植物灰霉病菌中新型双分体病毒的分子生物学特性研究
发布时间：2021-11-16 10:06
灰霉病是由死体营养型真菌灰葡萄孢（Botrytis cinerea）引起的一种重要植物病害。它的寄主范围广泛,可侵染1400多种植物,包括一些重要的经济作物,包括葡萄、草莓和番茄等。在作物收获前和收获后灰葡萄孢均可危害,并造成相当大的经济损失。由于大多数栽培的水果和蔬菜作物缺乏抗病品种,因此,灰霉病防治在很大程度上依赖反复使用杀菌剂。虽然可以达到预期的控制效果,但反复使用杀菌剂会引起环境污染,并产生抗杀菌剂菌株。因此,探索安全、可持续控制灰霉病措施（例如利用真菌病毒进行生物防治）日渐重要。目前,在灰葡萄孢中发现了很多弱毒相关真菌病毒,但还没有成功应用于灰葡萄孢生物防治。可能的限制因素包括以下两点:（i）由于菌株之间菌丝营养体不亲和,限制真菌病毒从含毒菌株向无毒菌株的传播;（ii）由于含毒菌株菌丝生长较慢,竞争力较低。因此有必要筛选更多含有真菌病毒的灰葡萄孢菌株。在本研究中,从灰葡萄孢QT5-19中发现了一种新的分体病毒属（Partitivirus）真菌病毒,对其分子特性,生物学特性以及生防潜力进行了评估。得到以下几点研究结果:1.获得了一株弱毒灰葡萄孢菌株QT5-19,从中鉴定出一种新...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：208 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
Chapter1.Literature review
1 Botrytis cinerea:a noble plant pathogen
1.1 Taxonomy and phylogeny
1.2 Biological characteristics of B.cinerea
1.3 Genetic diversity and adaptability
1.4 Infection mechanisms
1.5 Disease cycle and epidemiology
1.6 Economic importance of B.cinerea
1.7 Management of B.cinerea
1.7.1 Cultural management
1.7.2 Biological control
1.7.3 Chemical control
2 Mycoviruses
2.1 Taxonomy and classification of mycoviruses
2.1.1 DsRNA mycoviruses
(1)Totiviridae
(2)Partitiviridae
(a)Alphapartitivirus
(b)Betpartitivirus
(c)Gammapartitivirus
(d)Deltapartitivirus
(e)Cryspovirus
(3)Chrysoviridae
(4)Reoviridae
(5)Endornaviridae
(6)Megabirnaviridae
(7)Quadriviridae
2.1.2 Positive single-stranded RNA viruses
(1)Hypoviridae
(2)Narnaviridae
(3)Alphaflexiviridae
(4)Gammaflexiviridae
(5)Barnaviridae
(6)Fusariviridae
2.1.3 Reverse transcriptase RNA virus
(1)Pseudoviridae
(2)Metaviridae
2.1.4 Negative single-stranded RNA virus
2.1.5 Fungal DNA virus
2.3 Transmission of mycoviruses
2.4 Significance of mycoviruses
2.4.1 Symptomless or cryptic infection
2.4.2 Debilitation effects and hypovirulence
2.5 Interactions between fungal host and mycoviruses
2.6 Mycoviruses in B.cinerea
3 Competitive saprophytic ability
3.1 Saprophytic behavior of the plant pathogens
4 Volatile Organic Compounds(VOCs)
4.1 Detection of VOCs
4.2 VOCs emitted from fungi
4.2.1 Antifungal activity of the VOCs
4.2.2 Plant growth promotion activity of the VOCs
5 Study proposal
Chapter2 The hypovirulent strain QT5-19 of B.cinerea
1 Materials and Methods
1.1 Collection of plant diseased samples
1.2 Cultural media
1.3 Isolation,purification,and preservation of fungal strains
1.3.1 Preparation of conidial suspensions
1.3.2 Isolation
1.4 Screening of hypovirulent strains
1.4.1 Plantation of tobacco plants
1.4.2 Pathogenicity test
1.5 Determination of mycelial growth rate
1.6 Extraction of genomic DNA
1.7 Total RNA extraction and cDNA synthesis
1.8 PCR amplification
1.9 DNA sequencing
1.10 Determination of the bikaverin-biosynthesis gene cluster
1.11 Phylogenetic analysis
1.12 Culturing conditions for QT5-19
(1)Lighting period
(2)pH
(3)Temperature
(4)Media
(5)Carbon/nitrogen sources
1.13 Hypovirulence-associated physiological traits
(1)Infection cushions
(2)Laccase activity assay
(3)Pectinase activity assay
(4)Cellulase activity assay
(6)Determination of hyphal hydrophobicity
1.14 Statistical analysis
2 Results
2.1 Isolation and screening of hypovirulent strains
2.2 Pathogenicity,cultural and morphological characteristics of QT5-19
2.3 The bikaverin-biosynthesis gene cluster in B.cinerea
2.4 Molecular identification
2.5 Cultural features of QT5-19
2.6 Infection cushions
2.7 Hypovirulence-associated physiological traits in QT5-19
3 Discussion
Chapter3 A novel partitivirus in B.cinerea QT5-9
1 Materials and methods
1.1 Fungal strains
1.2 Bacterial Strains and Media
1.3 Vectors and primers
1.4 Enzymes and reagents
1.5 Equipments
1.6 Collection of mycelium
1.7 Extraction of the dsRNAs
1.7.1 Purification of the dsRNAs
1.8 cDNA synthesis
1.8.1 Ligation of the target cDNA fragments with pMD18-T
(1)A-Tailing of the cDNA fragments and ligation to pMD18-T
(2)Transformation of E.coli competent cells
(3)Detection of recombinant plasmids
1.9 Northern hybridization
1.9.1 Required reagents
1.9.2 Gel electrophoresis,transfer to the membrane and fixation
1.9.3 Probe Marking
1.9.4 Pre-hybridization and hybridization
1.9.5 GE film washing and CDP-star color development
1.10 Analysis of the mycovirus genome
1.11 Purification of virus particles
1.12 SDS-PAGE analysis
1.13 Phylogenetic analysis
1.14 RT-PCR detection of BcPV2
1.15 Detection of natural distribution of BcPV2 in B.cinerea strains
1.16 Horizontal transmission of BcPV2
1.17 Elimination of BcPV2 in QT5-19
1.17.1 Protoplast regeneration
1.17.2 Hyphal tipping
1.17.3 Thermal treatment
1.17.4 Chemical therapy
1.18 Statistical analysis
2 Results
2.1 Detection of the dsRNAs in QT5-19
2.2 The full-length cDNAs of the dsRNAs in QT5-19
2.3 The genomic structure of BcPV2 and northern hybridization
2.4 Sequence analysis of BcPV2
2.5 Virus Particles
2.6 Horizontal transmission of BcPV2
2.7 Elimination of BcPV2 in QT5-19
2.8 Natural distribution of BcPV2 in China
3 Discussion
Chapter4 Saprophytic Competitive Ability in QT5-19
1 Materials and Methods
1.1 Fungal strains and cultural conditions
1.2 Determination of competitive saprophytic ability
1.2.1 Preparation of mycelium fragments
1.2.2 Phenotypic observation of the mixed fungal cultures
(1)Intra-species competition trial
(2)Inter-species competition trial
1.2.3 Quantification of saprophytic colonization
1.3 Biocontrol assay:efficacy of the hyphal fragments of QT5-19
1.4 Statistical analysis
2 Results
2.1 Competitive saprophytic ability in QT5-19
2.1.2 Quantification of the saprophytic colonization on PDA
2.2 Suppression of infection by the hyphal fragments of QT5-19
2.2.1 Suppression of infection by B.cinerea
2.2.2 Suppression of disease infection against S.sclerotiorum A
3 Discussion
Chapter5 Antifungal volatiles produced by QT5-19
1 Materials and Methods
1.1 Fungal and bacterial strains
1.2 Efficacy of the VOCs of QT5-19
1.2.1 Dual cultures
1.2.2 Dish-inside-dish cultures
(1)Antifungal activity of the VOCs of QT5-19
(2)Antifungal activity of the QT5-19 VOCs from the AWG cultures
(3)Determination of the antifungal spectrum of the QT5-19 VOCs
1.2.4 Determination of antibacterial activity
1.2.5 Control of postharvest fruit rot of strawberry by the QT5-19 VOCs
1.3 Headspace collection and analysis of the QT5-19 VOCs
1.4 Statistical analysis
2 Results
2.1 QT5-19 has no direct antagonistic effect in dual cultures
2.2 QT5-19 produces antifungal volatiles
2.2.1 Antifungal spectrum of the QT5-19 VOCs
2.3 VOCs of QT5-19 has no antibacterial activity
2.4 Efficacy of the QT5-19 volatiles in the suppression of strawberry fruit rot
2.5 Suppression of Mucor fruit rot of strawberry
2.6 Suppression of Rhizopus fruit rot of strawberry
2.7 Suppression of Sclerotinia fruit rot of strawberry
2.8 Suppression of Penicillium fruit rot of strawberry
2.9 GC/MS identification of the QT5-19 VOCs
3 Discussion
Chapter6 Plant growth promotion by the QT5-19 VOCs
1 Materials and Methods
1.1 Fungal strains and cultural conditions
1.2 Preparation of plant materials
1.3 A plant growth-promotion assay in cultural media
1.4 A plant growth-promotion assay on pots
1.5 Determination of the minimum VOCs treatment duration
1.6 Effect of the QT5-19 VOCs on basic plant physiology and the chlorophyll content on tomato
1.7 Extraction of nucleic acids and qRT-PCR
1.8 Statistical analysis
2 Results
2.1 Response of tomato growth to the QT5-19 VOCs
2.2 The QT5-19 VOCs affected growth of tomato seedlings in a dose-dependent manner
2.3 Duration for the treatments with the QT5-19 VOCs
2.4 Physiological features and chlorophyll contents in tomato seedlings
2.5 Expression of the phytohormone biosynthesis-related genes
3 Discussion
Chapter7 Conclusions and Prospects
7.1 Conclusions
7.2 Future prospects
References
Supplementary information
Appendix 1:Reagents and cultural media
Appendix 2:Genomic DNA extraction
Appendix 3:Gel recovery and purification
Appendix 4:PCR product cleaning and purification
Appendix 5:Total RNA extraction from Botrytis cinerea mycelium
Appendix 6:cDNA synthesis,cloning,and sequencing
Appendix 7:qRT-PCR
List of publications during Ph.D.study
Acknowledgements
【参考文献】：
期刊论文
[1]农杆菌介导灰葡萄孢菌株RoseBc-3的遗传转化[J]. 范雷,张静,杨龙,李国庆. 华中农业大学学报. 2013(02)
[2]番茄几种有机挥发组分对尖镰孢的抑制作用[J]. 张鹏英,何培青,陈靠山,谢寒冰. 植物病理学报. 2006(01)
[3]来源于佳木斯茄子上的核盘菌菌株多样性的研究[J]. 李国庆,王道本,黄鸿章,周启. 植物病理学报. 1996(03)
本文编号：3498667
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：208 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
Chapter1.Literature review
1 Botrytis cinerea:a noble plant pathogen
1.1 Taxonomy and phylogeny
1.2 Biological characteristics of B.cinerea
1.3 Genetic diversity and adaptability
1.4 Infection mechanisms
1.5 Disease cycle and epidemiology
1.6 Economic importance of B.cinerea
1.7 Management of B.cinerea
1.7.1 Cultural management
1.7.2 Biological control
1.7.3 Chemical control
2 Mycoviruses
2.1 Taxonomy and classification of mycoviruses
2.1.1 DsRNA mycoviruses
(1)Totiviridae
(2)Partitiviridae
(a)Alphapartitivirus
(b)Betpartitivirus
(c)Gammapartitivirus
(d)Deltapartitivirus
(e)Cryspovirus
(3)Chrysoviridae
(4)Reoviridae
(5)Endornaviridae
(6)Megabirnaviridae
(7)Quadriviridae
2.1.2 Positive single-stranded RNA viruses
(1)Hypoviridae
(2)Narnaviridae
(3)Alphaflexiviridae
(4)Gammaflexiviridae
(5)Barnaviridae
(6)Fusariviridae
2.1.3 Reverse transcriptase RNA virus
(1)Pseudoviridae
(2)Metaviridae
2.1.4 Negative single-stranded RNA virus
2.1.5 Fungal DNA virus
2.3 Transmission of mycoviruses
2.4 Significance of mycoviruses
2.4.1 Symptomless or cryptic infection
2.4.2 Debilitation effects and hypovirulence
2.5 Interactions between fungal host and mycoviruses
2.6 Mycoviruses in B.cinerea
3 Competitive saprophytic ability
3.1 Saprophytic behavior of the plant pathogens
4 Volatile Organic Compounds(VOCs)
4.1 Detection of VOCs
4.2 VOCs emitted from fungi
4.2.1 Antifungal activity of the VOCs
4.2.2 Plant growth promotion activity of the VOCs
5 Study proposal
Chapter2 The hypovirulent strain QT5-19 of B.cinerea
1 Materials and Methods
1.1 Collection of plant diseased samples
1.2 Cultural media
1.3 Isolation,purification,and preservation of fungal strains
1.3.1 Preparation of conidial suspensions
1.3.2 Isolation
1.4 Screening of hypovirulent strains
1.4.1 Plantation of tobacco plants
1.4.2 Pathogenicity test
1.5 Determination of mycelial growth rate
1.6 Extraction of genomic DNA
1.7 Total RNA extraction and cDNA synthesis
1.8 PCR amplification
1.9 DNA sequencing
1.10 Determination of the bikaverin-biosynthesis gene cluster
1.11 Phylogenetic analysis
1.12 Culturing conditions for QT5-19
(1)Lighting period
(2)pH
(3)Temperature
(4)Media
(5)Carbon/nitrogen sources
1.13 Hypovirulence-associated physiological traits
(1)Infection cushions
(2)Laccase activity assay
(3)Pectinase activity assay
(4)Cellulase activity assay
(6)Determination of hyphal hydrophobicity
1.14 Statistical analysis
2 Results
2.1 Isolation and screening of hypovirulent strains
2.2 Pathogenicity,cultural and morphological characteristics of QT5-19
2.3 The bikaverin-biosynthesis gene cluster in B.cinerea
2.4 Molecular identification
2.5 Cultural features of QT5-19
2.6 Infection cushions
2.7 Hypovirulence-associated physiological traits in QT5-19
3 Discussion
Chapter3 A novel partitivirus in B.cinerea QT5-9
1 Materials and methods
1.1 Fungal strains
1.2 Bacterial Strains and Media
1.3 Vectors and primers
1.4 Enzymes and reagents
1.5 Equipments
1.6 Collection of mycelium
1.7 Extraction of the dsRNAs
1.7.1 Purification of the dsRNAs
1.8 cDNA synthesis
1.8.1 Ligation of the target cDNA fragments with pMD18-T
(1)A-Tailing of the cDNA fragments and ligation to pMD18-T
(2)Transformation of E.coli competent cells
(3)Detection of recombinant plasmids
1.9 Northern hybridization
1.9.1 Required reagents
1.9.2 Gel electrophoresis,transfer to the membrane and fixation
1.9.3 Probe Marking
1.9.4 Pre-hybridization and hybridization
1.9.5 GE film washing and CDP-star color development
1.10 Analysis of the mycovirus genome
1.11 Purification of virus particles
1.12 SDS-PAGE analysis
1.13 Phylogenetic analysis
1.14 RT-PCR detection of BcPV2
1.15 Detection of natural distribution of BcPV2 in B.cinerea strains
1.16 Horizontal transmission of BcPV2
1.17 Elimination of BcPV2 in QT5-19
1.17.1 Protoplast regeneration
1.17.2 Hyphal tipping
1.17.3 Thermal treatment
1.17.4 Chemical therapy
1.18 Statistical analysis
2 Results
2.1 Detection of the dsRNAs in QT5-19
2.2 The full-length cDNAs of the dsRNAs in QT5-19
2.3 The genomic structure of BcPV2 and northern hybridization
2.4 Sequence analysis of BcPV2
2.5 Virus Particles
2.6 Horizontal transmission of BcPV2
2.7 Elimination of BcPV2 in QT5-19
2.8 Natural distribution of BcPV2 in China
3 Discussion
Chapter4 Saprophytic Competitive Ability in QT5-19
1 Materials and Methods
1.1 Fungal strains and cultural conditions
1.2 Determination of competitive saprophytic ability
1.2.1 Preparation of mycelium fragments
1.2.2 Phenotypic observation of the mixed fungal cultures
(1)Intra-species competition trial
(2)Inter-species competition trial
1.2.3 Quantification of saprophytic colonization
1.3 Biocontrol assay:efficacy of the hyphal fragments of QT5-19
1.4 Statistical analysis
2 Results
2.1 Competitive saprophytic ability in QT5-19
2.1.2 Quantification of the saprophytic colonization on PDA
2.2 Suppression of infection by the hyphal fragments of QT5-19
2.2.1 Suppression of infection by B.cinerea
2.2.2 Suppression of disease infection against S.sclerotiorum A
3 Discussion
Chapter5 Antifungal volatiles produced by QT5-19
1 Materials and Methods
1.1 Fungal and bacterial strains
1.2 Efficacy of the VOCs of QT5-19
1.2.1 Dual cultures
1.2.2 Dish-inside-dish cultures
(1)Antifungal activity of the VOCs of QT5-19
(2)Antifungal activity of the QT5-19 VOCs from the AWG cultures
(3)Determination of the antifungal spectrum of the QT5-19 VOCs
1.2.4 Determination of antibacterial activity
1.2.5 Control of postharvest fruit rot of strawberry by the QT5-19 VOCs
1.3 Headspace collection and analysis of the QT5-19 VOCs
1.4 Statistical analysis
2 Results
2.1 QT5-19 has no direct antagonistic effect in dual cultures
2.2 QT5-19 produces antifungal volatiles
2.2.1 Antifungal spectrum of the QT5-19 VOCs
2.3 VOCs of QT5-19 has no antibacterial activity
2.4 Efficacy of the QT5-19 volatiles in the suppression of strawberry fruit rot
2.5 Suppression of Mucor fruit rot of strawberry
2.6 Suppression of Rhizopus fruit rot of strawberry
2.7 Suppression of Sclerotinia fruit rot of strawberry
2.8 Suppression of Penicillium fruit rot of strawberry
2.9 GC/MS identification of the QT5-19 VOCs
3 Discussion
Chapter6 Plant growth promotion by the QT5-19 VOCs
1 Materials and Methods
1.1 Fungal strains and cultural conditions
1.2 Preparation of plant materials
1.3 A plant growth-promotion assay in cultural media
1.4 A plant growth-promotion assay on pots
1.5 Determination of the minimum VOCs treatment duration
1.6 Effect of the QT5-19 VOCs on basic plant physiology and the chlorophyll content on tomato
1.7 Extraction of nucleic acids and qRT-PCR
1.8 Statistical analysis
2 Results
2.1 Response of tomato growth to the QT5-19 VOCs
2.2 The QT5-19 VOCs affected growth of tomato seedlings in a dose-dependent manner
2.3 Duration for the treatments with the QT5-19 VOCs
2.4 Physiological features and chlorophyll contents in tomato seedlings
2.5 Expression of the phytohormone biosynthesis-related genes
3 Discussion
Chapter7 Conclusions and Prospects
7.1 Conclusions
7.2 Future prospects
References
Supplementary information
Appendix 1:Reagents and cultural media
Appendix 2:Genomic DNA extraction
Appendix 3:Gel recovery and purification
Appendix 4:PCR product cleaning and purification
Appendix 5:Total RNA extraction from Botrytis cinerea mycelium
Appendix 6:cDNA synthesis,cloning,and sequencing
Appendix 7:qRT-PCR
List of publications during Ph.D.study
Acknowledgements
【参考文献】：
期刊论文
[1]农杆菌介导灰葡萄孢菌株RoseBc-3的遗传转化[J]. 范雷,张静,杨龙,李国庆. 华中农业大学学报. 2013(02)
[2]番茄几种有机挥发组分对尖镰孢的抑制作用[J]. 张鹏英,何培青,陈靠山,谢寒冰. 植物病理学报. 2006(01)
[3]来源于佳木斯茄子上的核盘菌菌株多样性的研究[J]. 李国庆,王道本,黄鸿章,周启. 植物病理学报. 1996(03)
本文编号：3498667
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