肯尼亚Nakuru地区马铃薯软腐病原菌分离鉴定及噬菌体防治研究

发布时间:2023-10-21 10:54
  溶果胶细菌能够引起马铃薯黑胫病和其他重要植物软腐病,严重影响食品的质量和产量,阻碍经济发展。本研究的目的主要是分离鉴定肯尼亚Nakuru地区感染马铃薯的溶果胶细菌,并针对分离的溶果胶细菌来分离裂解性噬菌体,从而探究合适的噬菌体混合制剂防治马铃薯软腐病。首先,从Nakuru的Molo和Mau Narok地区的不同农场收集有软腐病病症的马铃薯块茎,用结晶紫果胶酸盐培养基培养并分离病原菌,共获得71株病原菌。致病性测试结果显示其中的36株病原菌能浸染马铃薯块茎。用PCR扩增这36株致病菌的16S rDNA,通过测序对致病菌进行初步鉴定,再用BIOLOG微生物鉴定仪进行精准鉴定。鉴定结果显示36株致病菌中有20株软腐果胶杆菌亚种carotovorum,7株荧光假单胞菌B和9株荧光假单胞菌A,用特异性引物Y1和Y2扩增出了20株软腐果胶杆菌的果胶裂解酶编码基因,进一步证明了这20株致病菌为软腐果胶杆菌。以这20株软腐果胶杆菌作为目标菌株,从湖北省武汉市周边采集的土壤和污水中分离到11株噬菌体,其中有7株来属于肌尾噬菌体科,4株属于长尾噬菌体科。通过监测噬菌体和细菌悬液OD600的变化和一步生长曲...

【文章页数】:87 页

【学位级别】:硕士

【文章目录】:
摘要
ABSTRACT
CHAPTER ONE: INTRODUCTION
    1.1 Background of the study
    1.2 Literature review
        1.2.1 Importance of the Potato
        1.2.2 Pectolytic bacterial pathogens of potatoes
        1.2.3 Bacteriophages
    1.3 Problem statement
    1.4 Justification
    1.5 Objectives
    1.6 Hypotheses
CHAPTER TWO: ISOLATION AND CHARACTERIZATION OF PECTOLYTIC BACTERIAL PATHOGENS INFECTING POTATOES
    2.1 Materials and methods
        2.1.1 Bacterial isolation and culture conditions
        2.1.2 Pathogenicity tests
        2.1.3 Pectolytic activity tests
        2.1.4 Ribosomal DNA (16S rDNA) sequencing and Y1/Y2 primers DNA amplification
        2.1.5 Identification of the bacterial strains using BIOLOG microbial identification system (BIOLOG)
        2.1.6 Phylogenetic Analysis
    2.2 Results
        2.2.1 Bacteria sample isolation
        2.2.2 16S rDNA Sequencing and Y1/Y2 primers DNA amplification
        2.2.3 BIOLOG microbial identification
        2.2.4 Phylogenetic analysis
        2.2.5 Pathogenicity test results
        2.2.6 Pectolytic test results
    2.3 Discussion
        2.3.1 Phytopathogenic pectolytic bacteria infecting potatoes in Nakuru county, Kenya
        2.3.2 Pectolytic activity of isolated bacteria at different incubation temperatures
CHAPTER THREE: ISOLATION, CHARACTERIZATION AND USE OF BACTERIOPHAGES TO CONTROL PECTOBACTERIUM CAROTOVORUM
    3.1 Materials and Methods
        3.1.1 Phage isolation and purification
        3.1.2 Phage amplification
        3.1.3 Morphology of host bacteria
        3.1.4 Phage morphology assessment by transmission electron microscope
        3.1.5 Lytic activity of phages against Pectobacterium carotovorum
        3.1.6 Bacteriophage one step growth curve
        3.1.7 Antibacterial activity and phage specificity
        3.1.8 Development of phage resistance in Pectobacterium carotovorum
        3.1.9 Bacteriophage efficacy tests in controlling potato soft rot
    3.2 Results
        3.2.1 Phage isolation and purification
        3.2.2 Phhage morphoology
        3.2.3 Lyytic activityy of phagess against Peectobacterium carotov vorum
        3.2.4 Bacteriophage one step growth curve
        3.2.5 Antibacterial activity and phage specificity
        3.2.6 Development of resistance against bacteriophage infection
        3.2.7 Bacteriophage efficacy tests in controlling potato soft rot
    3.3 Disccussion
        3.3.1 Isolated bacteriophages for the control of Pectobacterium carotovorum
        3.3.2 Bacteriophage cocktail to overcome phage-resistance in Pectobacterium carotovorum
CHAPTER FOUR: CONCLUSION AND RECOMMENDATIONS
    4.1 Conclusion
    4.2 Recommendations and future prospects
REFERENCES
LIST OF ABBREVIATIONS AND ACRONYMS
APPENDICES
ACKNOWLEDGEMENTS
RéSUMé and PUBLISHED PAPERS



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