植物灰霉病菌中新型双分体病毒的分子生物学特性研究

论文摘要

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,从中鉴定出一种新的双分体病毒,发现该病毒与灰葡萄孢致病力衰退及发育缺陷（不能产生分生孢子和菌核）相关。在马铃薯葡萄糖琼脂培养基（potato dextrose agar,PDA）培养基上（20℃）,菌株QT5-19生长旺盛,产生粉红色色素,但不产菌核和分生孢子。QT5-19在苹果、黄瓜、油菜、草莓、葡萄、烟草和番茄等寄主上致病力极弱。通过病毒粒体提取,获得了一种球形病毒粒子,直径约为36 nm,包含两条双链RNA（dsRNA）片段,即dsRNA-1和dsRNA-2。对它们进行cDNA克隆,结果表明:dsRNA-1和dsRNA-2大小分别为1,909 bp和1,883 bp,分别编码依赖RNA的RNA聚合酶（RNA-dependent RNA polymerase,RdRp）和衣壳蛋白（coat protein,CP）。基于RdRp和CP序列进行系统进化分析,结果表明:dsRNA-1和dsRNA-2与Alphapartitivirus属病毒成员聚成一类,是一个新成员,命名为Botrytis cinerea partitivirus 2（BcPV2）。BcPV2可以通过菌丝接触（融合）传染到3个强毒灰葡萄孢菌株08168,B05.10和XN-1,传染后的衍生菌株08168T、B05.10T和XN-1T生长迅速,但不能产生分生孢子和菌核。且在番茄上表现出弱致病力。可见,BcPV2与灰葡萄孢弱毒特性及产孢和产菌核缺陷密切相关。2.发现菌株QT5-19具有较强的腐生竞争能力,通过腐生竞争QT5-19可抑制强毒灰葡萄孢和核盘菌对植物的侵染。在PDA培养基上测定了QT5-19和灰葡萄孢强毒菌株08168以及QT5-19与强毒核盘菌EP-1PNA367之间的腐生竞争能力。结果表明:QT5-19较菌株08168和EP-1PNA367腐生竞争能力更强。可能机制包括:（i）QT5-19菌丝旺盛生长;（ii）产生挥发性抗真菌物质。对QT5-19腐生竞争能力的生物防治潜力进行了评估。结果表明:QT5-19菌丝碎片可以抑制强毒灰葡萄孢菌株08168和菌株Ep-1PNA367侵染油菜叶片。3.菌株QT5-19产生挥发性物质具有广谱的抗真菌活性。除了灰葡萄孢之外,QT5-19产生的挥发性物质对B.aclada、B.pyriformis、B.sinoallii、Rhizopus stolonifer、Sclerotinia minor、S.sclerotiorum和Streptobotrys caulophylli等真菌菌丝生长具有抑制作用。用小麦粒培养QT5-19,作为物质挥发性物质来源,测试其对草莓果实腐烂病的控制效果。结果表明:QT5-19产生的挥发性物质能有效抑制由灰葡萄孢、核盘菌、冻土毛霉和匍枝根霉引起的草莓果实腐烂病。通过GC-MS检测及数据库比对分析,共鉴定出26种挥发性物质,包括乙醛、庚醛、1-辛烯-3-醇、1-octen-3-ol、反-2-辛烯醛、反-2-壬烯醛、2-十一烷醇、2-癸烯醛、苯并噻唑、1-（2-羟基-5-甲苯基）-邻氨基苯乙酮和十四醇等。4.菌株QT5-19挥发性物质对番茄生长有显著促生作用。在密闭的生长容器中,用PDA培养QT5-19,用其产生的挥发性物质熏蒸处理番茄幼苗,以新鲜PDA熏蒸处理为阴性对照,以PDA培养的菌株RoseBc-3熏蒸处理为阳性对照。结果表明:与阴性对照和阳性对照相比,QT5-19挥发性物质显著（P<0.05）促进番茄幼苗的生长。QT5-19的挥发物质物质处理产生促生效应的最短时间为1周,利用qRT-PCR探究了番茄体内植物生长素（检测SlIAA9基因）、细胞分裂素（检测SlCKX1基因）、赤霉素（检测SlGAox1基因）和乙烯（检测SlACO1基因）等植物激素相关合成基因的表达水平。结果表明:QT5-19挥发性物质处理后,番茄幼苗叶片中SlIAA9和SlCKX1表达量显著上升。这一结果表明:QT5-19挥发性物质促进番茄幼苗生长可能是生长素和细胞分裂素合成增加有关。本研究表明感染BcPV2的灰葡萄孢菌株QT5-19对灰葡萄孢和核盘菌具有良好的生物防治的潜力。QT5-19产生的挥发性物质不仅具有抗真菌活性,而且对植物具有促生作用。

论文目录

• 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

文章来源

类型: 博士论文

作者: 卡玛瑞（KAMARUZZAMAN）

导师: 李国庆

关键词: 灰葡萄孢,弱毒,生物防治

来源: 华中农业大学

年度: 2019

分类: 基础科学,农业科技

专业: 生物学,农业基础科学,植物保护

单位: 华中农业大学

分类号: S432.4

DOI: 10.27158/d.cnki.ghznu.2019.000153

总页数: 208

文件大小: 22675K

下载量: 99