纳他霉素对灰葡萄孢的活性及作用机理研究
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摘要
灰葡萄孢(Botrytis cinerea Pers.)是设施栽培和果蔬采后保鲜中的重要病原真菌,目前主要通过使用化学杀菌剂进行防治,由此产生的残留污染受到日益密切的关注,迫切需要寻找更加安全、高效的药剂。本研究以敏感灰葡萄孢菌株为对象,测定了纳他霉素(Natamycin)及多菌灵、乙霉威、腐霉利和嘧霉胺等药剂的抑制活性,同时,研究了纳他霉素对菌株生物学性状的影响、作用机理及不同试验条件对纳他霉素稳定性的影响,研究结果表明:
1、采用菌丝生长速率法测定了25℃下供试灰葡萄孢对纳他霉素等药剂的敏感性。结果表明,纳他霉素对灰葡萄孢有较高的活性,其EC50值为37.4212 mg/L;多菌灵、异菌脲和腐霉利的抑制效果最佳,EC50值分别为0.6973 mg/L、0.5315 mg/L和0.4411 mg/L;咪鲜胺、噻菌灵和嘧霉胺次之,EC50值分别为1.7181 mg/L、1.8432 mg/L和3.2956 mg/L;乙霉威的活性最差,EC50值达151.2110 mg/L。
2、研究了不同温度下纳他霉素等药剂对不同生育阶段灰葡萄孢的活性。结果表明,随温度下降药剂活性均有不同程度提高。其中纳他霉素在低温下对菌丝生长有很高的抑制活性,5℃时EC50值为0.9840 mg/L,活性较25℃提高了38倍。5℃时乙霉威活性较25℃提高了6倍,多菌灵活性提高了10.8倍,嘧霉胺、腐霉利、噻菌灵、异菌脲、咪鲜胺的活性分别提高了9.6倍、8.2倍、4.5倍、4.1倍和30.6倍;10℃时纳他霉素对分生孢子萌发的抑制活性是25℃时的2.5倍,对菌核萌发的抑制活性是25℃时的1.7倍。不同生育阶段菌体对药剂的敏感性差异显著,分生孢子对药剂最敏感,25℃时纳他霉素对分生孢子萌发的EC50是3.3239 mg/L,对菌丝生长的EC50是37.4212 mg/L,对菌核萌发的抑制活性极微弱,EC50大于2000 mg/L。
3、研究了纳他霉素和多菌灵对灰葡萄孢生物学性状的影响。两种药剂对菌株产孢、产菌核时间无显著影响,但纳他霉素较多菌灵可显著抑制菌株产孢、产菌核,多菌灵对菌株产孢、产菌核有明显的刺激作用。
4、研究了纳他霉素在不同试验条件下的稳定性。结果表明,紫外线照射可显著促进纳他霉素分解,照射0.5 h后,降解率达54.72%。低温能有效缓解纳他霉素的降解,25℃下放置24h后,纳他霉素浓度仅相当于初始配制浓度的51.2%,而5℃下放置24h后,是初始配制时的82.3%,降解率仅为17.7%。加入抗氧剂后,可显著抑制纳他霉素的降解,放置20d后,加入抗氧剂的处理中纳他霉素有效成分含量降解率为34.84%,而空白处理的降解率为68.16%。通过研究不同PH值下纳他霉素对灰葡萄孢菌丝生长和分生孢子萌发的影响发现,纳他霉素在弱酸性条件下有较好的作用效果。
5、初步探讨了纳他霉素对灰葡萄孢的作用机理。研究表明,纳他霉素对菌丝体内含物渗漏的影响明显。25℃下经纳他霉素处理8 h后,单位重量菌丝体可溶性糖渗漏量达到4 mg/g,较初始时提高了9倍;可溶性蛋白渗漏量达到177μg/g,为初始时的2倍;电导率达到700μs/cm,为初始时的6倍,而其它药剂如多菌灵、乙霉威等处理的变化均不明显。不同温度处理之间,温度越低,菌丝体内含物的渗漏量越少,纳他霉素处理的变化最为明显,5℃处理8h后可溶性糖渗漏量仅为25℃处理8h后的31%。经纳他霉素处理后,可显著降低菌丝体麦角甾醇含量,25℃下20 mg/L纳他霉素处理菌丝体中麦角甾醇含量为46.78μg/g,是空白对照处理的55%,培养温度越低,菌丝体中麦角甾醇含量越少。纳他霉素对菌株胞外酶活性没有显著影响。
6、在番茄活体叶片上接种了供试菌株。试验结果证明,供试药剂对灰葡萄孢病斑扩展的抑制作用与离体试验结果一致,经浸渍处理后,纳他霉素对灰葡萄孢病斑在叶片上扩展的抑制作用较多菌灵差,但显著好于乙霉威。
Botrytis cinerea Pers is an important pathogenic fungus of plant in greenhouse and postharvest storage. Now fungicide is the main prevent method. But people pay close attention to remaining problem. Search more safer and high-performance fungicide is an urgent task. Use sensitive strain as object, effects of natamycin, carbendazim, diethofencarb, procymidone and pyrimethani against B.cinerea were tested. The activity change of fungicides under different temperatures was also tested. We have also studied the effect of natamycin on biological characteristics of B. cinerea and action mechanism of natamycin. Stability of natamycin under different test conditions was also studied. The results showed that:
1 The sensitivity of B. cinerea to fungicides under 25℃was tested through mycelial growth rate method. Natamycin has good effect, EC50 value was 37.4212 mg/L, efficacy of carbendazim, iprodione and procymidone were remarkable, EC50 value were 0.6973 mg/L, 0.5315 mg/L and 0.4411 mg/L. Prochloraz, thiabendazole and pyrimethanil take the second place, EC50 value were 1.7181mg/L, 1.8432mg/L and 3.2956mg/L. The activity of diethofencarb was the lowest, EC50 value was 151.2110 mg/L.
2 The sensitivity of B. cinerea to fungicides under different temperatures was tested. With temperature decrease, the effect of fungicides to B. cinerea is increase. Natamycin have good effect on mycelial growth at low temperature, EC50 value at 5℃was 0.9840 mg/L, increased 38-folds compared with 25℃. The activity of diethofencarb, carbendazim, pyrimethanil, procymidone, thiabendazole, iprodione and prochloraz to B. cinerea increased 6-folds, 10.8-folds, 9.6-folds, 8.2-folds, 4.5-folds, 4.1-folds and 30.6-folds. The activity of natamycin to spore germination at 10℃increased 2.5-folds compared with 25℃, and to sclerote germination increased 1.7-folds. The distinction of activity to B. cinerea at different growth stages was significant. Conidium was most sensitive. EC50 value of natamycin to spore germination and mycelial growth were 3.3239mg/L and 37.4212 mg/L. Activity of natamycin to sclerote germination was very weak, EC50 value exceeded 2000 mg/L.
3 The toxicity to B. cinerea at different growth stages was also studied. The two fungicides have no effect on the time of spore and sclerote formation, but natamycin can restrain sporulation and sclerotigenic significantly compared with carbendazim. Carbendazim can stimulate sporulation and sclerotigenic.
4 The stability of natamycin under different conditions was studied. The result indicated that ultraviolet irradiation can promote decomposition. After irradiated half an hour, utility content degradation rate was 60%. Natamycin decompose slowly in low temperature. After twenty-four hours, utility content decreased to 51.2% compared with incipient under 25℃. While, under 5℃the utility content decreased to 82.3%, degradation rate was only 17.7%. Oxidation-resistant can restrain decomposition of natamycin. After twenty days, degradation rate of CK was 68.16%. While, the degradation rate of treatment which contain oxidation-resistant was 34.84%. The result of effect on mycelial growth and spore germination under different PH value indicated that, natamycin has better effect in weak acid condition.
5 Studies on action mechanism indicated that, influence of natamycin to mycelial leakage was significant. 8 hours after treated by natamycin, sugar leakage was 4 mg/g, increased 9-folds compared with incipient. Albumen leakage was 177μg/g. Conductivity was 700μs/cm. Other fungicides did not have so significant change. Among treatments of different temperatures, mycelial leakage decreased as temperature decreased. The change of treatment by natamycin was most significant. Sugar leakage after 8 hours under 5℃was 31 percents of treatment under 25℃. After treated by natamycin, the content of ergostero1 decreased. Ergostero1 content of treatment by 20 mg/L natamycin was 46.78μg/g, 55 percents of CK. Ergostero1 content decrease with temperature drop. But exocellular enzyme activity has no change.
6 The result of live leaf test was identical to the toxicity of mycelium growth. The inhibitory effect of natamycin on spot expand was worse than carbendazim, but better than diethofencarb remarkably.
1、采用菌丝生长速率法测定了25℃下供试灰葡萄孢对纳他霉素等药剂的敏感性。结果表明,纳他霉素对灰葡萄孢有较高的活性,其EC50值为37.4212 mg/L;多菌灵、异菌脲和腐霉利的抑制效果最佳,EC50值分别为0.6973 mg/L、0.5315 mg/L和0.4411 mg/L;咪鲜胺、噻菌灵和嘧霉胺次之,EC50值分别为1.7181 mg/L、1.8432 mg/L和3.2956 mg/L;乙霉威的活性最差,EC50值达151.2110 mg/L。
2、研究了不同温度下纳他霉素等药剂对不同生育阶段灰葡萄孢的活性。结果表明,随温度下降药剂活性均有不同程度提高。其中纳他霉素在低温下对菌丝生长有很高的抑制活性,5℃时EC50值为0.9840 mg/L,活性较25℃提高了38倍。5℃时乙霉威活性较25℃提高了6倍,多菌灵活性提高了10.8倍,嘧霉胺、腐霉利、噻菌灵、异菌脲、咪鲜胺的活性分别提高了9.6倍、8.2倍、4.5倍、4.1倍和30.6倍;10℃时纳他霉素对分生孢子萌发的抑制活性是25℃时的2.5倍,对菌核萌发的抑制活性是25℃时的1.7倍。不同生育阶段菌体对药剂的敏感性差异显著,分生孢子对药剂最敏感,25℃时纳他霉素对分生孢子萌发的EC50是3.3239 mg/L,对菌丝生长的EC50是37.4212 mg/L,对菌核萌发的抑制活性极微弱,EC50大于2000 mg/L。
3、研究了纳他霉素和多菌灵对灰葡萄孢生物学性状的影响。两种药剂对菌株产孢、产菌核时间无显著影响,但纳他霉素较多菌灵可显著抑制菌株产孢、产菌核,多菌灵对菌株产孢、产菌核有明显的刺激作用。
4、研究了纳他霉素在不同试验条件下的稳定性。结果表明,紫外线照射可显著促进纳他霉素分解,照射0.5 h后,降解率达54.72%。低温能有效缓解纳他霉素的降解,25℃下放置24h后,纳他霉素浓度仅相当于初始配制浓度的51.2%,而5℃下放置24h后,是初始配制时的82.3%,降解率仅为17.7%。加入抗氧剂后,可显著抑制纳他霉素的降解,放置20d后,加入抗氧剂的处理中纳他霉素有效成分含量降解率为34.84%,而空白处理的降解率为68.16%。通过研究不同PH值下纳他霉素对灰葡萄孢菌丝生长和分生孢子萌发的影响发现,纳他霉素在弱酸性条件下有较好的作用效果。
5、初步探讨了纳他霉素对灰葡萄孢的作用机理。研究表明,纳他霉素对菌丝体内含物渗漏的影响明显。25℃下经纳他霉素处理8 h后,单位重量菌丝体可溶性糖渗漏量达到4 mg/g,较初始时提高了9倍;可溶性蛋白渗漏量达到177μg/g,为初始时的2倍;电导率达到700μs/cm,为初始时的6倍,而其它药剂如多菌灵、乙霉威等处理的变化均不明显。不同温度处理之间,温度越低,菌丝体内含物的渗漏量越少,纳他霉素处理的变化最为明显,5℃处理8h后可溶性糖渗漏量仅为25℃处理8h后的31%。经纳他霉素处理后,可显著降低菌丝体麦角甾醇含量,25℃下20 mg/L纳他霉素处理菌丝体中麦角甾醇含量为46.78μg/g,是空白对照处理的55%,培养温度越低,菌丝体中麦角甾醇含量越少。纳他霉素对菌株胞外酶活性没有显著影响。
6、在番茄活体叶片上接种了供试菌株。试验结果证明,供试药剂对灰葡萄孢病斑扩展的抑制作用与离体试验结果一致,经浸渍处理后,纳他霉素对灰葡萄孢病斑在叶片上扩展的抑制作用较多菌灵差,但显著好于乙霉威。
Botrytis cinerea Pers is an important pathogenic fungus of plant in greenhouse and postharvest storage. Now fungicide is the main prevent method. But people pay close attention to remaining problem. Search more safer and high-performance fungicide is an urgent task. Use sensitive strain as object, effects of natamycin, carbendazim, diethofencarb, procymidone and pyrimethani against B.cinerea were tested. The activity change of fungicides under different temperatures was also tested. We have also studied the effect of natamycin on biological characteristics of B. cinerea and action mechanism of natamycin. Stability of natamycin under different test conditions was also studied. The results showed that:
1 The sensitivity of B. cinerea to fungicides under 25℃was tested through mycelial growth rate method. Natamycin has good effect, EC50 value was 37.4212 mg/L, efficacy of carbendazim, iprodione and procymidone were remarkable, EC50 value were 0.6973 mg/L, 0.5315 mg/L and 0.4411 mg/L. Prochloraz, thiabendazole and pyrimethanil take the second place, EC50 value were 1.7181mg/L, 1.8432mg/L and 3.2956mg/L. The activity of diethofencarb was the lowest, EC50 value was 151.2110 mg/L.
2 The sensitivity of B. cinerea to fungicides under different temperatures was tested. With temperature decrease, the effect of fungicides to B. cinerea is increase. Natamycin have good effect on mycelial growth at low temperature, EC50 value at 5℃was 0.9840 mg/L, increased 38-folds compared with 25℃. The activity of diethofencarb, carbendazim, pyrimethanil, procymidone, thiabendazole, iprodione and prochloraz to B. cinerea increased 6-folds, 10.8-folds, 9.6-folds, 8.2-folds, 4.5-folds, 4.1-folds and 30.6-folds. The activity of natamycin to spore germination at 10℃increased 2.5-folds compared with 25℃, and to sclerote germination increased 1.7-folds. The distinction of activity to B. cinerea at different growth stages was significant. Conidium was most sensitive. EC50 value of natamycin to spore germination and mycelial growth were 3.3239mg/L and 37.4212 mg/L. Activity of natamycin to sclerote germination was very weak, EC50 value exceeded 2000 mg/L.
3 The toxicity to B. cinerea at different growth stages was also studied. The two fungicides have no effect on the time of spore and sclerote formation, but natamycin can restrain sporulation and sclerotigenic significantly compared with carbendazim. Carbendazim can stimulate sporulation and sclerotigenic.
4 The stability of natamycin under different conditions was studied. The result indicated that ultraviolet irradiation can promote decomposition. After irradiated half an hour, utility content degradation rate was 60%. Natamycin decompose slowly in low temperature. After twenty-four hours, utility content decreased to 51.2% compared with incipient under 25℃. While, under 5℃the utility content decreased to 82.3%, degradation rate was only 17.7%. Oxidation-resistant can restrain decomposition of natamycin. After twenty days, degradation rate of CK was 68.16%. While, the degradation rate of treatment which contain oxidation-resistant was 34.84%. The result of effect on mycelial growth and spore germination under different PH value indicated that, natamycin has better effect in weak acid condition.
5 Studies on action mechanism indicated that, influence of natamycin to mycelial leakage was significant. 8 hours after treated by natamycin, sugar leakage was 4 mg/g, increased 9-folds compared with incipient. Albumen leakage was 177μg/g. Conductivity was 700μs/cm. Other fungicides did not have so significant change. Among treatments of different temperatures, mycelial leakage decreased as temperature decreased. The change of treatment by natamycin was most significant. Sugar leakage after 8 hours under 5℃was 31 percents of treatment under 25℃. After treated by natamycin, the content of ergostero1 decreased. Ergostero1 content of treatment by 20 mg/L natamycin was 46.78μg/g, 55 percents of CK. Ergostero1 content decrease with temperature drop. But exocellular enzyme activity has no change.
6 The result of live leaf test was identical to the toxicity of mycelium growth. The inhibitory effect of natamycin on spot expand was worse than carbendazim, but better than diethofencarb remarkably.
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