金龟子绿僵菌对根结线虫病和灰霉病的生物防治及其机理研究
发布时间：2024-03-04 00:52
金龟子绿僵菌是一种分布广泛的土壤习居真菌,可以感染大约200种昆虫和某些节肢动物,但对哺乳动物没有侵染性。因此,绿僵菌已经被开发为一种重要的植物有害昆虫防治资源。本论文利用番茄与植物病原物互作体系,探究了绿僵菌对根结线虫(Meloidogyne incognita)引起的根结线虫病和灰葡萄孢(Botrytis cinerea)引起的灰霉病的生物防治潜力。此外,对绿僵菌的生防机制进行了研究。主要研究结果如下:1. 金龟子绿僵菌可以寄生根结线虫。南方根结线虫是重要的世界性分布土传植物病原线虫,可以侵染许多蔬菜和大田作物。但是对环境友好的根结线虫防治方法有限。本论文探究了金龟子绿僵菌菌株IBCCM321.93对南方根结线虫引起的线虫病害防控潜力。通过扫描电镜和透射电镜观察,及利用绿色荧光标记的绿僵菌侵染线虫的卵,都表明绿僵菌可以寄生线虫的卵。此外,显微镜观察表明绿僵菌还可以寄生线虫的成熟雌虫。与水对照相比,1×108/ml绿僵菌孢子在处理线虫卵8天后,对卵孵化的抑制率可达75%。2. 金龟子绿僵菌的代谢产物具有杀根结线虫活性。绿僵菌的发酵滤液可以显著降低卵的孵化率,并对...
【文章页数】：192 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
Chapter 1.Literature Review
1.Root-knot nematode
1.1 Economic important
1.2 Damage symptoms
1.3 Biological characteristics of root-knot nematodes
1.4 Management strategies
1.4.1 Cultural control
1.4.2 Chemical control
2.Nematode biocontrol resources
2.1 Nematophagous bacteria
2.2 Nematophagous fungi
2.3 Molecular mechanism of nematophagous fungi infection of nematodes
3.Botrytis cinerea
3.1 Introduction
3.2 Nomenclature and Phylogeny
3.3 Ecology and physiology
3.4 Biological characteristics of B.cinerea
3.5 Desease development mechanisms
3.6 Disease cycle and epidemiology
3.7 Economic importance of B.cinerea
3.8 Current strategies to control B.cinerea
3.8.1 Cultural control
3.8.2 Chemical control
3.8.3 Biological control
3.9 Problems associated in controlling B.cinerea
4.Metarhizium anisopliae
4.1 Taxonomy and biology
4.2 Biocontrol mechanism of M.anisopliae
4.3 Researches on biocontrol agents of M.anisopiae
5.Progress in transcriptome research
6.Plant growth promotion with endophytic as well as biocontrol fungi
7.Significance and objectives of the study
Chapter 2.The entomopathogenic fungus Metarhizium anisopliae as a biocontrol agent of Meloidogyne incognita and plant growth promoter
1.Materials and Methods
1.1 Preparation of nematode inocula
1.2 Fungus culture
1.3 Pathogenicity test on eggs and adult females in vitro
1.4 Observation of M.anisopliae eggs parasitized by GFP labeled M.anisopliae
1.5 Evaluation of conidia impact on egg hatching
1.6 Preparation of eggs for scanning electron microscopy(SEM)
1.7 Preparation of eggs for transmission electron microscopy(TEM)
1.8 Assessment of M.anisopliae control efficacy on tomato root knot nematode in pot experiment
1.9 The application of M.anisopliae in the greenhouse unsterilized soil
1.10 The density-dependent effect of initial nematode inoculum levels on the efficacy of M.anisopliae against M.incognita on tomato
1.11 Impacts of M.anisopliae on plant growth
1.11.1 Recovery of M.anisopliae from plant roots
1.11.2 Molecular detection of M.anisopliae DNA within tomatoes root by PCR
1.11.3 Inoculation of tomato seedlings with M.anisopliae and sample preparation for confocal microscopy
1.12 Statistical analysis
2.Results
2.1 In vitro pathogenicity of M.anisopliae conidia against eggs and adult females of M.incognita
2.2 Eggs parasitism with GFP labelled M.anisopliae
2.3 Observation of M.incognita eggs parasitism with M.anisopliae by Transmission electron microscopy(TEM)
2.4 Biocontrol efficacy of M.anisopliae against tomato root knot nematode in pot experiment
2.5 Biocontrol efficacy of M.anisopliae on tomato root knot nematode in naturally M.incognita infested greenhouse soil
2.6 The density-dependent effect of initial nematode inoculum levels on the efficacy of M.anisopliae against M.incognita on tomato
2.6.1 Main effect of M.incognita inoculum levels on plant growth parameters and nematode's population
2.6.2 Effect of initial nematode inoculum levels on biocontrol efficacy of M.anisopliae against tomato root knot nematode
2.7 Impacts of M.anisopliae on plant growth
2.8 Evaluation of endophytic root colonization of tomato plants by M.anisopliae
3.Discussion
Chapter 3.Evaluation of in vitro and in vivo nematicidal potentiality of culture filtrate of Metarhizium anisopliae against Meloidogyne incognita
1.Methods and Materials
1.1 Fungus culture and preparation of culture filtrates
1.2 Nematode inocula
1.3 Effect of culture filtrate on egg hatch
1.4 Effect of culture filtrate on J2 mortality and heat stability of culture filtrate
1.5 Greenhouse pot experiment
1.6 Statistical analysis
2.Results
2.1 Effects of culture filtrate of M.anisopliae on eggs hatch of M.incognita
2.2 Effects of culture filtrate of M.anisopliae against M.incognita
2.3 Effect of heat treatment on toxic activity of M.anisopliae culture filtrate on J2 mortality
2.4 Evaluation the biocontrol efficiency of M.anisopliae culture filtrate on tomato root knot in pot study
3.Discussion
Chapter 4.Transcriptome Analysis of the Entomopathogenic Fungus M.anisopliae during Early egg infection of M.incognita
1.Materials and Method
1.1 Sample preparation for RNA extraction
1.2 RNA extraction,library preparation and sequencing
1.3 Data analysis
1.3.1 Quality control
1.3.2 Assembly and annotation of transcriptomes
1.3.3 Quantification of gene expression level
1.3.4 Differential expression analysis
1.3.5 GO,KEGG and GSEA enrichment analysis of DEGs
1.3.6 Phylogenetic analysis
1.3.7 qRT-PCR analysis
2.Results
2.1 Sequence assembly and general features of RNA-seq data
2.2 Verification of gene expression levels of transcriptomic data by qRT-PCR
2.3 GO enrichment analysis of DEGs
2.4 KEGG pathway analysis of DEGs
2.5 Gene set enrichment analysis(GSEA)
2.6 Gene expression profiles of M.anisopliae during early infection of M.incognita eggs
2.6.1 Peptides genes
2.6.2 Chitin metabolism related genes
2.6.3 Appressoria formation related genes
2.6.4 Signal transduction genes
2.6.5 Transcription factor genes
2.6.6 Transporter and cytochrome genes
2.6.7 Secondary metabolites synthesis genes
2.7 Functional analysis of some others up regulated single gene during infection
3.Discussion
Chapter 5.Biological Control of Tomato Gray Mold Caused by Botrytis cinerea with the Entomopathogenic Fungus Metarhizium anisopliae
1.Materials and methods
1.1 Fungal isolate and cultural condition
1.2 Assay inhibitory effect of M.anisopliae
1.3 Preparation of the Culture Filtrate of M.anisopliae
1.4 Effect of M.anisopliae culture filtrate against the growth of B.cinerea
1.5 Effect of culture filtrate of M.anisopliae against the germination of conidia and sclerotia of B.cinerea
1.6 Effect of culture filtrate of M.anisopliae on the plasma membrane integrity of B.cinerea conidia
1.7 Heat stability of culture filtrate activity of M.anisopliae
1.8 Sample preparation for Transmission Electron Microscopy
1.9 Effect of culture filtrate on gray mold on detached tomato leaves
1.10 Effect of culture filtrate of M.anisopliae on the gray mold of ripe tomato fruits
1.11 Effect of culture filtrate of M.anisopliae against tomato plant gray mold
1.12 Effect of volatile organic molecules(VOCs)of M.anisopliae on B.cinerea
1.13 Effect of M.anisopliae VOCs on Botrytis gray mold of postharvest tomatoes
1.14 Data analysis
2.Results
2.1 Assessment inhibitory effect of M.anisopliae to B.cinerea
2.2 M.anisopliae culture filtrate influenced the growth and morphology of B.cinerea
2.3 The culture filtrate of M.anisopliae affected the sclerotia germination of B.cinerea
2.4 The culture filtrate of M.anisopliae inhibited conidia germination of B.cinerea
2.5 The antifungal substance produced by M.anisopliae retains its activity at high temperatures
2.6 The culture filtrate of M.anisopliae reduced the gray mold severity of detached leaves,fruits and whole plants of the tomato
2.7 The Volatile organic compounds of M.anisopliae reduced the gray mold severity of postharvest tomatoes
3.Discussion
Chapter 6.Conclusion and Future Prospects
References
Supplementary information
Appendix
1.Reagents
2.DNA extraction
3.Total RNA extraction
4.cDNA synthesis
5.qRT-PCR
Acknowledgements
本文编号：3918628
【文章页数】：192 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of Abbreviations
Chapter 1.Literature Review
1.Root-knot nematode
1.1 Economic important
1.2 Damage symptoms
1.3 Biological characteristics of root-knot nematodes
1.4 Management strategies
1.4.1 Cultural control
1.4.2 Chemical control
2.Nematode biocontrol resources
2.1 Nematophagous bacteria
2.2 Nematophagous fungi
2.3 Molecular mechanism of nematophagous fungi infection of nematodes
3.Botrytis cinerea
3.1 Introduction
3.2 Nomenclature and Phylogeny
3.3 Ecology and physiology
3.4 Biological characteristics of B.cinerea
3.5 Desease development mechanisms
3.6 Disease cycle and epidemiology
3.7 Economic importance of B.cinerea
3.8 Current strategies to control B.cinerea
3.8.1 Cultural control
3.8.2 Chemical control
3.8.3 Biological control
3.9 Problems associated in controlling B.cinerea
4.Metarhizium anisopliae
4.1 Taxonomy and biology
4.2 Biocontrol mechanism of M.anisopliae
4.3 Researches on biocontrol agents of M.anisopiae
5.Progress in transcriptome research
6.Plant growth promotion with endophytic as well as biocontrol fungi
7.Significance and objectives of the study
Chapter 2.The entomopathogenic fungus Metarhizium anisopliae as a biocontrol agent of Meloidogyne incognita and plant growth promoter
1.Materials and Methods
1.1 Preparation of nematode inocula
1.2 Fungus culture
1.3 Pathogenicity test on eggs and adult females in vitro
1.4 Observation of M.anisopliae eggs parasitized by GFP labeled M.anisopliae
1.5 Evaluation of conidia impact on egg hatching
1.6 Preparation of eggs for scanning electron microscopy(SEM)
1.7 Preparation of eggs for transmission electron microscopy(TEM)
1.8 Assessment of M.anisopliae control efficacy on tomato root knot nematode in pot experiment
1.9 The application of M.anisopliae in the greenhouse unsterilized soil
1.10 The density-dependent effect of initial nematode inoculum levels on the efficacy of M.anisopliae against M.incognita on tomato
1.11 Impacts of M.anisopliae on plant growth
1.11.1 Recovery of M.anisopliae from plant roots
1.11.2 Molecular detection of M.anisopliae DNA within tomatoes root by PCR
1.11.3 Inoculation of tomato seedlings with M.anisopliae and sample preparation for confocal microscopy
1.12 Statistical analysis
2.Results
2.1 In vitro pathogenicity of M.anisopliae conidia against eggs and adult females of M.incognita
2.2 Eggs parasitism with GFP labelled M.anisopliae
2.3 Observation of M.incognita eggs parasitism with M.anisopliae by Transmission electron microscopy(TEM)
2.4 Biocontrol efficacy of M.anisopliae against tomato root knot nematode in pot experiment
2.5 Biocontrol efficacy of M.anisopliae on tomato root knot nematode in naturally M.incognita infested greenhouse soil
2.6 The density-dependent effect of initial nematode inoculum levels on the efficacy of M.anisopliae against M.incognita on tomato
2.6.1 Main effect of M.incognita inoculum levels on plant growth parameters and nematode's population
2.6.2 Effect of initial nematode inoculum levels on biocontrol efficacy of M.anisopliae against tomato root knot nematode
2.7 Impacts of M.anisopliae on plant growth
2.8 Evaluation of endophytic root colonization of tomato plants by M.anisopliae
3.Discussion
Chapter 3.Evaluation of in vitro and in vivo nematicidal potentiality of culture filtrate of Metarhizium anisopliae against Meloidogyne incognita
1.Methods and Materials
1.1 Fungus culture and preparation of culture filtrates
1.2 Nematode inocula
1.3 Effect of culture filtrate on egg hatch
1.4 Effect of culture filtrate on J2 mortality and heat stability of culture filtrate
1.5 Greenhouse pot experiment
1.6 Statistical analysis
2.Results
2.1 Effects of culture filtrate of M.anisopliae on eggs hatch of M.incognita
2.2 Effects of culture filtrate of M.anisopliae against M.incognita
2.3 Effect of heat treatment on toxic activity of M.anisopliae culture filtrate on J2 mortality
2.4 Evaluation the biocontrol efficiency of M.anisopliae culture filtrate on tomato root knot in pot study
3.Discussion
Chapter 4.Transcriptome Analysis of the Entomopathogenic Fungus M.anisopliae during Early egg infection of M.incognita
1.Materials and Method
1.1 Sample preparation for RNA extraction
1.2 RNA extraction,library preparation and sequencing
1.3 Data analysis
1.3.1 Quality control
1.3.2 Assembly and annotation of transcriptomes
1.3.3 Quantification of gene expression level
1.3.4 Differential expression analysis
1.3.5 GO,KEGG and GSEA enrichment analysis of DEGs
1.3.6 Phylogenetic analysis
1.3.7 qRT-PCR analysis
2.Results
2.1 Sequence assembly and general features of RNA-seq data
2.2 Verification of gene expression levels of transcriptomic data by qRT-PCR
2.3 GO enrichment analysis of DEGs
2.4 KEGG pathway analysis of DEGs
2.5 Gene set enrichment analysis(GSEA)
2.6 Gene expression profiles of M.anisopliae during early infection of M.incognita eggs
2.6.1 Peptides genes
2.6.2 Chitin metabolism related genes
2.6.3 Appressoria formation related genes
2.6.4 Signal transduction genes
2.6.5 Transcription factor genes
2.6.6 Transporter and cytochrome genes
2.6.7 Secondary metabolites synthesis genes
2.7 Functional analysis of some others up regulated single gene during infection
3.Discussion
Chapter 5.Biological Control of Tomato Gray Mold Caused by Botrytis cinerea with the Entomopathogenic Fungus Metarhizium anisopliae
1.Materials and methods
1.1 Fungal isolate and cultural condition
1.2 Assay inhibitory effect of M.anisopliae
1.3 Preparation of the Culture Filtrate of M.anisopliae
1.4 Effect of M.anisopliae culture filtrate against the growth of B.cinerea
1.5 Effect of culture filtrate of M.anisopliae against the germination of conidia and sclerotia of B.cinerea
1.6 Effect of culture filtrate of M.anisopliae on the plasma membrane integrity of B.cinerea conidia
1.7 Heat stability of culture filtrate activity of M.anisopliae
1.8 Sample preparation for Transmission Electron Microscopy
1.9 Effect of culture filtrate on gray mold on detached tomato leaves
1.10 Effect of culture filtrate of M.anisopliae on the gray mold of ripe tomato fruits
1.11 Effect of culture filtrate of M.anisopliae against tomato plant gray mold
1.12 Effect of volatile organic molecules(VOCs)of M.anisopliae on B.cinerea
1.13 Effect of M.anisopliae VOCs on Botrytis gray mold of postharvest tomatoes
1.14 Data analysis
2.Results
2.1 Assessment inhibitory effect of M.anisopliae to B.cinerea
2.2 M.anisopliae culture filtrate influenced the growth and morphology of B.cinerea
2.3 The culture filtrate of M.anisopliae affected the sclerotia germination of B.cinerea
2.4 The culture filtrate of M.anisopliae inhibited conidia germination of B.cinerea
2.5 The antifungal substance produced by M.anisopliae retains its activity at high temperatures
2.6 The culture filtrate of M.anisopliae reduced the gray mold severity of detached leaves,fruits and whole plants of the tomato
2.7 The Volatile organic compounds of M.anisopliae reduced the gray mold severity of postharvest tomatoes
3.Discussion
Chapter 6.Conclusion and Future Prospects
References
Supplementary information
Appendix
1.Reagents
2.DNA extraction
3.Total RNA extraction
4.cDNA synthesis
5.qRT-PCR
Acknowledgements
本文编号：3918628
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