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辣椒疫霉对双炔酰菌胺抗药性风险评估及抗药性生理生化机制研究

标题: 辣椒疫霉对双炔酰菌胺抗药性风险评估及抗药性生理生化机制研究
英文标题: Risk Evaluation of Resistance to Mandipropamid of Phytophthora Capsici and Studies on Its Physiological and Biochemical Mechanism
作者: 赵卫松
出版时间: 2011-01-01
所在大学: 河北农业大学
关键词: 辣椒疫霉,双炔酰菌胺,交互抗性,抗药性风险评估,生理生化机制
英文关键词: Phytophthora capsici,mandipropamid,cross-resistant,resistance risk,physiological and biochemical mechanism
论文级别: 硕士
学位: 学位论文
导师: 张小风%张金林
专业: 农药学
提交时间: 2011
摘要: 辣椒疫病是一种毁灭性病害,常对辣椒生产造成严重的经济损失.目前生产上仍以化学防治为主.长期单一地使用同类药剂,可能引起病原菌对药剂产生抗药性,导致药剂防效降低.羧酸酰胺类杀菌剂双炔酰菌胺(mandipropamid)是一种新型卵菌病害杀菌剂,在国内尚未大面积应用.本研究进行了辣椒疫霉对甲霜灵和烯酰吗啉的敏感性及对不同作用机制药剂的交互抗性、双炔酰菌胺对辣椒疫病的生物活性、辣椒疫霉对双炔酰菌胺的敏感基线、室内抗药性风险评估和抗药性生理生化机制等方面的研究.主要研究结果如下:1.测定了河北省不同地区的64株辣椒疫霉对甲霜灵和烯酰吗啉的敏感性.结果表明,甲霜灵对辣椒疫霉具有明显的抑制作用,EC50值为0.1661~27.5572μg/mL,最高与最低相差165.91倍,不同地区采集的菌株对甲霜灵的敏感性存在着很大差异,对烯酰吗啉均敏感.对EC50值进行线性回归分析,发现对甲霜灵的敏感性与对上述杀菌剂的敏感性之间的相关系数很低,不存在交互抗性.2.采用菌丝生长速率法测定了双炔酰菌胺、烯酰吗啉、嘧菌酯和霜脲氰对11个甲霜灵敏感菌株和3个甲霜灵抗性菌株的毒力;采用离体叶片法和小株喷雾法分别测定了双炔酰菌胺对辣椒疫病的保护作用和治疗作用;并采用菌饼靠接法测定了其对辣椒疫病的持效期.结果表明:双炔酰菌胺和烯酰吗啉对辣椒疫霉的抑制效果较好,嘧菌酯次之,霜脲氰最差;离体叶片法测定双炔酰菌胺10μg/mL对辣椒疫病的保护、治疗效果分别为86.67%、60.00%;小株喷雾法测定双炔酰菌胺100μg/mL对辣椒疫病的保护、治疗效果分别为95.25%、77.81%.用药14d后100μg/mL相对防效依旧高达65.92%.双炔酰菌胺对辣椒疫霉具有很好的抑制作用,对辣椒疫病具有很好的防治效果,其中保护效果高于治疗效果,且持效期较长,明显的治疗效果也说明该药剂具有一定的内吸传导性.3.采用菌丝生长速率法测定了分别采自河北定兴、藁城和山东寿光、青州等地的173株辣椒疫霉对双炔酰菌胺的敏感性,结果表明,其EC50值介于0.00270.0499μg/mL之间,最高与最低相差18.41倍,不同菌株的敏感性频率呈连续的单峰分布,因此可视为野生敏感菌株,可把173株辣椒疫霉的平均EC50值(0.0219±0.0102)μg/mL作为对双炔酰菌胺的敏感基线.4.采用紫外诱导+菌落角变和药剂驯化的方法分别处理10个菌株,分别获得7株和1株突变体,其抗性水平在41.50~394437.77倍之间,突变频率分别为1.46%和1.43%.通过不同抗性水平菌株对5种药剂的敏感性测定和相关性分析发现,双炔酰菌胺与烯酰吗啉、甲霜灵、嘧菌酯和霜脲氰之间不存在交互抗性.8个抗性突变体在无药的CA培养基平板上继代培养11代后,6个突变体抗药性不能稳定遗传;2个突变体能稳定遗传,EC50值维持在很高的水平,分别为1429.5237μg/mL和5000μg/mL以上.突变体的生物学性状研究表明:与亲本菌株相比,抗性突变体在菌丝生长速率、致病性、产孢能力、适合度指数和竞争力几个性状方面均具不同程度的差异;抗性突变体DX 2-3 20m和DX 2-3 F7的产孢量、适合度和竞争性均高于亲本菌株,有利于抗性群体的形成.综上所述,辣椒疫霉对双炔酰菌胺存在中等抗性风险.因此,双炔酰菌胺的连续使用有利于抗药群体的发展,为避免和延缓抗药性的产生,生产上应将其与其它无交互抗性的杀菌剂交替使用.5.比较辣椒疫霉对双炔酰菌胺抗性菌株及其亲本菌株的生理生化指标.结果表明:亲本菌株和抗性菌株的菌丝生长受NaCl和葡萄糖影响较小,且NaCl和葡萄糖不同浓度处理后的所有菌株之间渗透压均不存在显著性差异,故得知NaCl和葡萄糖均不能为辣椒疫霉提供营养和抑制其渗透.双炔酰菌胺低浓度处理能使抗性菌株菌体内渗漏出较多的内含物,但随着处理时间的延长和浓度的提高内含物渗漏反而减少;亲本菌株菌体内的可溶性蛋白含量和β-1,3-葡聚糖酶的活力均高于抗性菌株.随着双炔酰菌胺处理时间的延长,亲本菌株和抗性菌株的可溶性蛋白含量和β-1,3-葡聚糖酶的活力都呈下降趋势.因此推测,辣椒疫霉对双炔酰菌胺抗性产生可能是由于抗性菌株内含物通过细胞膜外渗,致使药液在菌体内的积累减少,最终使到达作用靶标药剂的实际浓度下降;同时菌体的可溶性蛋白含量降低、β-1,3-葡聚糖酶的活力下降也可能是对双炔酰菌胺产生抗性的原因.
英文摘要: Pepper Phytophthora blight is a devastating diseases, which can bring huge losses of pepper.So far, chemical control is still a main means of control of the disease in the production, but the long-term use of a single class of fungicides may result in occurrence of resistance to fungicides and low control efficacy. Carboxylic acid amide fungicide mandipropamid is a new type of Oomycetes fungicide and has not been used at large scale in China. Therefore, this study was undertaken on detection of sensitivity to metalaxyl and dimethomorph and cross-resistance relationship between metalaxyl or dimethomorph and the other fungicides with different mechanisms in Phytophthora capsici, the biological activity of mandipropamid against P. capsici, the sensitivity baseline to mandipropamid in P. capsici, risk assessment of resistance to mandipropamid and the mechanism of the physiological and biochemical mechanism of resistance to mandipropamid. The main results are as follows:1 Sixty-four isolates of P. capsici collected from different regions of Hebei province and Shijiazhuang were tested by measuring mycelial growth rate in order to explicit the sensitivity of P. capsici to metalaxyl and dimethomorph. Sensitivity of 11 relatively sensitive isolates and 3 relatively resistant isolates to metalaxyl, mandipropamid, dimethomorph, azoxystrobin, and cymoxanil were determined by the same method to make sure that it existed cross resistance or not between metalaxyl and other diverse active mechanism fungicides. The results showed that metalaxyl to P. capsici has obvious inhibition, EC50 of 0.1661 27.5572μg /mL, the highest and lowest difference of 165.91 times. Collected from different regions strains of P. capsici sensitive to metalaxyl there were very different.The linear regression showed that there were no positively-correlated cross resistance between metalaxy and other diverse active mechanism fungicide. Dimethomorph on in the fully sensitive state.2 Sensitivity of 11 relatively sensitive isolates and 3 relatively resistant isolates to metalaxyl, mandipropamid, dimethomorph, azoxystrobin, and cymoxanil were determined by measuring mycelial growth rate;Protective effect ,curative of control of mandipropamid against pepper blight were separately determined by detached leaves test and potted plant spray test, and duration of control of mandipropamid against pepper blight were determined connection by fungus cake. The results showed that, mandipropamid and dimethomorph of the CAA fungicides have the best inhibition and the highest toxicity of P. capsici, followed by azoxystrobin of the strobilurin fungicides, cymoxanil has the worst. at the concentration of 10μg/mL, the preventive and curative action of mandipropamid against pepper blight were 86.67%、60.00%by detached leaves test, at the concentration of 100μg/mL, the preventive and curative action of mandipropamid against pepper blight were 95.25%、77.81%by potted plant spray test. With the 100μg/mL relative efficacy is still as high as 65.92%after 14days, and intervals up to 14days. General way to analyze, mandipropamid has good inhibition, but also has good control effect and duration. Its good effects also shows that mandipropamid has good absorption within the conduction.3 The sensitivity to mandipropamid of 173 P. capsici strains in Dingxing and Gaocheng of Hebei Province and Shouguang and Qingzhou of Shandong Province were determined by mycelia growth inhibition method. The results demonstrated that EC50 value ranged from 0.0027 to 0.0499μg /mL, the difference between maximum and minimum are 18.48 times. With a mean value of (0.0219±0.0102)μg/mL. The sensitivity frequency of P. capsici to mandipropamid distributed as a single peak curve. The data could be used as baseline sensitivity of P. capsici to mandipropamid.4 The 10 strains were separately treated by UV- irradiating with colony point change and taming, with 7 and 1 resistant mutants to mandipropamid separately obtained in vitro by UV-irradiating and taming; The resistant levels ranged from 41.50 to 394437.77 times. Mutation frequencies were separately 1.46%and 1.43%. It was proved that mandipropamid has not cross resistance with dimethomorph, metalaxyl, azoxystrobin, and cymoxanil by comparing the sensitivity of different isolates to different fungicides and analysis their linear relation. 8 resistant mutants to mandipropamid were cultured in the CA medium without fungicide for 11 generations, the resistant trait were stably inheritable in 2 isolates, and their EC50 were 1429.5237μg/mL and more than 5000μg/mL , but not stably inheritable in 6 isolates. Some biological characteristics of mutants were studied in laboratory. The mutants exhibited diversity in mycelial growth rate, pathogenicity, sporulation ability, fitness and competition ability with their parental strains. The sporulation, fitness and competition of DX 2-3 20m and DX 2-3 F7 were higher than the parental strains, contributing to the formation