广谱拮抗菌WB3的激光诱变筛选与复合菌肥的生物防治应用
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摘要
目的:鉴定细菌WB3的种属并确认其拮抗范围;通过激光对拮抗菌WB3进行诱变筛选,以筛选出拮抗能力得到有效提高并可稳定遗传的突变菌株;使其与另一株具有广谱拮抗性的枯草芽孢杆菌QM3进行混合培养发酵,将发酵菌液与生化黄腐酸菌肥配制成可应用于田间病害防治的复合生物菌肥。
方法:通过形态学观察、生理生化实验检测以及16SrRNA序列分析对已分离得到的WB3进行菌种鉴定;做WB3针对12株常见植物病害的平板拮抗实验来初步确认其拮抗范围;采用He-Ne激光照射具有拮抗能力的枯草芽孢杆菌WB3,同时以番茄早疫病菌作为指示菌,以期获得诱变效果最佳且能稳定遗传的正向突变菌株;尝试将QM3与WB3进行混合培养,包括平板交叉划线、液体摇瓶培养等方法,观察混合培养下的细菌总数以及拮抗能力的变化;通过改进玉米粉液体培养基来摸索适合二者混合培养的工业发酵培养基;将混合拮抗菌液与生化黄腐酸菌肥进行复配获得复合生物防治菌肥,并进行田间应用试验来检验其防治效果。
结果:WB3经鉴定是一株枯草芽孢杆菌,且对12株植物致病真菌均具有拮抗效应,是一株广谱拮抗细菌;30min He-Ne激光辐照对WB3有明显的诱变作用,所筛选出的最佳突变株WB3'对指示菌的拮抗能力有显著的提高并能稳定遗传;QM3与WB3'可以较好地共同生长,且混合拮抗菌发酵液在拮抗能力方面出现了互补提高的现象;复合生物菌肥相较于化学农药,在番茄等植物大田试验的防病、治病以及促生等效应方面具有全面优势。
结论:运用He-Ne激光诱变方法可以提高WB3的拮抗能力;广谱拮抗菌WB3与QM3能共存于同一生长环境,且二者在生长与拮抗两方面均出现互相促进的效应;复合生防菌肥在实际应用中的防效和肥效均令人满意。
Purpose:To identify the genus of WB3 and affirm its antagonistic range.Induce WB3 by laser in order to pick out the mutation strain whoes antagonistic ability has improved mostly, and who can inherit stably.Do mixed culture with WB3 and another broad-spectrum antagonistic B.subtilis QM3,then match the mixed fermentation broth with biochemical fulvic acid(BFA) bacteria fertilizer together to form a new product,multiple bacteria fertilizer,which would be applied in field experiments.
Method:Identify WB3 through the way of morphology observation,physio-biochemical detection and 16SrRNA sequence analysis.Affirm the strain's antagonistic range initially by doing flat plate(FP) inhibition tests of WB3 on 12 common fungous diseases.Use He-Ne laser to irradiate WB3 who has a broad-spectrum antagonistic range,during the course,making the Alternaria solani as an indication,hoping one mutation strain whose antagonistic ability can be improved a lot and could inherit stably would be obtained.Try to cultivate WB3 and QM3 together through plate crossing streaking,fluid shake flask cultivation,observing the total number of the bacteria and the variation of the antagonistic ability after the mixed fermentation.Furthermore,try to fumble out a fermentation culture fit for the mixed strains basing on corn flour fluid culture.In the end,match the mixed antagonistic bacteria liquor and BFA bacteria fertilizer to form the multiple biological controlling bacteria fertilizer in order to be used in field experiments and application.
Result:WB3 belongs to Bucillus subtilis after the identification,which has an antagonistic ability against the 12 plant pathomycetes.30minutes He-Ne laser irradiation has an apparent mutagenesis effect on WB3.The best mutation strain WB3',whose antagonistic ability against the indication has been improved obviously and could inherit stably.WB3' and QM3 could get with each other well in the same living environment,and their antagonistic abilities can be improved and complement each other.Comparing to the chemical pesticide,multiple bacteria fertilizer has overall advantages in many fields,including disease preventing,curing, and growth promoting.
Conclusion:Using He-Ne laser to irradiate WB3 could improve the antagonistic ability of broad-spectrum antagonistic B.subtilis WB3,which can grow with QM3 together in the same nutrition environment.At the same time,there is a promoting effect both in the growing part and inhibition part.Multiple bacteria fertilizer has a satisfactory prevention effect in the field application experiments as well as its manurial effect.
方法:通过形态学观察、生理生化实验检测以及16SrRNA序列分析对已分离得到的WB3进行菌种鉴定;做WB3针对12株常见植物病害的平板拮抗实验来初步确认其拮抗范围;采用He-Ne激光照射具有拮抗能力的枯草芽孢杆菌WB3,同时以番茄早疫病菌作为指示菌,以期获得诱变效果最佳且能稳定遗传的正向突变菌株;尝试将QM3与WB3进行混合培养,包括平板交叉划线、液体摇瓶培养等方法,观察混合培养下的细菌总数以及拮抗能力的变化;通过改进玉米粉液体培养基来摸索适合二者混合培养的工业发酵培养基;将混合拮抗菌液与生化黄腐酸菌肥进行复配获得复合生物防治菌肥,并进行田间应用试验来检验其防治效果。
结果:WB3经鉴定是一株枯草芽孢杆菌,且对12株植物致病真菌均具有拮抗效应,是一株广谱拮抗细菌;30min He-Ne激光辐照对WB3有明显的诱变作用,所筛选出的最佳突变株WB3'对指示菌的拮抗能力有显著的提高并能稳定遗传;QM3与WB3'可以较好地共同生长,且混合拮抗菌发酵液在拮抗能力方面出现了互补提高的现象;复合生物菌肥相较于化学农药,在番茄等植物大田试验的防病、治病以及促生等效应方面具有全面优势。
结论:运用He-Ne激光诱变方法可以提高WB3的拮抗能力;广谱拮抗菌WB3与QM3能共存于同一生长环境,且二者在生长与拮抗两方面均出现互相促进的效应;复合生防菌肥在实际应用中的防效和肥效均令人满意。
Purpose:To identify the genus of WB3 and affirm its antagonistic range.Induce WB3 by laser in order to pick out the mutation strain whoes antagonistic ability has improved mostly, and who can inherit stably.Do mixed culture with WB3 and another broad-spectrum antagonistic B.subtilis QM3,then match the mixed fermentation broth with biochemical fulvic acid(BFA) bacteria fertilizer together to form a new product,multiple bacteria fertilizer,which would be applied in field experiments.
Method:Identify WB3 through the way of morphology observation,physio-biochemical detection and 16SrRNA sequence analysis.Affirm the strain's antagonistic range initially by doing flat plate(FP) inhibition tests of WB3 on 12 common fungous diseases.Use He-Ne laser to irradiate WB3 who has a broad-spectrum antagonistic range,during the course,making the Alternaria solani as an indication,hoping one mutation strain whose antagonistic ability can be improved a lot and could inherit stably would be obtained.Try to cultivate WB3 and QM3 together through plate crossing streaking,fluid shake flask cultivation,observing the total number of the bacteria and the variation of the antagonistic ability after the mixed fermentation.Furthermore,try to fumble out a fermentation culture fit for the mixed strains basing on corn flour fluid culture.In the end,match the mixed antagonistic bacteria liquor and BFA bacteria fertilizer to form the multiple biological controlling bacteria fertilizer in order to be used in field experiments and application.
Result:WB3 belongs to Bucillus subtilis after the identification,which has an antagonistic ability against the 12 plant pathomycetes.30minutes He-Ne laser irradiation has an apparent mutagenesis effect on WB3.The best mutation strain WB3',whose antagonistic ability against the indication has been improved obviously and could inherit stably.WB3' and QM3 could get with each other well in the same living environment,and their antagonistic abilities can be improved and complement each other.Comparing to the chemical pesticide,multiple bacteria fertilizer has overall advantages in many fields,including disease preventing,curing, and growth promoting.
Conclusion:Using He-Ne laser to irradiate WB3 could improve the antagonistic ability of broad-spectrum antagonistic B.subtilis WB3,which can grow with QM3 together in the same nutrition environment.At the same time,there is a promoting effect both in the growing part and inhibition part.Multiple bacteria fertilizer has a satisfactory prevention effect in the field application experiments as well as its manurial effect.
引文
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