水稻细菌性褐条病菌（Acidovorax oryzae）分泌系统相关基因的毒力鉴定及对其生物防治策略的研究
发布时间：2023-07-27 08:45
水稻细菌性褐条病(BBS)是由种传细菌Acidovorax oryzae(Ao)所引起,属于革兰氏阴性菌,对世界范围内的水稻生产造成了严重的经济损失。传统的防治方法对该种传病害的防治效果较为有限。植物病原细菌分泌的效应蛋白在感染宿主植物的过程中发挥重要作用,从分泌系统的角度来阐明Ao的致病机制,不仅有利于对该病害进行有效的防控,而且对抗性育种也很有帮助。例如利用天然拮抗微生物和生物合成的纳米颗粒等基于生物防治的植物保护方法,对BBS的防治中有可能替代传统的化学防治手段。生物防治具有天然、无污染、高效率和特异性等好处,使其成为控制植物细菌病害的重要候选。有趣的是,Ao菌株RS-1的全基因组分析已经鉴定出大量分泌系统相关基因,我们课题组最近的研究表明T6SS和T4SS基因在Ao菌株RS-2的毒力方面发挥作用。然而,Ⅲ型分泌系统(T3SS)基因簇的基因在Ao菌株RS-2中的作用尚未研究。因此,对菌株RS-2的T3SS基因的研究为揭示水稻BBS病害的致病机制提供了基础;并且耐盐性根际细菌和生物银纳米粒子也为该水稻病害的防治提供了新的选择。基于这些问题,本研究项目开展了一系列研究工作,为水稻BB...
【文章页数】：174 页
【学位级别】：博士
【文章目录】：
acknowledgements
Abstract
摘要
Chapter 1 Introduction
Chapter 2 Literature review
2.1 Bacterial brown stripe disease of rice
2.2 Virulence factors in Acidovorax oryzae
2.2.1 Bacterial surface structures
2.2.2 Bacterial secretion system
2.3 Application of halotolerant bacteria in biocontrol
2.3.1 Halophilic and halotolerant bacteria in hypersaline environments
2.3.2 Potential of halotolerant Bacillus species in biocontrol and promotion of plant growth
2.3.3 Mechanisms involved in growth promotion of plant and biocontrol of halotolerantBacillus species
2.4 Aspects of nanotechnology in control of plant bacterial disease
2.4.1 Synthesis of silver nanoparticles
2.4.2 Nano-biofertilizer approaches to improve plant health
2.4.3 Nanoparticles-based plant disease management for plant bacterial diseases
2.4.4 Potential mechanism of antimicrobial effects and plant promotion of AgNPs
Chapter 3 Role of type Ⅲ secretion system genes in virulence of rice bacterial brown stripepathogen Acidovorax oryzae strain RS-2
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.3.1 Experimental materials
3.3.1.1 Bacterial strains and phage
3.3.1.2 Culture medium, solution, and antibiotics
3.3.2 Routine experimental methods
3.3.2.1 Primers design
3.3.2.2 Conventional PCR
3.3.2.3 Enzyme digestion and ligation reaction
3.3.2.4 Heat shock and electroporation transformation experiment
3.3.2.5 Bacterial genomic and plasmid DNA extraction
3.3.3 Prediction of T3SS gene cluster in Ao strain RS-2
3.3.4 Phylogenetic analysis
3.3.5 Construction of T3SS mutants
3.3.6 Construction of T3SS complementation
3.3.7 Seedling pathogenicity of brown stripe bacteria in rice
3.3.8 Determination of bacterial growth
3.3.9 Determination of biofilm formation
3.3.10 Determination of motility
3.3.11 Determination of EPS and extracellular enzyme activities
3.3.12 Detection of bacterial sensitivity to phage
3.3.13 Statistical analysis
3.4 Results
3.4.1 In silico identification of T3SS genes
3.4.2 Validation of T3SS genes mutants and complementation
3.4.3 T3SS involved in virulence of Ao strain RS-2 to rice seedlings
3.4.4 T3SS involved in growth of Ao strain RS-2
3.4.5 T3SS involved in swimming motility of Ao strain RS-2
3.4.6 T3SS involved in biofilm formation of Ao strain RS-2
3.4.7 T3SS involved in production of EPS
3.4.8 T3SS genes did not affect API phage infection and extracellular enzyme production
3.5 Discussion
3.6 Conclusions
Chapter 4 Halotolerant bacteria belonging to operational group Bacillus amyloliquefaciens in biocontrol of the rice bacterial brown stripe pathogen Acidovorax oryzae
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Bacterial pathogen, growth media and inoculum preparation
4.3.2 Isolation of halotolerant bacteria
4.3.3 In vitro screening for antagonism
4.3.4 In planta screening for antagonism
4.3.5 Bacteriological,physiological and biochemical tests
4.3.6 Fatty acids analysis of halotolerant bacteria
4.3.7 Phylogenetic analyses of 16S rRNA gene sequence
4.3.8 Phylogenetic analyses of gyrA and rpoB gene sequences
4.3.9 Siderophore production of halotolerant bacteria
4.3.10 Lipopeptides detection by MALDI-TOF-MS analysis of halotolerant bacteria
4.3.11 Determination of antibacterial activity of Bacillus culture filtrates
4.3.12 Motility Assays
4.3.13 Biofilm formation assay
4.3.14 Transmission electron microscopy analysis
4.3.15 Statistical analysis
4.4 Results
4.4.1 Isolation of halotolerant bacteria
4.4.2 In vitro antagonistic activities against Ao strains
4.4.3 In planta antagonistic activities against Ao strains
4.4.4 Phylogenetic identification based on the 16S rRNA, gyrA,and rpoB genes sequences
4.4.5 Bacteriological,physiological,biochemical, fatty acids characterization and siderophore production
4.4.6 MALDI-TOF identification of lipopeptides of isolates K5-3 and PPB6
4.4.7 Growth inhibition by the metabolites
4.4.8 Inhibition of swimming motility by the metabolites
4.4.9 Inhibition of biofilm formation by the metabolites
4.4.10 Damage of bacterial cells by the metabolites
4.5 Discussion
4.6 Conclusions
Chapter 5 Biogenic synthesis of silver nanoparticles using Phyllanthus emblica fruit extractand its inhibitory effect against the rice bacterial brown stripe pathogen Acidovorax oryzaestrain RS-2
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.3.1 Materials
5.3.2 Preparation of extract from P. emblica fresh fruit
5.3.3 Biosynthesis of AgNPs using P. emblica fruit extract
5.3.4 Characterization of the synthesized AgNPs
5.3.5 In vitro antibacterial activity of AgNPs
5.3.6 Minimum inhibitory concentration (MIC)of AgNPs
5.3.7 Effect of contact time of AgNPs on cell survival of Ao strain RS-2
5.3.8 Swarming motility assay
5.3.9 Biofilm inhibition assay
5.3.10 Live/dead cell staining
5.3.11 Transmission electron microscopy (TEM) analysis
5.3.12 Secretion of effectorsprotein Hcp (hemolysin coregulated protein)
5.3.13 Statistical analysis
5.4 Results
5.4.1 Synthesis and characterization of the synthesized nanoparticles
5.4.2 In vitro antimicrobial activity of AgNPs
5.4.3 Minimum inhibitory concentration (MIC) of AgNPsagainst Ao strain RS-2
5.4.4 Effect of contact time of AgNPs on cell survival of Ao strain RS-2
5.4.5 Swarming motility
5.4.6 Biofilm formation
5.4.7 Live/dead cell staining
5.4.8 Damage of bacterial cells by the synthesized AgNPs
5.4.9 Secretion of effector protein Hcp
5.5 Discussion
5.6 Conclusions
Research Conclusions and Prospects
References
Appendices
List of Publications
本文编号：3837703
【文章页数】：174 页
【学位级别】：博士
【文章目录】：
acknowledgements
Abstract
摘要
Chapter 1 Introduction
Chapter 2 Literature review
2.1 Bacterial brown stripe disease of rice
2.2 Virulence factors in Acidovorax oryzae
2.2.1 Bacterial surface structures
2.2.2 Bacterial secretion system
2.3 Application of halotolerant bacteria in biocontrol
2.3.1 Halophilic and halotolerant bacteria in hypersaline environments
2.3.2 Potential of halotolerant Bacillus species in biocontrol and promotion of plant growth
2.3.3 Mechanisms involved in growth promotion of plant and biocontrol of halotolerantBacillus species
2.4 Aspects of nanotechnology in control of plant bacterial disease
2.4.1 Synthesis of silver nanoparticles
2.4.2 Nano-biofertilizer approaches to improve plant health
2.4.3 Nanoparticles-based plant disease management for plant bacterial diseases
2.4.4 Potential mechanism of antimicrobial effects and plant promotion of AgNPs
Chapter 3 Role of type Ⅲ secretion system genes in virulence of rice bacterial brown stripepathogen Acidovorax oryzae strain RS-2
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.3.1 Experimental materials
3.3.1.1 Bacterial strains and phage
3.3.1.2 Culture medium, solution, and antibiotics
3.3.2 Routine experimental methods
3.3.2.1 Primers design
3.3.2.2 Conventional PCR
3.3.2.3 Enzyme digestion and ligation reaction
3.3.2.4 Heat shock and electroporation transformation experiment
3.3.2.5 Bacterial genomic and plasmid DNA extraction
3.3.3 Prediction of T3SS gene cluster in Ao strain RS-2
3.3.4 Phylogenetic analysis
3.3.5 Construction of T3SS mutants
3.3.6 Construction of T3SS complementation
3.3.7 Seedling pathogenicity of brown stripe bacteria in rice
3.3.8 Determination of bacterial growth
3.3.9 Determination of biofilm formation
3.3.10 Determination of motility
3.3.11 Determination of EPS and extracellular enzyme activities
3.3.12 Detection of bacterial sensitivity to phage
3.3.13 Statistical analysis
3.4 Results
3.4.1 In silico identification of T3SS genes
3.4.2 Validation of T3SS genes mutants and complementation
3.4.3 T3SS involved in virulence of Ao strain RS-2 to rice seedlings
3.4.4 T3SS involved in growth of Ao strain RS-2
3.4.5 T3SS involved in swimming motility of Ao strain RS-2
3.4.6 T3SS involved in biofilm formation of Ao strain RS-2
3.4.7 T3SS involved in production of EPS
3.4.8 T3SS genes did not affect API phage infection and extracellular enzyme production
3.5 Discussion
3.6 Conclusions
Chapter 4 Halotolerant bacteria belonging to operational group Bacillus amyloliquefaciens in biocontrol of the rice bacterial brown stripe pathogen Acidovorax oryzae
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Bacterial pathogen, growth media and inoculum preparation
4.3.2 Isolation of halotolerant bacteria
4.3.3 In vitro screening for antagonism
4.3.4 In planta screening for antagonism
4.3.5 Bacteriological,physiological and biochemical tests
4.3.6 Fatty acids analysis of halotolerant bacteria
4.3.7 Phylogenetic analyses of 16S rRNA gene sequence
4.3.8 Phylogenetic analyses of gyrA and rpoB gene sequences
4.3.9 Siderophore production of halotolerant bacteria
4.3.10 Lipopeptides detection by MALDI-TOF-MS analysis of halotolerant bacteria
4.3.11 Determination of antibacterial activity of Bacillus culture filtrates
4.3.12 Motility Assays
4.3.13 Biofilm formation assay
4.3.14 Transmission electron microscopy analysis
4.3.15 Statistical analysis
4.4 Results
4.4.1 Isolation of halotolerant bacteria
4.4.2 In vitro antagonistic activities against Ao strains
4.4.3 In planta antagonistic activities against Ao strains
4.4.4 Phylogenetic identification based on the 16S rRNA, gyrA,and rpoB genes sequences
4.4.5 Bacteriological,physiological,biochemical, fatty acids characterization and siderophore production
4.4.6 MALDI-TOF identification of lipopeptides of isolates K5-3 and PPB6
4.4.7 Growth inhibition by the metabolites
4.4.8 Inhibition of swimming motility by the metabolites
4.4.9 Inhibition of biofilm formation by the metabolites
4.4.10 Damage of bacterial cells by the metabolites
4.5 Discussion
4.6 Conclusions
Chapter 5 Biogenic synthesis of silver nanoparticles using Phyllanthus emblica fruit extractand its inhibitory effect against the rice bacterial brown stripe pathogen Acidovorax oryzaestrain RS-2
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.3.1 Materials
5.3.2 Preparation of extract from P. emblica fresh fruit
5.3.3 Biosynthesis of AgNPs using P. emblica fruit extract
5.3.4 Characterization of the synthesized AgNPs
5.3.5 In vitro antibacterial activity of AgNPs
5.3.6 Minimum inhibitory concentration (MIC)of AgNPs
5.3.7 Effect of contact time of AgNPs on cell survival of Ao strain RS-2
5.3.8 Swarming motility assay
5.3.9 Biofilm inhibition assay
5.3.10 Live/dead cell staining
5.3.11 Transmission electron microscopy (TEM) analysis
5.3.12 Secretion of effectorsprotein Hcp (hemolysin coregulated protein)
5.3.13 Statistical analysis
5.4 Results
5.4.1 Synthesis and characterization of the synthesized nanoparticles
5.4.2 In vitro antimicrobial activity of AgNPs
5.4.3 Minimum inhibitory concentration (MIC) of AgNPsagainst Ao strain RS-2
5.4.4 Effect of contact time of AgNPs on cell survival of Ao strain RS-2
5.4.5 Swarming motility
5.4.6 Biofilm formation
5.4.7 Live/dead cell staining
5.4.8 Damage of bacterial cells by the synthesized AgNPs
5.4.9 Secretion of effector protein Hcp
5.5 Discussion
5.6 Conclusions
Research Conclusions and Prospects
References
Appendices
List of Publications
本文编号：3837703
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