枯草芽孢杆菌D221发酵条件优化及对辣椒根腐病防治效果初探
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摘要
枯草芽孢杆菌D221是由东北农业大学农学院农药实验室从辣椒根际土壤中分离筛选得到的。通过离体和活体试验测定,该菌株对辣椒根腐病病原菌有良好的拮抗活性。本文在研究的基础上,对D221菌株的发酵条件优化、抗菌谱测定、无菌发酵液稳定性测定以及防治辣椒根腐病室内盆栽试验等进行了初步的研究,主要研究结果如下:
1.对枯草芽孢杆菌D221的摇瓶发酵营养成分和发酵条件进行了优化,确定了最佳营养成分和发酵条件。试验结果表明:培养基以蔗糖2.0%,胰蛋白胨0.5%,牛肉膏2.0%,NaCl 0.5%,MgSO4 0.1%为最佳。最适的发酵条件为摇床转速150 r/min,接种量5%,初始PH为7,在250 mL三角瓶的装液量为30 mL,30℃恒温条件下发酵48 h。
2.对枯草芽孢杆菌D221菌株无菌发酵液的紫外光和自然光稳定性、热稳定性、酸碱稳定性和耐储藏性进行了研究。结果表明:枯草芽孢杆菌D221发酵液对紫外光和自然光均不敏感;该发酵液在80℃以下稳定性较强,在100℃环境中,随着时间的延长,稳定性有所下降;在中性和弱酸的环境下能保持良好的抗菌活性;且耐储性较强。
3.测定了枯草芽孢杆菌D221无菌发酵液的抑菌谱,发现该菌株无菌发酵液对核盘菌(Sclerotinia sclerotinorum)、尖镰孢菌(Fusarium oxysporum)、立枯丝核菌(Rhizoctonia solani)、大丽花轮枝孢(Verticillium dahliae)、麦根腐平脐蠕孢(Bipolaris sorokiniana)、大豆疫霉(Phytophthora sojae)等植物病原真菌有明显的抑制作用,为该生防菌的应用提供了依据。
4.对枯草芽孢杆菌D221对辣椒根腐病的防治效果进行室内盆栽试验。试验结果表明:D221菌株发酵液对辣椒根腐病有良好的防治效果。接种辣椒根腐病前48 h、24 h和0 h以20 mL/盆浇灌D221菌株发酵液,接种病原菌7天后调查,防效分别达96.8%、88.4%和77.4%,接种14天后防效分别为80.3%、56.6%和37.5%。
Bacillus subtilis strain D221 is isolated from rhizosphere of pepper by Pesticides lab of Northeast Agricultural Univerdity. Strain D221 showed antagonistic activity though plate inhibition method and pot culture trails against hot pepper root rot. On the basis of studies,this disseration is focused particularly on studies on fermentation condition optimization,test of stability of fermentation broth,control efficacy of Bacillus subtilis D221 against hot pepper root rot in the greenhouse.The main results are as follows:
1.Optimized the liquid fermentation medium and technological conditions of Bacillus subtilis D221,The results showed that optimum medium formula was Sucrose 2.0%,Trgprotein 0.5%,Beef 2.0%,NaCl 0.5%,MgSO4 0.1%. The favorable fermentation condition was determined: The optimal fermentation parameters were 30℃,150 r/min, 5% inoculation volume,initial pH value of medium 7, and 30mL culture in 250mL flask for 48h,which had the strongest antagonistic avtivity. Under this optimized condition,the inhibition zone diameter increased 66.7%to the initial condition.
2.Studied on the stability of UV、natural light、different temperature、different pH、reservation.The results for antibiotic physicochemical property of strain D221 showed that the active substance was stable to natural light and ultraviolet irradiation; It was also relatively stable to the hyperthermia around 80℃while the inhibition activity falls down at 100℃; .Prtial stability to acid or alkali, unstability to high alkali condition; and Storage stronger .
3.By means of mycelium growth rate we measured the antibiogram of D221 strain, its fermentation products were against Sclerotinia sclerotinorum, Fusarium oxysporum, Rhizoctonia solani, Verticillium dahliae, Bipolaris sorokiniana, Phytophthora sojae.The results showed that the fermentation products of D221 can resist many plant pathogenic fungus.
4. Pot experments in greenhouse showed that the cell-free filtrate of D221 fermentation have a good control efficacy to the hot pepper root rot in seedings after inoculated with pathogen. Using strain D221 cell-free filtrate 48h, 24h and 0h defore inoculation, the biocontrol efficacy were 96.8%,88.4% and 77.4% respectively investigated 7d after inoculation. The biocontrol efficacy was 80.3%,56.6% and 37.5% respectively 14d after inoculation. The result showed that strain D221 was a potential biocontrol agent in hot pepper root rot control.
1.对枯草芽孢杆菌D221的摇瓶发酵营养成分和发酵条件进行了优化,确定了最佳营养成分和发酵条件。试验结果表明:培养基以蔗糖2.0%,胰蛋白胨0.5%,牛肉膏2.0%,NaCl 0.5%,MgSO4 0.1%为最佳。最适的发酵条件为摇床转速150 r/min,接种量5%,初始PH为7,在250 mL三角瓶的装液量为30 mL,30℃恒温条件下发酵48 h。
2.对枯草芽孢杆菌D221菌株无菌发酵液的紫外光和自然光稳定性、热稳定性、酸碱稳定性和耐储藏性进行了研究。结果表明:枯草芽孢杆菌D221发酵液对紫外光和自然光均不敏感;该发酵液在80℃以下稳定性较强,在100℃环境中,随着时间的延长,稳定性有所下降;在中性和弱酸的环境下能保持良好的抗菌活性;且耐储性较强。
3.测定了枯草芽孢杆菌D221无菌发酵液的抑菌谱,发现该菌株无菌发酵液对核盘菌(Sclerotinia sclerotinorum)、尖镰孢菌(Fusarium oxysporum)、立枯丝核菌(Rhizoctonia solani)、大丽花轮枝孢(Verticillium dahliae)、麦根腐平脐蠕孢(Bipolaris sorokiniana)、大豆疫霉(Phytophthora sojae)等植物病原真菌有明显的抑制作用,为该生防菌的应用提供了依据。
4.对枯草芽孢杆菌D221对辣椒根腐病的防治效果进行室内盆栽试验。试验结果表明:D221菌株发酵液对辣椒根腐病有良好的防治效果。接种辣椒根腐病前48 h、24 h和0 h以20 mL/盆浇灌D221菌株发酵液,接种病原菌7天后调查,防效分别达96.8%、88.4%和77.4%,接种14天后防效分别为80.3%、56.6%和37.5%。
Bacillus subtilis strain D221 is isolated from rhizosphere of pepper by Pesticides lab of Northeast Agricultural Univerdity. Strain D221 showed antagonistic activity though plate inhibition method and pot culture trails against hot pepper root rot. On the basis of studies,this disseration is focused particularly on studies on fermentation condition optimization,test of stability of fermentation broth,control efficacy of Bacillus subtilis D221 against hot pepper root rot in the greenhouse.The main results are as follows:
1.Optimized the liquid fermentation medium and technological conditions of Bacillus subtilis D221,The results showed that optimum medium formula was Sucrose 2.0%,Trgprotein 0.5%,Beef 2.0%,NaCl 0.5%,MgSO4 0.1%. The favorable fermentation condition was determined: The optimal fermentation parameters were 30℃,150 r/min, 5% inoculation volume,initial pH value of medium 7, and 30mL culture in 250mL flask for 48h,which had the strongest antagonistic avtivity. Under this optimized condition,the inhibition zone diameter increased 66.7%to the initial condition.
2.Studied on the stability of UV、natural light、different temperature、different pH、reservation.The results for antibiotic physicochemical property of strain D221 showed that the active substance was stable to natural light and ultraviolet irradiation; It was also relatively stable to the hyperthermia around 80℃while the inhibition activity falls down at 100℃; .Prtial stability to acid or alkali, unstability to high alkali condition; and Storage stronger .
3.By means of mycelium growth rate we measured the antibiogram of D221 strain, its fermentation products were against Sclerotinia sclerotinorum, Fusarium oxysporum, Rhizoctonia solani, Verticillium dahliae, Bipolaris sorokiniana, Phytophthora sojae.The results showed that the fermentation products of D221 can resist many plant pathogenic fungus.
4. Pot experments in greenhouse showed that the cell-free filtrate of D221 fermentation have a good control efficacy to the hot pepper root rot in seedings after inoculated with pathogen. Using strain D221 cell-free filtrate 48h, 24h and 0h defore inoculation, the biocontrol efficacy were 96.8%,88.4% and 77.4% respectively investigated 7d after inoculation. The biocontrol efficacy was 80.3%,56.6% and 37.5% respectively 14d after inoculation. The result showed that strain D221 was a potential biocontrol agent in hot pepper root rot control.
引文
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