辽宁葡萄炭疽病菌对多菌灵抗药性研究
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摘要
葡萄炭疽病是辽宁省葡萄生产上的重要病害之一,已对多菌灵产生抗性。本文在广泛调查和病原采集的基础上,应用了传统的形态学与分子生物学相结合的方法进行病原鉴定;建立了葡萄炭疽病菌对多菌灵的敏感基线并测定其敏感性,评估了该病菌对多菌灵的抗药性风险;进行了室内药剂的筛选,从而为农业生产上提高药效,减少损失,病害防治上提供理论依据。获得研究结果如下:
1、经对辽宁省主要葡萄产区24个葡萄炭疽菌株致病性测定、病原菌形态鉴定和进一步用rRNA-ITS区段进行序列分析的分子鉴定后,结果表明:引起辽宁省不同地区葡萄炭疽病的病原鉴定为半知菌亚门中炭疽菌属的胶孢炭疽菌(Colletotrichum gloeosporioides(Penz.)Sacc.)。并用检测出来的ITS序列与GenBank中的核酸数据进行比对。结果显示该菌与已发表的胶孢炭疽菌同源性相似度很高,达到99%以上。这与生物学鉴定的结果一致。
2、建立了葡萄炭疽菌对多菌灵的敏感基线,采用生长速率法测定了这24种葡萄炭疽菌株对多菌灵的敏感性,结果表明:该病菌对多菌灵的敏感性测定值EC_(50)的范围是0.2678μg/mL～2.7312μg/mL,平均值1.14492μg/mL。大部分菌株的抗药性表现为中抗水平(EC_(50)为0.4598μg/mL～2.299μg/mL),仅有一个菌株抗性较高(EC_(50)为2.7312μg/mL),而不同敏感性频率分布呈不规则的正态分布,存在敏感性下降的风险。
3、对葡萄炭疽菌在室内进行了多菌灵风险评估。试验结果表明:采用紫外诱导与药剂驯化的方法获得三株抗药性突变体,抗药水平分别是6431.96倍、46.76倍和11.72倍。紫外诱导的两个突变菌株与自然抗药菌株经过继代培养后的稳定性较高,而药剂驯化菌株经继代培养后的稳定性下降的幅度较大。在致病力比较当中,各诱导菌株没有亲本菌株和自然抗药菌株致病力强。在测定各菌株菌丝生长速率、产孢量与孢子萌发率等适合度方面,SJX-U2、SJX-Y1、FML菌株比敏感菌株SJX有良好的适合度,而SJX-U1的适合度较弱。亲本菌株室内竞争力优于各抗药性突变体,在混合条件下有一定的竞争力。菌株SJX和FML对各药剂均无交互抗性,而诱导菌株SJX-U2和SJX-Y1对退菌特没有交互抗性,而对福美双、百菌清、甲基托布津药剂存在正交互抗性。
4、对SJX、FML、SJX-Y1这三种菌株进行室内药效试验,结果表明:70%甲基托布津WP、50%退菌特WP、50%多菌灵WP、50%咪鲜胺WP四种杀菌剂的药效抑制率相对较好。考虑价格、毒素、药效等方面,进行不同浓度药剂筛选,结果敏感菌株用1000倍50%多菌灵抑菌率较好,抗药菌株用4000倍50%咪鲜胺抑菌较好。
Grape anthracnose is one of the important diseases on the grape production in Liaoning Province,have been resistant to carbendazim.This article based on extensive investigation and collection of pathogens,application of the traditional morphology and molecular biology methods for pathogen identification;established baseline- sensitivity of grape anthracnose to carbendazim and determined sensitivity;assessed the risk of carbendazim on the drug-resistant;conducted indoor pharmaceutical screening,so as to provide a theoretical basis to improve the pharmacodynamic of agricultural production、reduce losses and disease prevention.The results obtained are as follows:
Ⅰ.As the 24 anthrax strains of grapes of the main grape producing areas in Liaoning Province determination of pathogenic,identification of pathogen morphology and further use of rRNA-ITS sequence analysis section for molecular identification,the results show that:Arising from different regions of Liaoning Province of grape anthracnose pathogen was identified as semi-known in the anthrax cotina genus Colletotrichum gloeosporioides (ColIetotrichum gloeosporioides(Penz.) Sacc.).And detected with the ITS sequences in the GenBank nucleic acid data showed that bacteria,than has been published with the Colletotrichum gloeosporioides high homology similarity,identification and biology of the results were consistent.
Ⅱ.Established grape anthrax baseline-sensitivity to carbendazim,the growth rate method for the determination of 24 kinds of grapes in this strain of anthrax carbendazim sensitivity,and results showed that:the sensitivity of bacteria on the determination of carbendazim EC_(50) value of scope 0.2678μg/mL～2.7312μg/mL,average 1.14492μg/mL. Most of the drug-resistant strains resistant to the level of performance(EC_(50) for 0.4598μg/mL～2.299μg/mL),high resistance is only one strain(EC_(50) for 2.7312μg/mL), and different frequency distribution of the sensitivity of the normal distribution was irregular,there is the risk of decreased sensitivity.
Ⅲ.Grape anthrax had indoor risk assessment to carbendazim.The experimental results show that:the use of UV-induced acclimation and pharmaceutical methods of three drug-resistant mutants,resistance level is 6431.96 times the 46.76 percent and 11.72 percent.The two UV-induced mutant strains with natural resistance after subculture through the stability of higher pharmaceutical domesticated strains after subculture by the stability of the larger drop.Comparisons in the pathogenicity,the strains did not pro-induced and natural resistance of the strain of highly pathogenic strains.Determination in terms of fit of the strains in the mycelial growth rate,sporulation and spore germination rate,SJX-U2,SJX-Y1,FML strain than the sensitive strains SJX have a good fit,and SJX-U1 weaker fitness.Pro-competitiveness of this strain is superior to the interior of drug-resistant mutant in the mixed conditions,a certain degree of competitiveness.SJX strain on the Pharmacy and FML no cross-resistance,and the strain induced by SJX-U2 and SJX-Y1 special bacteria to retire there is no cross-resistance,while thiram, chlorothalonil,thiophanate-methyl Pharmacy there are cross-resistant sexual.
Ⅳ.On SJX,FML,SJX-Y1 of the three strains to carry out efficacy testing,results showed that:70%thiophanate-methyl WP,50%retired special bacteria WP,50% carbendazim WP,50%amine Prochloraz WP efficacy of four fungicides relatively good inhibitory rate.Taking into account the price,toxins,effects,etc.,for different concentrations of pharmaceutical screening,the results of the sensitive strain with 1,000 times the rate of 50%Carbendazim Bacteriostasis better,with 4000-fold resistant strains 50%better Prochloraz Bacteriostasis amine.
1、经对辽宁省主要葡萄产区24个葡萄炭疽菌株致病性测定、病原菌形态鉴定和进一步用rRNA-ITS区段进行序列分析的分子鉴定后,结果表明:引起辽宁省不同地区葡萄炭疽病的病原鉴定为半知菌亚门中炭疽菌属的胶孢炭疽菌(Colletotrichum gloeosporioides(Penz.)Sacc.)。并用检测出来的ITS序列与GenBank中的核酸数据进行比对。结果显示该菌与已发表的胶孢炭疽菌同源性相似度很高,达到99%以上。这与生物学鉴定的结果一致。
2、建立了葡萄炭疽菌对多菌灵的敏感基线,采用生长速率法测定了这24种葡萄炭疽菌株对多菌灵的敏感性,结果表明:该病菌对多菌灵的敏感性测定值EC_(50)的范围是0.2678μg/mL～2.7312μg/mL,平均值1.14492μg/mL。大部分菌株的抗药性表现为中抗水平(EC_(50)为0.4598μg/mL～2.299μg/mL),仅有一个菌株抗性较高(EC_(50)为2.7312μg/mL),而不同敏感性频率分布呈不规则的正态分布,存在敏感性下降的风险。
3、对葡萄炭疽菌在室内进行了多菌灵风险评估。试验结果表明:采用紫外诱导与药剂驯化的方法获得三株抗药性突变体,抗药水平分别是6431.96倍、46.76倍和11.72倍。紫外诱导的两个突变菌株与自然抗药菌株经过继代培养后的稳定性较高,而药剂驯化菌株经继代培养后的稳定性下降的幅度较大。在致病力比较当中,各诱导菌株没有亲本菌株和自然抗药菌株致病力强。在测定各菌株菌丝生长速率、产孢量与孢子萌发率等适合度方面,SJX-U2、SJX-Y1、FML菌株比敏感菌株SJX有良好的适合度,而SJX-U1的适合度较弱。亲本菌株室内竞争力优于各抗药性突变体,在混合条件下有一定的竞争力。菌株SJX和FML对各药剂均无交互抗性,而诱导菌株SJX-U2和SJX-Y1对退菌特没有交互抗性,而对福美双、百菌清、甲基托布津药剂存在正交互抗性。
4、对SJX、FML、SJX-Y1这三种菌株进行室内药效试验,结果表明:70%甲基托布津WP、50%退菌特WP、50%多菌灵WP、50%咪鲜胺WP四种杀菌剂的药效抑制率相对较好。考虑价格、毒素、药效等方面,进行不同浓度药剂筛选,结果敏感菌株用1000倍50%多菌灵抑菌率较好,抗药菌株用4000倍50%咪鲜胺抑菌较好。
Grape anthracnose is one of the important diseases on the grape production in Liaoning Province,have been resistant to carbendazim.This article based on extensive investigation and collection of pathogens,application of the traditional morphology and molecular biology methods for pathogen identification;established baseline- sensitivity of grape anthracnose to carbendazim and determined sensitivity;assessed the risk of carbendazim on the drug-resistant;conducted indoor pharmaceutical screening,so as to provide a theoretical basis to improve the pharmacodynamic of agricultural production、reduce losses and disease prevention.The results obtained are as follows:
Ⅰ.As the 24 anthrax strains of grapes of the main grape producing areas in Liaoning Province determination of pathogenic,identification of pathogen morphology and further use of rRNA-ITS sequence analysis section for molecular identification,the results show that:Arising from different regions of Liaoning Province of grape anthracnose pathogen was identified as semi-known in the anthrax cotina genus Colletotrichum gloeosporioides (ColIetotrichum gloeosporioides(Penz.) Sacc.).And detected with the ITS sequences in the GenBank nucleic acid data showed that bacteria,than has been published with the Colletotrichum gloeosporioides high homology similarity,identification and biology of the results were consistent.
Ⅱ.Established grape anthrax baseline-sensitivity to carbendazim,the growth rate method for the determination of 24 kinds of grapes in this strain of anthrax carbendazim sensitivity,and results showed that:the sensitivity of bacteria on the determination of carbendazim EC_(50) value of scope 0.2678μg/mL～2.7312μg/mL,average 1.14492μg/mL. Most of the drug-resistant strains resistant to the level of performance(EC_(50) for 0.4598μg/mL～2.299μg/mL),high resistance is only one strain(EC_(50) for 2.7312μg/mL), and different frequency distribution of the sensitivity of the normal distribution was irregular,there is the risk of decreased sensitivity.
Ⅲ.Grape anthrax had indoor risk assessment to carbendazim.The experimental results show that:the use of UV-induced acclimation and pharmaceutical methods of three drug-resistant mutants,resistance level is 6431.96 times the 46.76 percent and 11.72 percent.The two UV-induced mutant strains with natural resistance after subculture through the stability of higher pharmaceutical domesticated strains after subculture by the stability of the larger drop.Comparisons in the pathogenicity,the strains did not pro-induced and natural resistance of the strain of highly pathogenic strains.Determination in terms of fit of the strains in the mycelial growth rate,sporulation and spore germination rate,SJX-U2,SJX-Y1,FML strain than the sensitive strains SJX have a good fit,and SJX-U1 weaker fitness.Pro-competitiveness of this strain is superior to the interior of drug-resistant mutant in the mixed conditions,a certain degree of competitiveness.SJX strain on the Pharmacy and FML no cross-resistance,and the strain induced by SJX-U2 and SJX-Y1 special bacteria to retire there is no cross-resistance,while thiram, chlorothalonil,thiophanate-methyl Pharmacy there are cross-resistant sexual.
Ⅳ.On SJX,FML,SJX-Y1 of the three strains to carry out efficacy testing,results showed that:70%thiophanate-methyl WP,50%retired special bacteria WP,50% carbendazim WP,50%amine Prochloraz WP efficacy of four fungicides relatively good inhibitory rate.Taking into account the price,toxins,effects,etc.,for different concentrations of pharmaceutical screening,the results of the sensitive strain with 1,000 times the rate of 50%Carbendazim Bacteriostasis better,with 4000-fold resistant strains 50%better Prochloraz Bacteriostasis amine.
引文
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