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黄瓜白粉病菌对己唑醇抗性诱导及抗性机制的初步研究

Studies on Resistance-induced and Mechanism of Sphaerotheca Fuliginea to Hexaconazole

【作者】 杨连来

【导师】 慕卫;

【作者基本信息】 山东农业大学 , 农药学, 2007, 硕士

【摘要】 本文以黄瓜白粉病菌为研究对象,通过紫外线照射和药剂驯化相结合的方法获得了黄瓜白粉病菌对己唑醇的抗性菌株。并比较了黄瓜白粉病菌敏感菌株和抗性菌株的生物学特性;同时对其抗性的生理生化机制进行了初步研究。主要内容如下:1.通过子叶叶盘法测定了山东地区50个黄瓜白粉病菌菌株对己唑醇的敏感性,结果发现,不同地区的病原菌对己唑醇表现出不同的敏感性,其中EC50值最高达0.16μg·ml-1,最低EC50只有0.0030μg·ml-1。相差倍数53.3倍。50个菌株对己唑醇不同敏感性菌株的频率分布呈一个近似的正态分布。根据野生敏感型病原群体对药剂敏感性为正态分布的原理,故认定此试验测定得到50个菌株对己唑醇的平均EC50值0.028μg·ml-1可作为山东地区黄瓜白粉菌对己唑醇的敏感性基线。2.利用紫外线诱导、药剂驯化以及两者相结合的方法获得了黄瓜白粉病菌对己唑醇的抗性菌株,其中,以紫外线诱导和药剂驯化相结合的方法获得的抗性菌株的抗性倍数最高,为105倍。并以此菌株作为研究对象。3.敏感菌株分生孢子萌发较抗性菌株分生孢子萌发早8h,但其菌丝体产孢的时间要比抗性菌株菌丝体产孢的时间要晚24h。从萌发时间上看,敏感菌株和抗性菌株的分生孢子萌发时间都在接种40h后开始萌发,且两者萌发率为10%~20%,都比较低。虽然萌发率比较低,但敏感菌株和抗性菌株的产孢能力都非常强,抗性菌株接种6d,新生长的菌丝体上已经开始产生分生孢子梗,接种8d,在菌丝体上观察到大量的成串分生孢子,接种9d后,在外部形态上,就开始观察到白粉病病斑。4.在涂抹相同孢子量的情况下,发病10d后抗性菌株产孢量为1.51×105分生孢子/ml,而敏感菌株的产孢量为1.18×105分生孢子/ml,表明抗性菌株的产孢量要高于敏感菌株,但两者差异不显著(p=0.1665>0.05)。从病情指数来看,接种抗性菌株的黄瓜病指为30,而接种敏感菌株的黄瓜病指为25,表明抗性菌株的致病力略强于敏感菌株,但两者也无显著差异(p=0.2662>0.05)。5.室内获得的对己唑醇高抗的黄瓜白粉病抗性菌株,对戊唑醇、腈菌唑、多抗霉素和嘧菌酯的抗性倍数均略高于1,表明室内获得的抗性菌株对戊唑醇、腈菌唑、多抗霉素和嘧菌酯都不表现交互抗性,而与醚菌酯的抗性倍数为5.85,表现交互抗性。6.抗性菌株在孢子萌发率、芽管个数和产孢量方面都比敏感菌株略高,但三者无显著差异。抗性菌株的菌丝分枝略低于敏感菌株,两者无显著差异。表明抗性菌株的适合度与敏感菌株相比没有显著变化。7.抗性菌株与敏感菌株分生孢子等量混合培养7代后,抗性菌株的频率在混合群体中先逐渐降低后略有上升;混合比例在20:80的处理培养7代后,只有少量的抗性菌株存在。在80:20的混合比例处理中,抗性菌株的频率的变化趋势为先上升后下降再略有上升,但变化幅度不大,表明抗性频率基本保持稳定。8.敏感菌株和抗性菌株在酯酶同工酶图谱、过氧化物酶图谱和可溶性蛋白电泳图谱中基本无明显的差异,敏感菌株和抗性菌株的条带数相同。但敏感菌株和抗性菌株的条带颜色不一样,说明二者之间存在量的差异。

【Abstract】 In this dissertation, Hexaconazole-resistant strains to Sphaerotheca fuliginea were obtained by chemical taming and UV irradiation in the laboratory, And studied on biological characteristic and resistant mechanism of sensitive strain and resistant strain of S. fuliginea.1. The sensitivity of Sphaerotheca fuliginea to hexaconazole was tested in vitro culture of cotyledon to 50 strains collected from shandong in lab. The results showed that the sensitivity of strains ranged from 0.0030μg/ml to 0.16μg/ml, the mean EC50 value was 0.028μg/ml,it could be as baseline sensitivity of Sphaerotheca fuliginea to hexaconazole because frequencies of fifty strains accords with Gaussian distribution.2. The hexaconazole-resistant strains were obtained by UV irradiation or chemical taming or UV irradiation and chemical taming in the laboratory. Resistance factor of a highly resistant strain,10~5 fold, was obtained by UV irradiation and chemical taming.3. Time of pycnidiospore germination of the HR strains were later than their parent strains without significant difference, But contrary to time of mycelia sporulation. Germinating time of all spores was 40 hours after inoculation, ratio of pycnidiospore germination of sensitive and resistant strains was very low, 10%~20%. But Sporulation capacity of spore was very high. Spores appeared six days after inoculation,A mass of bunched spore was observed eight days, And symptom appeared nine days after inoculation.4. Sporulation quantity of resistant strain was 1.51×10~5 spores/ml ten days after inoculation. and sensitive strain was 1.18×10~5 spores/ml at the same condition.There was no significant difference between that. Disease index of cucumber that inoculated resistant strain was 30,and disease index of cucumber that inoculated sensitive strain was 25, that showed pathogenicity of resistant strain was higher than sensitive strain but no significant difference.5. Hexaconazole-resistant strains exhibit no cross-resistance to tebuconazole, myclobutanil, polyoxin and azoxystrobin because resistant factor was nearly 1, and positive resistance to kresoxim-methyl for resistant factor was 5.85.6. The germination percentage of aonidia, the number of germmination tubes per conidium, and the number of penetration sites per conidium of highly resistant strain were higher than sensitive strain. And the number of branches per hypha was contrary to that. Fitness parameters of resistant and sensitive strains had no significant difference without fungicides.7. Competition ability of resistant and sensitive strains were measured. The results showed that frequency of resistant strains was no significant difference when spores of resistant and sensitive strains mixed with 80:20 were incubated after 7 generation with no fungicide treatment; when mixed with 50:50, and frequency of high resistant strain was slowly descent; When mixed with 20:80, frequency of resistant strain was lower than sensitive strain, it imply that the resistance of Sphaerotheca fuliginea may be disappear without fungicide pressure.8. The contents of esterases isoenzymes, peroxidase isoenzymes and soluble proteins were compared between resistant strain and sensitive strain. The results showed that the electrophoretic patterns of resistant strain and sensitive strain was the same, but the quantity of that was not.

  • 【分类号】S436.421
  • 【被引频次】7
  • 【下载频次】383
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