【作者】 刘爽；

【导师】 王媛媛； 彭旭文；

【作者基本信息】 沈阳农业大学 ， 生物与医药硕士（专业学位）， 2024， 硕士

【摘要】 植物寄生线虫病害的发生是导致我国农作物产量减少的原因之一。微生物菌剂在防治病虫害的过程中,代谢产物也有着广泛的活性。本文利用研究室前期筛选获得、具有自主知识产权的拜氏梭菌(Clostridium beijerinckii)Sneb518发酵液为研究对象。以南方根结线虫(Meloidogyne incognita)和大豆胞囊线虫(Heterodera glycines)为靶标,测定Sneb518发酵液对两种植物寄生线虫的生物活性,采用凝胶HW-40柱层析、萃取法和SCIEX X500 OTOF高分辨质谱等方法,对菌株Sneb518代谢产物中的活性物质进行初步分离、纯化和鉴定。本研究为拜氏梭菌Sneb518开展植物线虫病害的开发利用奠定理论和应用基础。研究结果如下:1.拜氏梭菌Sneb518杀线虫活性物质的初步分离。利用5种有机溶剂进行萃取,明确乙酸乙酯萃取水相物质毒杀供试线虫较高活性。通过凝胶HW-40柱层析法分离得到8个杀线虫活性物质,其中以组分1(Sample-1)、组分2(Sample-2)、组分4(Sample-3)杀线虫活性较高,处理M.incognita 24 h的校正死亡率分别达到了74.2%,62.2%,70.4%,处理H.glycines 24 h的校正死亡率分别达到了75.6%,66.5%,73.3%。2.拜氏梭菌Sneb518活性物质的鉴定。经SCIEX X500 OTOF高分辨质谱鉴定并通过Metabolite Pilot软件对比微生物天然产物数据库,在Sample-1中共鉴定到224个化合物,含特有化合物75个,4种代谢物母药,在Sample-2中共鉴定到239个化合物,含特有化合物97个,5种代谢物母药,在Sample-3中共鉴定到223个化合物,含特有化合物69个,4种代谢物母药。微生物代谢物相关包括脂多糖、聚酮、大环内酯类、萜烯、氨基酸、环肽、寡肽肽生物碱、苯和蒽环类药物。利用HPLC进一步对Sample-2进行验证分析,结果证明Sampl-2中含有匹马霉素。对匹马霉素处理M.incognita 24 h的校正死亡率可达到54.2%。3.拜氏梭菌Sneb518对番茄根结线虫病的防效测定。通过对拜氏梭菌Sneb518发酵液进行番茄灌根处理,结果表明:番茄经拜氏梭菌Sneb518灌根处理后具有良好的防效,处理30d防效达到30%,60d防效达到62.1%,可有效控制番茄根部根结的形成,且降低了根结指数和线虫的数量。同时,采用该菌株发酵液对番茄种子及黄瓜种子进行了萌发测验,当处理5d时,番茄种子及黄瓜种子几乎全都发芽,而对照组发芽数量并没有处理组的多。由此可见,Sneb518发酵液有催芽作用。更多还原

【Abstract】 Plant parasitic nematode diseases are one the of the reasons for China’s decreased crop yield.Mirobial agents also have a wide range of activities in the process of preventing and controlling diseases,and their metabolites.In the current research,Clostridium beijerinckii Sneb518 fermentation broth was employed as the research object,which had undergone preliminary screening in the laboratory.Using M.incognita and H.glycines as targets,determine the biological activity of Sneb518 fermentation broth against two plant parasitic nematodes.The active substances in the metabolites of the strain Sneb518 were preliminarily isolated,purified and identified by SCIEX X500 OTOF high-resolution mass spectrometry,gel HW-40 column chromatography,extraction,etc.,and the field control effect was determined by using the fermentation broth of Sneb518.The study lays the theoretical and practical foundation for the development and utilization of plant nematode diseases by C.beijerinckii Sneb518.The research results are as follows:1.Preliminary isolation of nematicidal active substances from C.beijerinckii Sneb518.The five organic solvents were use for extraction,namely petroleum ether,chloroform,ethyl acetate,n-butanol,and n-hexane.The optimal extraction solvent for nematicidal active substances from Clostridium beijerinckii Sneb518 was determined to be ethyl acetate.According to the adjusted mortality of nematodes,the water phase after ethyl acetate substances was separated by gel HW-40 column chromatography.Among them,component 1(Sample-1),component 2(Sample-2),and component 4(Sample-3)had higher nematicidal activity.The adjusted mortality of M.incognita after 24 h treatment reached 74.2%,62.2%,and 70.4%,respectively,and the corrected mortality of H.glycines after 24 h treatment reached 75.6%,65.5%,and 73.3%,respectively.2.Identification of the Sneb518 active substance from C.beijerinckii.The components in the three components were identified using the SCIEX X500 OTOF high-resolution mass spectrometry system.Several compounds were identified,such as 224 compounds in Sample-1,239 compounds in Sample-2,and 223 compounds in Sample-3.Microbial metabolite-related compounds include lipopolysaccharides,polyketones,macrolides,terpenes,amino acids,cyclic peptides,oligopepides,pepide alkaloids,benzene,and anthracyclines.Further validation analysis was conducted using HPLC on the standards of Sample-2 and pimamycin,and the results showed that Sample-2 contained pimamycin.After 24 hours of treatment with pimamycin,the corrected mortality rates of M.incognita reached 54.2%.3.Determination of the Control Effect of C.beijerinckii Sneb518 on Tomato Root knot Nematode Disease.Irrigated tomato root by C.beijerinckii Sneb518 fermentation broth during the seeding stage,the results showed that the prevention effect after 30 days of treatment was 30%,and the prevention effect after 60 days reached 62.1%,effectively control the formation of tomato root knot,and reduce the root knot index and the number of nematodes.The preliminary germination experiments of tomato and cucumber seeds were used in the study to determine the safety of Sneb518 fermentation broth,indicating that Sneb518 fermentation broth promotes the germination and growth of the two vegetable seeds.更多还原