梨黑斑病拮抗放线菌的选育及其抗菌物质研究
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摘要
梨黑斑病是一类危害严重的世界性气传真菌病害,它的有效防治已经成为鸭梨生产和贮存中有待解决的难题。本研究从生物防治的角度出发,分离筛选梨黑斑病菌拮抗放线菌株,通过诱变育种提高其生产性能,并对突变株产生的抗菌物质及生防效果进行研究,旨在为开发高效防治梨黑斑病的生防制剂奠定工作基础。
采用稀释平板法,从果园根际土壤中分离到372株放线菌。通过抑菌圈筛选,得到6株对梨链格孢菌(Alternaria kikuchiana)拮抗作用较强的菌株;进而对这6株菌进行果实活体筛选,其中B105菌株代谢物抑菌效果最好,28℃下的抑菌率为67.4%。抑菌谱实验表明它对苹果轮纹病菌(Physalospora piricola)、柑橘青霉病菌(Penicillium expansum Link),梨黑星病菌(Venturia pirina)、番茄灰霉病菌(Botrytis cinerea)等多种植物病原真菌有明显的抑制作用。
对B105菌株进行形态特征、培养特征、生理生化特性测试及16S rDNA序列分析,结果表明该菌株属于链霉菌属,在以16S rDNA序列为基础构建的系统发育树中,B105菌株与金色链霉菌(Streptomyces aureus)的同源性最高,达到99%以上。
利用紫外线、微波和化学试剂NTG三种理化因子对菌株B105进行诱变育种,结果发现NTG诱变效果最好,在其浓度为1mg/mL时获得了高产突变株N2,它的发酵液抑菌圈直径比出发菌株提高了52.2%,在果实上的抑菌效果比出发菌株提高了19.2%,并且遗传性能稳定。
对突变株N2所产生的抗菌物质的性质进行了研究,结果显示此物质可耐受高温,具有较宽的pH活性范围,同时对光照稳定,耐贮存;利用pH纸色谱、捷克八溶剂系统纸色谱等方法初步确认其抗菌物质为一种碱性水溶性抗生素。
生防测试结果表明N2菌株产生的抗菌物质能够显著抑制病原菌菌丝的生长和孢子的萌发;在果实及叶片上对梨黑斑病害的防效分别达到85.7%和82.3%,明显高于对照药剂多菌灵500倍稀释液的防效。
Pear black spot caused by Alternaria kikuchiana is a serious air spreading disease which distributes widely in the world. 1t is a difficult problem to control the disease effectively. The research focuses on the biocontrol of pear black spot. Therefore, mutagenic breeding and inhibition effects were studied after an antibiotic actionmycete strain was screened and identified. It provided the theory basis and the technical support for the exploiting agricultural antibiotics for biocontrol.
Three hundred and seventy two strains of actionmycetes were isolated from the rhizosphere soil samples using dilution method, from which six strains effectively inhibiting Alternaria kikuchiana were screened out by inhibitory zone method. After further screening on fruits, It was found that strain B105 was more effectivly than other antagonistic actinomycetes and its fermentation filtratethe inhibition rate got to 66.5% at 28℃. B105 strain also showed broad antagonism against pathogenic fungi in plant, such as: Physalospora piricola, Penicillium expansum Link, Venturia pirina, Botrytis cinerea et al.
The morphological, cultural physiological, biochemical characteristics and 16S rDNA sequences of strain B105 were studied. Its substrate mycelium had no partition, the aerial mycelium were ramose, the spores were oval and the surface was smooth, which consistented with the characteristics of the genus Streptomyces. The analysis of 16S rDNA sequence of strain B105 suggested that it was most closely related to Streptomyces aureus and the sequence identity was 99%. From the polyphasic taxonomical view, the strain B105 falled into Streptomyces aureus.
In order to improve the antibiotic-producing, UV, microwave and NTG were used to mut -agenize B105 spores, respectively. The results showed NTG has stronger mutagenic effect than UV and microwave. Under the optimum dosage of 1mg/ml, a high productive mutant strain named N2 was screened out. The diameter of antibiotic zone of N2 increased by 52.2% and the inhibitation effects to AlternAria kikuchiana increased by19.2% compared to B105 strain. Futhermore , its high capacity for producing antibiotic remained stable after 5 times of subcultures and its antifungal spectrum was not altered.
The antagonistic substance produced by the strain was considered as one of its antagonistic mechanisms against Alternaria kikuchiana. The antagonistic substance was stable to heat, pH, and illumination, its inhibitory activity could remain 76.5% even incubating at 100℃for 120mins. Accoding to the results of Jack's Eight Solvent System Chromatography and pH Paper Chromatography of the fermentation extracts indicated that the antagonistic substance was basic and water solubility-alkaline antibiotic.
The result of biocontrol test indicated that the fermentation filtrate had strong inhibition mycelium growth and spore germination of Alternaria kikuchiana, and the control effects on the diseased fruits and pear leaves were 85.7% and 82.3%, respectively, which were obviously better than 50% Duojunling (500 times dilution). The result indicated that strain B105 was really a potential biocontrol endophytic actinomycete.
采用稀释平板法,从果园根际土壤中分离到372株放线菌。通过抑菌圈筛选,得到6株对梨链格孢菌(Alternaria kikuchiana)拮抗作用较强的菌株;进而对这6株菌进行果实活体筛选,其中B105菌株代谢物抑菌效果最好,28℃下的抑菌率为67.4%。抑菌谱实验表明它对苹果轮纹病菌(Physalospora piricola)、柑橘青霉病菌(Penicillium expansum Link),梨黑星病菌(Venturia pirina)、番茄灰霉病菌(Botrytis cinerea)等多种植物病原真菌有明显的抑制作用。
对B105菌株进行形态特征、培养特征、生理生化特性测试及16S rDNA序列分析,结果表明该菌株属于链霉菌属,在以16S rDNA序列为基础构建的系统发育树中,B105菌株与金色链霉菌(Streptomyces aureus)的同源性最高,达到99%以上。
利用紫外线、微波和化学试剂NTG三种理化因子对菌株B105进行诱变育种,结果发现NTG诱变效果最好,在其浓度为1mg/mL时获得了高产突变株N2,它的发酵液抑菌圈直径比出发菌株提高了52.2%,在果实上的抑菌效果比出发菌株提高了19.2%,并且遗传性能稳定。
对突变株N2所产生的抗菌物质的性质进行了研究,结果显示此物质可耐受高温,具有较宽的pH活性范围,同时对光照稳定,耐贮存;利用pH纸色谱、捷克八溶剂系统纸色谱等方法初步确认其抗菌物质为一种碱性水溶性抗生素。
生防测试结果表明N2菌株产生的抗菌物质能够显著抑制病原菌菌丝的生长和孢子的萌发;在果实及叶片上对梨黑斑病害的防效分别达到85.7%和82.3%,明显高于对照药剂多菌灵500倍稀释液的防效。
Pear black spot caused by Alternaria kikuchiana is a serious air spreading disease which distributes widely in the world. 1t is a difficult problem to control the disease effectively. The research focuses on the biocontrol of pear black spot. Therefore, mutagenic breeding and inhibition effects were studied after an antibiotic actionmycete strain was screened and identified. It provided the theory basis and the technical support for the exploiting agricultural antibiotics for biocontrol.
Three hundred and seventy two strains of actionmycetes were isolated from the rhizosphere soil samples using dilution method, from which six strains effectively inhibiting Alternaria kikuchiana were screened out by inhibitory zone method. After further screening on fruits, It was found that strain B105 was more effectivly than other antagonistic actinomycetes and its fermentation filtratethe inhibition rate got to 66.5% at 28℃. B105 strain also showed broad antagonism against pathogenic fungi in plant, such as: Physalospora piricola, Penicillium expansum Link, Venturia pirina, Botrytis cinerea et al.
The morphological, cultural physiological, biochemical characteristics and 16S rDNA sequences of strain B105 were studied. Its substrate mycelium had no partition, the aerial mycelium were ramose, the spores were oval and the surface was smooth, which consistented with the characteristics of the genus Streptomyces. The analysis of 16S rDNA sequence of strain B105 suggested that it was most closely related to Streptomyces aureus and the sequence identity was 99%. From the polyphasic taxonomical view, the strain B105 falled into Streptomyces aureus.
In order to improve the antibiotic-producing, UV, microwave and NTG were used to mut -agenize B105 spores, respectively. The results showed NTG has stronger mutagenic effect than UV and microwave. Under the optimum dosage of 1mg/ml, a high productive mutant strain named N2 was screened out. The diameter of antibiotic zone of N2 increased by 52.2% and the inhibitation effects to AlternAria kikuchiana increased by19.2% compared to B105 strain. Futhermore , its high capacity for producing antibiotic remained stable after 5 times of subcultures and its antifungal spectrum was not altered.
The antagonistic substance produced by the strain was considered as one of its antagonistic mechanisms against Alternaria kikuchiana. The antagonistic substance was stable to heat, pH, and illumination, its inhibitory activity could remain 76.5% even incubating at 100℃for 120mins. Accoding to the results of Jack's Eight Solvent System Chromatography and pH Paper Chromatography of the fermentation extracts indicated that the antagonistic substance was basic and water solubility-alkaline antibiotic.
The result of biocontrol test indicated that the fermentation filtrate had strong inhibition mycelium growth and spore germination of Alternaria kikuchiana, and the control effects on the diseased fruits and pear leaves were 85.7% and 82.3%, respectively, which were obviously better than 50% Duojunling (500 times dilution). The result indicated that strain B105 was really a potential biocontrol endophytic actinomycete.
引文
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