番茄灰霉病菌生防细菌BAB-1的鉴定及发酵工艺的优化
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摘要
菌株BAB-1是从根际土壤中分离筛选到的一株对番茄灰霉病病原菌具有较强抑制作用的生防细菌,田间对番茄灰霉病(Tomato grey mold)表现出理想的防治效果。
本研究首先测定了菌株BAB-1无菌体培养液对番茄灰霉菌的抑菌活性和对番茄灰霉病的防治效果。双层平板试验中在盛有菌株BAB-1无菌体培养液的牛津杯周围出现大而透明的抑菌圈,说明该菌株可产生对番茄灰霉菌有抑制作用的物质,离体叶片的测定结果表明该菌株的无菌体培养液对番茄灰霉病的防效达到70.92%。
通过对该菌株进行形态特征观察、16S rDNA序列分析和API 50CHB细菌鉴定试剂盒鉴定,确定菌株BAB-1为枯草芽孢杆菌(Bacillus subtilis)。
采用16种培养基对菌株BAB-1进行了发酵培养,结果表明,3号培养基最适合其菌体和抑菌物质的产生,并且明确活菌数达到最高时最佳培养时间为28h,产生最多抑菌物质的最适培养时间为48h。进一步对3号培养基的培养条件进行了优化,发现在培养温度30℃,转速210rpm,初始pH为7.24,接种量3﹪,装样量为70mL/250 mL时菌体产量最高,活菌数达1.63×109cfu/mL;而在培养温度30℃,转速210rpm,初始pH为7.24,接种量2﹪,装样量为100mL/250mL时最适合抑菌物质的产生,对番茄灰霉病菌的抑菌圈直径达1.81 cm。
本文进一步对3号培养基的成分进行了优化,通过单因子试验及正交试验得到各成分的最佳添加量为淀粉4%,葡萄糖2.5%,蛋白胨0.05%,NaNO3 0.3%,CaCO3 0.12%,MgSO40.15%,K2HPO4 0.25%,MnSO4 0.035%。优化后的发酵培养基对菌株BAB-1活菌数影响不明显,抑菌圈直径比原发酵培养基提高了19%。
通过进一步比较菌株BAB-1在8号、3号、和优3号培养基培养后的活菌数、抑菌活性、防效以及脂肽类抑菌物质的产生,验证了优3号培养基是菌株BAB-1的最适合培养基。
Antigonistic bacterial strain BAB-1 was isolated from cotton rhizosphere and showed strong inhibition against Botrytis cinerea Pers. This strain also displayed significant control efficacy against tomato grey mold under the field condition.
In this study, the inhibitory activity against B.cinerea and biocontrol ability to tomato grey mold by strain BAB-1 was tested by using the cell free supernatant. In the double-layered plate test, large and clear inhibition zone was appeared round the oxford cup which contain the supernatant of strain BAB-1. The result indicated that strain BAB-1 could produce antifungal substance. The control effect of strain BAB-1 against tomato grey mold was 70.92% by using the detached leaf method.
Strain BAB-1 was identified as Bacillus subtilis according to its morphological characteristics, 16S rDNA sequence and the testing result of API 50CHB system.
16 different media for the bacterial growth and the antifungal substance producing was selected and tested. Results showed that the medium No. 3 was suitable for either bacterial growth or the antifungal substance production. The cell of strain BAB-1 reached the highest after 28 hours culturing, meanwhile the supernatant showed the strongest antifungal activity against B. cinerea after 48 hours culturing. Optimum fermentation condition for the cell growth of strain BAB-1 were as followed: temperature 30℃, 210 rpm, pH 7.24, inoculation amount 3% and amount of loading 70mL in 250mL flask, the cell density could reach 1.63×109cfu/mL. The optimum conditions for the antifungal substance production were temperature 30℃, 210 rpm, pH7.24, inoculation amount 2% and amount of loading 100mL in 250mL flask. Under the optimum conditions, the inhibition zone of strain BAB-1 against B. cinerea could reach 1.81cm width.
The medium components was further optimized by using single factor and the orthogonal testings. The optimum medium was as followings: starch 4%, glucose 2.5%, peptone 0.05%, NaNO3 0.3%, CaCO3 0.12%, MgSO4 0.15%, K2HPO4 0.25%, MnSO4 0.035%.
Finally, the cell production, antifungal activity and the lipopeptide production of strain BAB-1 was compared by using three kinds of media, Medium No.8, Medium No.3 and Medium modified 3. The result proved that the optimum medium of strain BAB-1 was Medium modified 3.
本研究首先测定了菌株BAB-1无菌体培养液对番茄灰霉菌的抑菌活性和对番茄灰霉病的防治效果。双层平板试验中在盛有菌株BAB-1无菌体培养液的牛津杯周围出现大而透明的抑菌圈,说明该菌株可产生对番茄灰霉菌有抑制作用的物质,离体叶片的测定结果表明该菌株的无菌体培养液对番茄灰霉病的防效达到70.92%。
通过对该菌株进行形态特征观察、16S rDNA序列分析和API 50CHB细菌鉴定试剂盒鉴定,确定菌株BAB-1为枯草芽孢杆菌(Bacillus subtilis)。
采用16种培养基对菌株BAB-1进行了发酵培养,结果表明,3号培养基最适合其菌体和抑菌物质的产生,并且明确活菌数达到最高时最佳培养时间为28h,产生最多抑菌物质的最适培养时间为48h。进一步对3号培养基的培养条件进行了优化,发现在培养温度30℃,转速210rpm,初始pH为7.24,接种量3﹪,装样量为70mL/250 mL时菌体产量最高,活菌数达1.63×109cfu/mL;而在培养温度30℃,转速210rpm,初始pH为7.24,接种量2﹪,装样量为100mL/250mL时最适合抑菌物质的产生,对番茄灰霉病菌的抑菌圈直径达1.81 cm。
本文进一步对3号培养基的成分进行了优化,通过单因子试验及正交试验得到各成分的最佳添加量为淀粉4%,葡萄糖2.5%,蛋白胨0.05%,NaNO3 0.3%,CaCO3 0.12%,MgSO40.15%,K2HPO4 0.25%,MnSO4 0.035%。优化后的发酵培养基对菌株BAB-1活菌数影响不明显,抑菌圈直径比原发酵培养基提高了19%。
通过进一步比较菌株BAB-1在8号、3号、和优3号培养基培养后的活菌数、抑菌活性、防效以及脂肽类抑菌物质的产生,验证了优3号培养基是菌株BAB-1的最适合培养基。
Antigonistic bacterial strain BAB-1 was isolated from cotton rhizosphere and showed strong inhibition against Botrytis cinerea Pers. This strain also displayed significant control efficacy against tomato grey mold under the field condition.
In this study, the inhibitory activity against B.cinerea and biocontrol ability to tomato grey mold by strain BAB-1 was tested by using the cell free supernatant. In the double-layered plate test, large and clear inhibition zone was appeared round the oxford cup which contain the supernatant of strain BAB-1. The result indicated that strain BAB-1 could produce antifungal substance. The control effect of strain BAB-1 against tomato grey mold was 70.92% by using the detached leaf method.
Strain BAB-1 was identified as Bacillus subtilis according to its morphological characteristics, 16S rDNA sequence and the testing result of API 50CHB system.
16 different media for the bacterial growth and the antifungal substance producing was selected and tested. Results showed that the medium No. 3 was suitable for either bacterial growth or the antifungal substance production. The cell of strain BAB-1 reached the highest after 28 hours culturing, meanwhile the supernatant showed the strongest antifungal activity against B. cinerea after 48 hours culturing. Optimum fermentation condition for the cell growth of strain BAB-1 were as followed: temperature 30℃, 210 rpm, pH 7.24, inoculation amount 3% and amount of loading 70mL in 250mL flask, the cell density could reach 1.63×109cfu/mL. The optimum conditions for the antifungal substance production were temperature 30℃, 210 rpm, pH7.24, inoculation amount 2% and amount of loading 100mL in 250mL flask. Under the optimum conditions, the inhibition zone of strain BAB-1 against B. cinerea could reach 1.81cm width.
The medium components was further optimized by using single factor and the orthogonal testings. The optimum medium was as followings: starch 4%, glucose 2.5%, peptone 0.05%, NaNO3 0.3%, CaCO3 0.12%, MgSO4 0.15%, K2HPO4 0.25%, MnSO4 0.035%.
Finally, the cell production, antifungal activity and the lipopeptide production of strain BAB-1 was compared by using three kinds of media, Medium No.8, Medium No.3 and Medium modified 3. The result proved that the optimum medium of strain BAB-1 was Medium modified 3.
引文
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