綠桃蚜(Myzus persicae)是一種在全球范圍對(duì)農(nóng)業(yè)造成重大經(jīng)濟(jì)損失的害蟲。昆蟲病原真菌如白僵菌和蠟蚧輪枝菌是環(huán)境友好、靶標(biāo)特異、能夠有效防治多種刺吸式昆蟲的生物農(nóng)藥。本文測(cè)定了兩株白僵菌(BB-72和BB-252)和一株蠟蚧輪枝菌(V-4)對(duì)綠桃蚜的抗性作用。采用葉浸法對(duì)BB-72、BB-252和V-4的濾液、孢子懸浮液及孢子懸浮液兩兩組合進(jìn)行生物測(cè)定。對(duì)濾液進(jìn)行生物測(cè)定時(shí)每株真菌用2 mL濾液,對(duì)孢子懸浮液生物測(cè)定時(shí)每株真菌使用三種不同濃度的孢子懸浮液(1×10~6、1×10~7和1×10~8 CFU·mL~(-1)),對(duì)孢子懸浮液兩兩組合及三種真菌孢子混合液進(jìn)行生物測(cè)定采用LC_(50)和LC_(33)。結(jié)果顯示,所有處理組在第10天對(duì)蚜蟲的拮抗作用最強(qiáng)。兩株白僵菌(BB-72和BB-252)菌株(死亡率分別為95%和91%)均顯示出比綠僵菌(V-4)菌株(死亡率87%)具有更好的蚜蟲致死效果。另外,菌株BB-72和BB-252的孢子懸浮液組合比其它兩兩組合和三種真菌孢子組合具有更高的蚜蟲致死率(94%),表明這兩株白僵菌菌株在抗蚜蟲作用上是相互兼容促進(jìn)的,可以作為新的生物防治的資源。隨后,從白僵菌BB-72(ARSEF 2860)菌株中提取一種新型的蛋白激發(fā)子PeBb1,該激發(fā)子可以誘導(dǎo)大白菜系統(tǒng)抗性以拮抗綠桃蚜。經(jīng)鑒定該蛋白激發(fā)子基因長(zhǎng)度為534 bp,編碼177個(gè)氨基酸,分子量約為19 kDa,并可誘導(dǎo)煙草葉片產(chǎn)生超敏反應(yīng)(HR)。用三種不同濃度的PeBb1蛋白(26、35、52μg·mL~(-1))對(duì)大白菜上的綠桃蚜進(jìn)行體外生物測(cè)定,在大白菜4葉期進(jìn)行蛋白激發(fā)子噴施處理,并將蚜蟲成蟲置于處理過(guò)的白菜葉子表面。結(jié)果顯示,激發(fā)子PeBb1對(duì)桃蚜具有顯著(p0.05)的半致死效果。另外,與生長(zhǎng)在對(duì)照植物上的蚜蟲相比,取食蛋白激發(fā)子PeBb1處理后白菜的蚜蟲,其繁殖力顯著降低。采用RT-qPCR分析植物防御相關(guān)基因的表達(dá),結(jié)果顯示,PeBb1處理組乙烯(ET)和茉莉酸(JA)通路相關(guān)基因表達(dá)上調(diào)。本研究不僅證明了新型昆蟲病原真菌對(duì)綠桃蚜的抗性作用,同時(shí)分離鑒定了能誘導(dǎo)白菜抗綠桃蚜的新蛋白激發(fā)子PeBb1。
【學(xué)位單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2019
【中圖分類】：S476.12
【文章目錄】：
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


本文編號(hào)：2853649