In order to clarify the effect of lipopeptide secreted by Bacillus subtilis WL2 on Phytophthora infestans and the prevention potential on potato late blight, the paper strip method was adopted to evaluate the inhibitory activity of lipopeptide crude extract on mycelial growth and spores germination of P. infestans. In addition, in vitro tissue method was used to evaluate the disease prevention effect of lipopeptide crude extract. And the types of lipopeptide were also identified by ESI-MS.The results showed that when the concentration of lipopeptide crude extract was at 25 μg/mL, the diameter of inhibitory zone was 5.9 mm and mycelial recovery growth rate of P. infestans after inhibition was only 5.2 mm/d. There was- DOI:10.3969/j.issn.1000-1565.2018.06.012枯草芽孢杆菌WL2脂肽粗提物对致病疫霉的抑制作用及其分离鉴定吴艳清1,王游游2,王畅1,沙梦莹1(1.保定学院 生物化工与环境工程学院,河北 保定 071000;2.河北大学 生命科学学院,河北 保定 071002)摘 要:为明确枯草芽孢杆菌(Bacillus subtilis)WL2产生的脂肽对致病疫霉菌丝体生长的影响及其预防马铃薯晚疫病的潜力,以滤纸片法检测脂肽粗提物对菌丝生长的抑制效果,显微镜观察脂肽粗提物对致病疫霉孢子萌发的影响,离体组织培养法明确脂肽粗提物对晚疫病的预防效果,高效液相色谱法(HPLC)纯化脂肽、电子喷雾质谱(ESI-MS)鉴定脂肽种类.结果表明,当脂肽粗提物质量浓度为25 μg/mL时,抑菌圈直径为5.9 mm,抑制后的菌丝恢复生长速度仅有5.2 mm/d,与对照组(11.2 mm/d)存在显著差异(P<0.05);当脂肽质量浓度为3 μg/mL时,孢子囊萌发比率只有9.7%,用质量浓度为4 μg/mL的脂肽抑制后,释放游动孢子比率只有7.6%,与对照组均存在显著差异(P<0.05);脂肽粗提物(20 μg/mL)对晚疫病的预防效果最好,病情指数最低(20.6),与对照组病情指数(73.1)有显著差异(P<0.05);经HPLC分离和ESI-MS鉴定,明确了WL2菌株可产iturinA和surfactin两类脂肽物质.以上结果说明WL2菌株产生的脂肽在预防马铃薯晚疫病方面具有较大的潜力.关键词:枯草芽孢杆菌;脂肽;抑制;致病疫霉;鉴定中图分类号:S435.32 文献标志码:A 文章编号:1000-1565(2018)06-0632-08Inhibitory effect of lipopeptide crude extract produced by Bacillus subtilis WL2 on Phytophthora infestans and its isolation and identificationWU Yanqing1, WANG Youyou2, WANG Chang1, SHA Mengying1(1.College of Biochemical and Environmental Engineering, Baoding University, Baoding 071000, China; 2. College of Life Sciences, Hebei University, Baoding 071002, China)Abstract: In order to clarify the effect of lipopeptide secreted by Bacillus subtilis WL2 on Phytophthora infestans and the prevention potential on potato late blight, the paper strip method was adopted to evaluate the inhibitory activity of lipopeptide crude extract on mycelial growth and spores germination of P. infestans. In addition, in vitro tissue method was used to evaluate the disease prevention effect of lipopeptide crude extract. And the types of lipopeptide were also identified by ESI-MS.The results showed that when the concentration of lipopeptide crude extract was at 25 μg/mL, the diameter of inhibitory zone was 5.9 mm and mycelial recovery growth rate of P. infestans after inhibition was only 5.2 mm/d. There was- 收稿日期:2018-04-10 基金项目:河北省青年自然科学基金资助项目(C2015104014);保定学院博士基金资助项目(2013Z01) 第一作者:吴艳清(1982—),男,河北河间人,保定学院讲师,博士,主要从事微生物资源开发及应用研究.E-mail:wuyanqing198212@126.com 通信作者:王游游(1992—),男,河北石家庄人,河北大学硕士研究生,主要从事植物病害生物防治研究.E-mail:15032257263@163.com第6期吴艳清等:枯草芽孢杆菌WL2脂肽粗提物对致病疫霉的抑制作用及其分离鉴定significant difference in comparison with control(11.2 mm/d, P<0.05). In addition, spores direct germination rate was 9.7% after inhibition with lipopeptide crude extract(3 μg/mL)and zoospore release rate was only 7.6% after inhibition with of lipopeptide crude extract(4 μg/mL). There were differences in comparison with control(P<0.05). In addition, lipopeptide crude extract(20 μg/mL)exhibited the best effect on disease prevention of potato late blight, and the disease index(20.6)was the lowest. There were differences in comparison with control(73.1, P<0.05). The lipopeptide produced by WL2 strain were iturinA and surfactin. These results indicate that lipopeptide produced by WL2 strain has great potential in controlling potato late blight in future.
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