin. The EC50 value ranged from 0.0351 to 0.1492μg/mL. There was no cross resistance between azoxystrobin and metalaxyl in P. infestans populations tessted.2. The baseline-sensitivity of P. infestans to azoxystrobin was found, with an everage EC₅₀ value 0.0604±0.0226μg/mL.3. One lower level mutant X-DC98-3 of P. infestans with azoxystrobin-resistance was isolated by treating mycelium with azoxystrobin and ultraviolet radiation. The resistance level of mutant X-DC98-3 was 20 times as original isolate. By validating resistance, the result suggested that the resistance to azoxystrobin of other low azoxystrobin-resistant mutants of P. infestans was decreased.4. The fingeprint of azoxystrobin-resistant mutants and wild isolates of P. infestans were amplified using AFLP. The differencial bands between azoxystrobin-resistant mutants and wild isolates of P. infestans were produced with 8 primers combination out of 144. Among them, 1 to 2 specific bands between isolates X-DC98-3 and DC98-3 were amplified by primer combination E4/M5, E4/M8, E7/M5, E15/M6, E16/M9, respectively. One 250bp specific band was amplified from X-DC98-3, one 170bp specific band was amplified from isolates X-DC98-3, X-DK98-1 and DK98-1 with primer combinations of E19/M4. One 280bp specific band was amplified with primer combination E15/M19 between isolates X-DK98-1 and DK98-1. One 200bp specific band was amplified with primer combination E20/M11 from isolates X-DC98-3, A-HH00-2, A-HW00-5 and UV-HD01-4.5. Two SSR primers of Pi4B and Pi4G were charged for amplifing the azoxystrobin-resistant mutants and wild isolates of P. infestans. The result showed that the SSR genotypes of azoxystrobin-resistant mutants were the same with their wild isolates of P. infestans. It mignt be infered that the SSR genotypes was not linked with the sensitivity to azoxystrobin of P. infestans.更多还原