Microbial Plant Pathogens-Detection and Disease Diagnosis: (eBook)

Preface 8
Acknowledgement 10
Contents 12
Table of Contents for Volumes 1 and 3 16
Chapter 1: Introduction 24
1.1 Microbial Plant Pathogens as a MajorLimiting Factor of Crop Production 24
1.2 Discovery of Bacteria as Plant Pathogens 25
1.3 Detection of Bacterial and Phytoplasmal Pathogens and Disease Diagnosis 25
References 27
Chapter 2: Detection of Bacterial and Phytoplasmal Pathogens 28
2.1 Detection of Bacterial Pathogens in Plant Organs 31
2.1.1 Biological Methods 31
2.1.1.1 Use of Media for Culturing Bacteria 35
2.1.1.2 Use of Diagnostic Hosts 37
2.1.1.3 Use of Bacteriophages (Phage Typing) 37
2.1.2 Biochemical Methods 38
2.1.2.1 Properties of Bacteria in Culture 38
2.1.2.2 Metabolic Fingerprinting 39
2.1.2.3 Properties of Bacterial Cells 40
Direct Colony Thin Layer Chromatography 40
Polyacrylamide Gel Electrophoresis 41
Conductimetric Assay 43
Isozyme Analysis 43
Use of Fluorescent Markers/Bioluminescent Strains of Pathogens 44
2.1.3 Immunoassays 44
2.1.3.1 Agglutination Test 46
2.1.3.2 Gel Diffusion Test 47
2.1.3.3 Enzyme-Linked Immunosorbent Assay 47
2.1.3.4 Dot Immunobinding Assay 52
2.1.3.5 Immunofluorescence Test 52
2.1.3.6 Immunolabeling and Electron Microscopy 54
2.1.3.7 Immunomagnetic Fishing/Isolation of Bacterial Pathogens 55
2.1.3.8 Flow Cytometry 55
2.1.4 Nucleic Acid-Based Techniques 57
2.1.4.1 Nucleic Acid Hybridization Methods 57
2.1.4.2 Restriction Fragment Length Polymorphism 58
2.1.4.3 Polymerase Chain Reaction 59
Detection by Targeting Specific Gene Sequences 65
Detection by Targeting Non-nuclear Nucleic Acids 68
Detection by Using Variants of PCR 70
Integration of PCR with Other Detection Methods 76
2.1.4.4 Real-Time Polymerase Chain Reaction 81
2.1.4.5 Loop-Mediated Isothermal Amplification Technique 86
2.1.4.6 Isothermal and Chimeric Primer-Mediated Amplificationof Nucleic Acids (ICAN) Technique 87
2.1.4.7 DNA Array Technology 88
2.1.4.8 Multilocus Sequence Typing System 89
2.2 Detection of Phytoplasmal Pathogens in Plant Organs 89
2.2.1 Histochemical Methods 92
2.2.2 Immunoassays 95
2.2.2.1 Rapid Slide Test 95
2.2.2.2 Enzyme-Linked Immunosorbent Assay 96
2.2.2.3 Immunofluorescence Technique 98
2.2.2.4 Immunosorbent Electron Microscopy and Gold-Labeled Antibody Decoration 98
2.2.3 Nucleic Acid-Based Techniques 100
2.2.3.1 Dot Blot Hybridization Assay 100
2.2.3.2 Restriction Fragment Length Polymorphism 103
2.2.3.3 Polymerase Chain Reaction-Based Assays 103
2.2.3.4 Real-Time Polymerase Chain Reaction 120
2.2.3.5 Heteroduplex Mobility Analysis 124
2.2.3.6 DNA Array Technology 125
2.3 Detection of Bacterial Pathogens in Seeds and Planting Materials 126
2.3.1 Detection of Bacterial Pathogens in Seeds 127
2.3.1.1 Biological Methods 127
2.3.1.2 Immunoassays 128
Agglutination-/Diffusion-Based Tests 128
Immunofluorescence Microscopy 128
Enzyme-Linked Immunosorbent Assay 129
2.3.1.3 Detection by Nucleic Acid-Based Assays 131
Nucleic Acid Hybridization Technique 132
Restriction Fragment Length Polymorphism Analysis 132
Polymerase Chain Reaction-Based Assays 132
Real-Time Polymerase Chain Reaction 135
2.3.2 Detection of Bacterial Pathogens in Propagative Planting Materials 137
2.3.2.1 Detection by Biological Methods 137
2.3.2.2 Detection by Immunoassays 137
Enzyme-Linked Immunosorbent Assay 137
Immunofluorescence Test 138
2.3.2.3 Detection by Nucleic Acid-Based Techniques 139
Nucleic Acid Hybridization Technique 139
Polymerase Chain Reaction-Based Assays 139
Real-Time Polymerase Chain Reaction 142
DNA Array Technology 144
Nucleic Acid Sequence-Based Amplification Assay 145
2.3.3 Detection of Bacterial Pathogens in Postharvest Produce 146
2.4 Detection of Phytoplasmal Pathogens in Planting Materials 148
Appendix 1: Media for Isolation of Bacterial Plant Pathogens 149
Appendix 2: Detection of Acidovorax avenae subsp. citrulli (Aac) by Pulsed-Field Gel Electrophoresis (PFGE) 152
Appendix 3: Detection of Bacterial Pathogens by Slide Agglutination Test (Lyons and Taylor 1990) 153
Preparation of Somatic Antigen 153
Production of Antiserum 153
Production of Staphylococcus aureus Reagent 153
Conjugation of Working Reagent 154
Slide Agglutination Test 154
Appendix 4: Detection of Xanthomonas campestris pv. vesicatoria by Enzyme-Linked Immunosorbent Assay (ELISA) Formats 154
Preparation of Bacterial Cells (Antigen) 154
Preparation of Plant Samples 155
ABC-Enzyme-Linked Immunosorbent Assay 155
Appendix 5: Detection of Erwinia chrysanthemi Using Fimbria-Specific Antibody in ELISA Technique (Singh et al. 2000) 156
Production of Antiserum 156
Screening Hybridomas for Specific MAbs 156
Detection of Target Pathogen in Plant Tissues 156
Appendix 6: Detection of Erwinia chrysanthemi by Immunogold Labeling and Electron Microscopy Technique 157
Immunogold Labeling 157
Electron Microscopy 157
Appendix 7: Detection of Clavibacter michiganensis subsp. michiganensis by Flow Cytometry (Chitaara et al. 2006) 157
Labeling of Target Bacterial Cells 157
Flow cytometric Analysis 158
Appendix 8: Detection of Erwinia amylovora by Polymerase Chain Reaction (PCR) (Stöger et al. 2006) 158
Extraction of DNA 158
Polymerase Chain Reaction 159
Appendix 9: Detection of Xanthomonas campestris pv. vesicatoria (Xcv) by PCR Assay (Park et al. 2009) 159
PCR Amplification 159
Dot Blot Analysis 159
Appendix 10: Detection of Xanthomonas axonopodis pv. citri (Xac) by Immuno-Capture (IC) and Nested (N)-PCR Assay 160
Magnetic Immunocapture of Target Pathogen Cells 160
PCR Reactions 160
Appendix 11: Detection of Xylella fastidiosa (Xf) in Xylem Exudates of Grapevine By IC-PCR Technique (Guo and Lu 2004 161
Extraction of Xylem Exudates 161
Preparation of Xf Suspension by Immunocapture Procedure 161
PCR Amplification 161
Appendix 12: Detection of Xanthomonas axonopodis pv. citri by Real Time PCR Assay (Mavrodieva et al. 2004) 162
Sample Preparation Using the IT 1-2-3 RAPID DNA Purification Kit 162
Real-Time PCR Assay 162
Appendix 13: Detection of Phytoplasmal Pathogens by Histological Techniques (Galvis et al. 2007) 163
DAPI Fluorescence Test 163
Transmission Electron Microscopy (TEM) 163
Appendix 14: Detection of Spiroplasma Infecting Carrot by Nested PCR Assay (Green et al. 1999 Lee et al. 2006)
Extraction of Pathogen DNA 163
Nested PCR and RFLP Analysis for Phytoplasmas 164
Nested PCR and RFLP Analysis for Spiroplasmas 165
Appendix 15: Detection of Phytoplasmas by Oligonucleotide Microarray-Based Assay (Nicolaisen and Bertaccini 2007) 165
PCR Amplification 165
Labeling of Hybridization Probes 165
Printing and Post-processing of Oligonucleotide Arrays 165
Microarray Hybridization 166
Microarray Scanning 166
Appendix 16: Detection of Clavibacter michiganensis subsp. michiganensis (Cmm) by Immunomagnetic Separation (IMS) – PlatingTechnique 166
Coating Magnetic Beads 166
Standardization of IMS Technique 167
Isolation of Target Pathogen from Naturally Infected Seeds by IMS 167
Appendix 17: Detection of Xanthomonas campestris pv. carotae by PCR Assay (Meng et al. 2004) 167
Preparation of Seed-wash 167
DNA Extraction from Seed-Wash 168
Appendix 18: Detection of Acidovorax avenae subsp. citrulli by Immuno-capture PCR Assay (Xiao et al. 2007) 168
Selection of Antibodies and Primers 168
Detection by Standard PCR Assay 168
Detection by Immuno-capture (IC)-PCR Assay 168
Appendix 19: Detection of Burkholderia glumae in Rice Seeds by Real-time PCR Assay (Sayler et al. 2006) 169
Preparation of Seed-wash 169
Real-time PCR Assay 169
References 169
Chapter 3: Detection of Bacterial and Phytoplasmal Pathogens in the Environment 193
3.1 Detection of Bacterial Pathogens in the Environment 194
3.1.1 Detection of Bacterial Pathogens in Soil 194
3.1.1.1 Biological Methods 194
3.1.1.2 Immunoassays 195
Immunofluorescence Tests 195
Enzyme-Linked Immunosorbent Assay 196
3.1.1.3 Nucleic Acid-Based Techniques 196
Polymerase Chain Reaction-Based Assays 196
3.1.2 Detection of Bacterial Pathogens in Water 200
3.1.2.1 Biological Methods 201
3.1.2.2 Immunoassays 202
3.1.2.3 Nucleic Acid-Based Methods 202
3.1.3 Detection of Bacterial Pathogens in Air 204
3.1.4 Detection of Bacterial Pathogens in Alternative Host Plants 205
3.1.5 Detection of Bacterial Pathogens in Vector Insects 206
3.1.5.1 Biological Methods 206
3.1.5.2 Immunoassays 207
3.1.5.3 Nucleic Acid-Based Techniques 207
Nucleic Acid Hybridization Techniques 207
Polymerase Chain Reaction 208
3.2 Detection of Phytoplasmal Pathogens in the Environment 211
3.2.1 Detection of Phytoplasmal Pathogens in Alternative Host Plants 211
3.2.1.1 Nucleic Acid-Based Techniques 211
3.2.2 Detection of Phytoplasmal Pathogens in Insect Vectors 212
3.2.2.1 Immunoassays 213
3.2.2.2 Nucleic Acid-Based Assays 213
Nucleic Acid Hybridization Technique 213
Appendix 1: Detection of Ralstonia solanacearum Race 1 Strains in Soils by BIO-PCR Assay (Lin et al. 2009) 217
Bacterial Enrichment Using MSM-1 Broth 217
Appendix 2: Detection of Xylella fastidiosa (Xf) in Vector Insects by PCR Assay (Krell et al. 2007) 218
Extraction of DNA 218
References 218
Chapter 4: Assessment of Variability in Bacterial and Phytoplasmal Pathogens 222
4.1 Assessment of Variability in Bacterial Pathogens 223
4.1.1 Assessment of Variations in Pathogenicity 223
4.1.2 Assessment of Variations in Phage Sensitivity 223
4.1.3 Assessment of Variations in Cultural Characteristics 224
4.1.4 Assessment of Variations in Biochemical Characteristics 225
4.1.4.1 Fatty Acid Analysis 225
4.1.4.2 Polyacrylamide Gel Electrophoretic Analysis 225
4.1.5 Assessment of Variations in Immunological Characteristics 227
4.1.6 Assessment of Variations in Nucleic Acid Characteristics 228
4.1.6.1 Restriction Fragment Length Polymorphism Method 228
4.1.6.2 Polymerase Chain Reaction-Based Techniques 229
4.1.6.3 Random Amplified Polymorphic DNA Technique 234
4.1.6.4 Amplified Fragment Length Polymorphism Technique 236
4.1.6.5 DNA Microarray Technology 237
4.1.7 Assessment of Variability in Resistance to Chemicals 238
4.2 Assessment of Variability in Phytoplasmal Pathogens 239
4.2.1 Assessment of Variability in Immunological Characteristics 240
4.2.2 Assessment of Variability in Genomic Characteristics 240
4.2.2.1 Polymerase Chain Reaction-Dependent Techniques 241
4.2.2.2 Subtractive Suppression Hybridization 244
4.2.2.3 Single-Strand Conformation Polymorphism Analysis 245
4.2.2.4 Heteroduplex Mobility Assay 246
Appendix 1: Differentiation of Strains of Clavibacter michiganensis subsp. michiganensis (Cmm) by Macrorestriction PulsedField Gel Electrophoresis (PFGE) Technique 247
References 248
Chapter 5: Diagnosis of Bacterial Diseases of Plants 254
5.1 Choice of Diagnostic Tests for Bacterial Diseases 255
5.1.1 Conventional Techniques 255
5.1.2 Biochemical Tests 256
5.1.3 Immunoassays 258
5.1.4 Nucleic Acid-Based Techniques 259
5.2 Choice of Diagnostic Tests for Phytoplasmal Diseases 261
5.2.1 Biological Methods 261
5.2.2 Nucleic Acid-Based Techniques 261
5.3 Agencies Involved in Disease Diagnosis 262
5.3.1 Disease Diagnostic Centers 263
5.3.2 Plant Quarantines 264
References 266
Index 268