昆虫病原真菌及蛋白激发子PeBb1对桃蚜的抗性作用
发布时间：2020-10-23 22:46
绿桃蚜(Myzus persicae)是一种在全球范围对农业造成重大经济损失的害虫。昆虫病原真菌如白僵菌和蜡蚧轮枝菌是环境友好、靶标特异、能够有效防治多种刺吸式昆虫的生物农药。本文测定了两株白僵菌(BB-72和BB-252)和一株蜡蚧轮枝菌(V-4)对绿桃蚜的抗性作用。采用叶浸法对BB-72、BB-252和V-4的滤液、孢子悬浮液及孢子悬浮液两两组合进行生物测定。对滤液进行生物测定时每株真菌用2 mL滤液,对孢子悬浮液生物测定时每株真菌使用三种不同浓度的孢子悬浮液(1×10~6、1×10~7和1×10~8 CFU·mL~(-1)),对孢子悬浮液两两组合及三种真菌孢子混合液进行生物测定采用LC_(50)和LC_(33)。结果显示,所有处理组在第10天对蚜虫的拮抗作用最强。两株白僵菌(BB-72和BB-252)菌株(死亡率分别为95%和91%)均显示出比绿僵菌(V-4)菌株(死亡率87%)具有更好的蚜虫致死效果。另外,菌株BB-72和BB-252的孢子悬浮液组合比其它两两组合和三种真菌孢子组合具有更高的蚜虫致死率(94%),表明这两株白僵菌菌株在抗蚜虫作用上是相互兼容促进的,可以作为新的生物防治的资源。随后,从白僵菌BB-72(ARSEF 2860)菌株中提取一种新型的蛋白激发子PeBb1,该激发子可以诱导大白菜系统抗性以拮抗绿桃蚜。经鉴定该蛋白激发子基因长度为534 bp,编码177个氨基酸,分子量约为19 kDa,并可诱导烟草叶片产生超敏反应(HR)。用三种不同浓度的PeBb1蛋白(26、35、52μg·mL~(-1))对大白菜上的绿桃蚜进行体外生物测定,在大白菜4叶期进行蛋白激发子喷施处理,并将蚜虫成虫置于处理过的白菜叶子表面。结果显示,激发子PeBb1对桃蚜具有显著(p0.05)的半致死效果。另外,与生长在对照植物上的蚜虫相比,取食蛋白激发子PeBb1处理后白菜的蚜虫,其繁殖力显著降低。采用RT-qPCR分析植物防御相关基因的表达,结果显示,PeBb1处理组乙烯(ET)和茉莉酸(JA)通路相关基因表达上调。本研究不仅证明了新型昆虫病原真菌对绿桃蚜的抗性作用,同时分离鉴定了能诱导白菜抗绿桃蚜的新蛋白激发子PeBb1。
【学位单位】：中国农业科学院
【学位级别】：博士
【学位年份】：2019
【中图分类】：S476.12
【文章目录】：
摘要
ABSTRACT
CHAPTER Ⅰ INTRODUCTION AND REVIEW OF LITERATURE
1.1 Microbial Biocontrol Agents
1.1.1 Entomopathogenic Viruses
1.1.2 Entomopathogenic Bacteria
1.1.3 Entomopathogenic Nematodes
1.1.4 Entomopathogenic Fungi
1.1.5 The Fungal Breaching Process
1.1.6 Host Defense Systems
1.1.7 Advantages of Using Fungi as Insecticides
1.1.8 Disadvantages of the Fungi as Biocontrol Agents
1.2 Plant Defense Responses
1.3 Elicitors Induce Resistance
1.4 Chinese Cabbage Defense Response
1.5 Abiotic and Biotic Stresses Related Pathways
1.5.1 Jasmonic Acid Signaling Pathway
1.5.2 Ethylene Signaling Pathway
1.6 Objectives
CHAPTER Ⅱ In vitro Pathogenicity of Some Entomopathogenic Fungal Strains against Green Peach Aphid Myzus persicae（Homoptera:Aphididae）
2.1 Introduction
2.2 Materials and Methods
2.2.1 Insect Culture
2.2.2 Fungal Isolates and Conidial Suspensions Preparation
2.2.3 Pathogenicity Bioassays
2.2.4 Data Analysis
2.3 Results
2.3.1 Pathogenicity of Filtrates by Different Strains of B.bassiana and L.lecanii against M.persicae
2.3.2 Virulence of Conidial Bioassay of Three Fungal Strains against M.persicae
2.3.3 Entomopathogenicity of Binary Combinations of Entomopathogenic Fungal Strains against M.persicae
2.4 Discussion
2.5 Summary
CHAPTER Ⅲ PeBb1,a Novel Protein Elicitor from Beauveria bassiana Strain ARSEF2860,Induces Systemic Resistance in Brassica rapa subsp.pekinensis against Myzus persicae（Homoptera:Aphididae）
3.1 Introduction
3.2 Materials and Methods
3.2.1 Rearing of Myzus persicae
3.2.2 Plant,Pathogen and Bacteria Culture
3.2.3 Isolation and Detection of Crude Protein
3.2.4 Purification of Protein
3.2.5 Mass Spectrometry Analysis and Gene Cloning
3.2.6 Expression and Purification of Recombinant Protein
3.2.7 Characterization of the PeBb1 Protein Elicitor
3.2.8 Bioassay of Elicitor Activity against M.persicae
3.2.9 Analysis of the Expression of Defense-related Genes Induced by PeBb1 using RT-qPCR
3.2.10 Protein Assay
3.2.11 Statistical Analysis
3.3 Results
3.3.1 Purification,Characterization and Identification of PeBb
3.3.2 PeBb1 Induced Hypersensitive Response HR in Tobacco Leaves
3.3.3 Effect of Elicitor PeBb1 on the Fecundity of M.persicae
3.3.4 Expression Level of Plant Defense-Associated Genes in Response to PeBb1 elicitor
3.4 Discussion
3.5 Summary
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
RESUME
本文编号：2853649
【学位单位】：中国农业科学院
【学位级别】：博士
【学位年份】：2019
【中图分类】：S476.12
【文章目录】：
摘要
ABSTRACT
CHAPTER Ⅰ INTRODUCTION AND REVIEW OF LITERATURE
1.1 Microbial Biocontrol Agents
1.1.1 Entomopathogenic Viruses
1.1.2 Entomopathogenic Bacteria
1.1.3 Entomopathogenic Nematodes
1.1.4 Entomopathogenic Fungi
1.1.5 The Fungal Breaching Process
1.1.6 Host Defense Systems
1.1.7 Advantages of Using Fungi as Insecticides
1.1.8 Disadvantages of the Fungi as Biocontrol Agents
1.2 Plant Defense Responses
1.3 Elicitors Induce Resistance
1.4 Chinese Cabbage Defense Response
1.5 Abiotic and Biotic Stresses Related Pathways
1.5.1 Jasmonic Acid Signaling Pathway
1.5.2 Ethylene Signaling Pathway
1.6 Objectives
CHAPTER Ⅱ In vitro Pathogenicity of Some Entomopathogenic Fungal Strains against Green Peach Aphid Myzus persicae（Homoptera:Aphididae）
2.1 Introduction
2.2 Materials and Methods
2.2.1 Insect Culture
2.2.2 Fungal Isolates and Conidial Suspensions Preparation
2.2.3 Pathogenicity Bioassays
2.2.4 Data Analysis
2.3 Results
2.3.1 Pathogenicity of Filtrates by Different Strains of B.bassiana and L.lecanii against M.persicae
2.3.2 Virulence of Conidial Bioassay of Three Fungal Strains against M.persicae
2.3.3 Entomopathogenicity of Binary Combinations of Entomopathogenic Fungal Strains against M.persicae
2.4 Discussion
2.5 Summary
CHAPTER Ⅲ PeBb1,a Novel Protein Elicitor from Beauveria bassiana Strain ARSEF2860,Induces Systemic Resistance in Brassica rapa subsp.pekinensis against Myzus persicae（Homoptera:Aphididae）
3.1 Introduction
3.2 Materials and Methods
3.2.1 Rearing of Myzus persicae
3.2.2 Plant,Pathogen and Bacteria Culture
3.2.3 Isolation and Detection of Crude Protein
3.2.4 Purification of Protein
3.2.5 Mass Spectrometry Analysis and Gene Cloning
3.2.6 Expression and Purification of Recombinant Protein
3.2.7 Characterization of the PeBb1 Protein Elicitor
3.2.8 Bioassay of Elicitor Activity against M.persicae
3.2.9 Analysis of the Expression of Defense-related Genes Induced by PeBb1 using RT-qPCR
3.2.10 Protein Assay
3.2.11 Statistical Analysis
3.3 Results
3.3.1 Purification,Characterization and Identification of PeBb
3.3.2 PeBb1 Induced Hypersensitive Response HR in Tobacco Leaves
3.3.3 Effect of Elicitor PeBb1 on the Fecundity of M.persicae
3.3.4 Expression Level of Plant Defense-Associated Genes in Response to PeBb1 elicitor
3.4 Discussion
3.5 Summary
CONCLUSION
REFERENCES
ACKNOWLEDGEMENTS
RESUME
本文编号：2853649
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