枯草芽孢杆菌12-041和12-059对玉米纹枯病生物防治的研究
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摘要
本试验旨在从花生和中药材植物体内分离的内生芽孢杆菌中筛选出对玉米纹枯病具有较好生防效果的优良菌株。并对该菌株进行鉴定、发酵培养基筛选和发酵条件优化以及对其产生的抑菌物质进行初步探索。
(1)通过平板对峙法、发酵液抑菌法初筛和盆栽防病促生试验,从132株内生芽孢杆菌中,筛选出对玉米纹枯病具有很好生防潜力菌株12-041和12-059。
(2)对菌株12-041和12-059发酵培养基进行了筛选并对发酵条件进行了优化,确定两菌株的最佳发酵培养基为改良SDAY培养基:蛋白胨10g,酵母膏10g,葡萄糖40g,MnSO_4·4H_2O 1.65g(5mmol/L)、蒸馏水1000ml,最佳培养条件为250ml三角瓶装液量为30ml,生长温度30℃,转速为200prm,培养基起始pH为7.5,培养时间为72h时产芽孢量和抑菌效果都达到了最佳。
(3)抑菌物质初探结果发现:两菌株基本不产生挥发性抑菌物质,无菌发酵液的不同梯度(NH_4)_2SO_4沉淀物粗提蛋白不具有抑菌活性,通过酸沉淀和75%乙醇提取的小分子物质具有很强的抑菌活性。
(4)田间试验表明:菌株12-041和12-059菌液对玉米纹枯病的防效分别为69.37%和68.42%,显著高于井岗霉素药液61.82%的防效。
(5)通过菌株形态、生理生化特征及16S rDNA序列分析,将菌株12-041和12-059鉴定为枯草芽孢杆菌(Bacillus subtilis)。
The objective of this research was aimed at screening the antagonistic effect of endophytic bacillus on the Maize Sheath Blight. The antagonistic strains were identified.The fermentation medium and the growth condition were optimized. The inhibitory substance produced by the antagonistic strains was studied preliminarily..
(1) Two antagonistic strains 12-041 and 12-059 were selected from one hundred and thirty-two endophytic bacillus strains isolated from peanuts and medicinal plants by primary screening of dual culture, the inhibitory ability of the filtrated fermentation cultures and pot inoculation experiment.
(2) The fermented medium and conditions for strains 12-041 and 12-059 was investigated.The medium was modified SDAY medium, which contained peptone 10g, yeast extract paste 10g, glucose 40g, MnSO_4·4H_2O 1.62g(5mmol/L), distilled water 1000ml. The optimal growth conditions were temperature 30℃, 200prm, intial pH7.5, culture time 72h with 30ml medium in 250ml triangular flask. The bacterial sporulation quantity and the inhibitory activity of the fermented filtrate could reach to the best.
(3) The antifungal compound produced by strains 12-041 and 12-059 was not the crude protein extracted by ammonium sulfate precipitation at different concentrations. The low weight molecules extracted by acid precipitation and 75% alcohol abstraction had strong inhibitory activity to R. solani.
(4) In pot experiment, 12-041 and 12-059 biocontrol efficacies against Maize Sheath Blight were 69.37% and 68.42%, respectively, which were significantly higher than that of Jinggangmycin.
(5) The strains 12-041 and 12-059 were both identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and sequences of 16S rDNA.
(1)通过平板对峙法、发酵液抑菌法初筛和盆栽防病促生试验,从132株内生芽孢杆菌中,筛选出对玉米纹枯病具有很好生防潜力菌株12-041和12-059。
(2)对菌株12-041和12-059发酵培养基进行了筛选并对发酵条件进行了优化,确定两菌株的最佳发酵培养基为改良SDAY培养基:蛋白胨10g,酵母膏10g,葡萄糖40g,MnSO_4·4H_2O 1.65g(5mmol/L)、蒸馏水1000ml,最佳培养条件为250ml三角瓶装液量为30ml,生长温度30℃,转速为200prm,培养基起始pH为7.5,培养时间为72h时产芽孢量和抑菌效果都达到了最佳。
(3)抑菌物质初探结果发现:两菌株基本不产生挥发性抑菌物质,无菌发酵液的不同梯度(NH_4)_2SO_4沉淀物粗提蛋白不具有抑菌活性,通过酸沉淀和75%乙醇提取的小分子物质具有很强的抑菌活性。
(4)田间试验表明:菌株12-041和12-059菌液对玉米纹枯病的防效分别为69.37%和68.42%,显著高于井岗霉素药液61.82%的防效。
(5)通过菌株形态、生理生化特征及16S rDNA序列分析,将菌株12-041和12-059鉴定为枯草芽孢杆菌(Bacillus subtilis)。
The objective of this research was aimed at screening the antagonistic effect of endophytic bacillus on the Maize Sheath Blight. The antagonistic strains were identified.The fermentation medium and the growth condition were optimized. The inhibitory substance produced by the antagonistic strains was studied preliminarily..
(1) Two antagonistic strains 12-041 and 12-059 were selected from one hundred and thirty-two endophytic bacillus strains isolated from peanuts and medicinal plants by primary screening of dual culture, the inhibitory ability of the filtrated fermentation cultures and pot inoculation experiment.
(2) The fermented medium and conditions for strains 12-041 and 12-059 was investigated.The medium was modified SDAY medium, which contained peptone 10g, yeast extract paste 10g, glucose 40g, MnSO_4·4H_2O 1.62g(5mmol/L), distilled water 1000ml. The optimal growth conditions were temperature 30℃, 200prm, intial pH7.5, culture time 72h with 30ml medium in 250ml triangular flask. The bacterial sporulation quantity and the inhibitory activity of the fermented filtrate could reach to the best.
(3) The antifungal compound produced by strains 12-041 and 12-059 was not the crude protein extracted by ammonium sulfate precipitation at different concentrations. The low weight molecules extracted by acid precipitation and 75% alcohol abstraction had strong inhibitory activity to R. solani.
(4) In pot experiment, 12-041 and 12-059 biocontrol efficacies against Maize Sheath Blight were 69.37% and 68.42%, respectively, which were significantly higher than that of Jinggangmycin.
(5) The strains 12-041 and 12-059 were both identified as Bacillus subtilis according to their morphological, physiological, biochemical characteristics and sequences of 16S rDNA.
引文
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