Preface ........................................................ xv
Acknowledgement .............................................. xvii
1. Introduction ................................................. 1
1.1. Molecular Biology as a Research Tool ....................... 1
1.2. Application of Molecular Techniques ........................ 3
References ...................................................... 6
2. Molecular Techniques for Detection of Microbial Pathogens .... 7
2.1. Detection of Microbial Pathogens by Biochemical
Techniques ................................................. 9
2.1.1 Electrophoresis ...................................... 9
2.2. Detection of Microbial Pathogens by Immunoassays .......... 14
2.2.1. Viral Pathogens .................................... 15
2.2.2. Bacterial Pathogens ................................ 25
2.2.3. Fungal Pathogens ................................... 29
2.3. Detection of Microbial Plant Pathogens by Nucleic
Acid-Based Techniques ..................................... 32
2.3.1. Detection of Viral Pathogens ....................... 34
2.3.2. Detection of Viroids ............................... 57
2.3.3. Detection of Bacterial Pathogens ................... 60
2.3.4. Detection of Phytoplasmal Pathogens ................ 80
2.3.5. Detection of Fungal Pathogens ...................... 85
Appendix 1: Electrophoretic Characterization of Strains of
Bacterial Pathogen Xanthomonas campestris pv.
vesicatoria (Xcv) (Bouzar et al. 1994) ................... 110
Appendix 2: Detection of Virus-Specific Protein in Infected
Leaves by Sodium Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis (SDS-PAGE) (Seifers et al. 1996,
2005) .................................................... 111
Appendix 3: Indirect ELISA for Assessing Titers
of PABs and MABs Specific to Callalily chlorotic
spot virus (CCSV) and Watermelon silver mottle
virus (WSMoV) (Lin et al. 2005) .......................... 112
Appendix 4: Detection of Citrus psorosis virus (CPsV)
by Direct Tissue Blot Immunoassay (DTBIA)
(Martin et al. 2002) ..................................... 113
Appendix 5: Detection of Potato virus Y (PVY) and
Cucumber mosaic virus (CMV) in Tobacco by
Immunostaining Technique (Ryang et al. 2004) ............. 113
Appendix 6: Detection of Citrus tristeza virus (CTV)
by In Situ Immunoassay (ISIA) (Lin et al. 2000) .......... 114
Appendix 7: Detection of Potyvirus by Western Blot
Analysis (Larsen et al. 2003) ............................ 115
Appendix 8: Detection of Bacterial Pathogens by
Enzyme-Linked Immunosorbent Assay (ELISA) in
Seeds (Lamka et al. 1991; Pataky et al. 2004) ............ 115
Appendix 9: Detection of Ultilago nuda Barley Seeds by
DAS-ELISA (Eibel et al. 2005) ............................ 116
Appendix 10: Detection of Plant Viruses by
Reverse-Transcription-Poiymerase Chain Reaction
(RT-PCR) Assay (Huang et al. 2004; Spiegel
et al. 2004) ............................................. 118
Appendix 11: Detection of Virus (Potato virus Y)
by Reverse Transcription - DIAPOPS System (Nicolaisen
et al. 2001) ............................................. 119
Appendix 12: Detection of Grape fan leaf virus (GFLV)
in Nematode Vector Xiphinema index by RT-PCR
(Finetti-Sialer and Ciancio 2005) ........................ 121
Appendix 13: Detection of Potato virus Y by Reverse
Transcription Loop-Mediated Isothermal Amplification
DNA (Nie 2005) ........................................... 122
Appendix 14: Detection of Fruit Tree Viroids by a Rapid
RT-PCR Test (Hassen et al. 2006) ......................... 123
Appendix 15: Membrane BIO-PCR Technique for Detection of
Bacterial Pathogen (Pseudomonas syringae pv.
phaseolicola) (Schaad et al. 2007) ....................... 125
Appendix 16: Detection of Bacterial Pathogens by DNA Array
Technology (Fessehaie et al. 2003; Scholberg et
al. 2005) ................................................ 126
Appendix 17: Extraction of Genomic DNA from Fungal
Pathogens (Phytophthora spp.) (Lamour and Finley 2006) ... 128
Appendix 18: Detection of Mycosphaerella graminicola
in Wheat Using Reverse Transcription (RT)-PCR
(Guo et al. 2005) ........................................ 129
References .................................................... 130
3. Molecular Variability of Microbial Plant Pathogens ......... 159
3.1. Molecular Basis of Variability of Fungal Pathogens ....... 160
3.1.1. Isozyme Variation ................................. 161
3.1.2. Immunological Assay ............................... 162
3.1.3. Dot-Blot Hybridization Assay ...................... 163
3.1.4. Restriction Fragment Length Polymorphism .......... 163
3.1.5. Polymerase Chain Reaction ......................... 168
3.1.6. Random Amplified Polymorphic DNA Technique ........ 175
3.1.7. Amplified Fragment Length Polymorphism
Technique ......................................... 179
3.1.8. DNA Fingerprinting ................................ 183
3.1.9. Microsatellite Amplification ...................... 183
3.1.10.Single-Strand Conformation Polymorphism
Analysis .......................................... 184
3.2. Molecular Basis of Variability of Bacterial Pathogens .... 185
3.2.1. Immunoassays ...................................... 185
3.2.2. Restriction Fragment Length Polymorphism .......... 186
3.2.3. Polymerase Chain Reaction ......................... 187
3.2.4. Random Amplified Polymorphic DNA .................. 191
3.2.5. DNA-DNA Hybridization ............................. 193
3.2.6. Amplified Fragment Length Polymorphism
Technique ......................................... 195
3.2.7. PCR-Based Suppression Subtractive
Hybridization ..................................... 195
3.3. Molecular Basis of Variability of Viral Pathogens ........ 196
3.3.1. Immunological Techniques .......................... 196
3.3.2. Nucleic Acid-Based Techniques ..................... 200
3.4. Molecular Basis of Variability of Viroid Pathogens ....... 208
Appendix 1: Microsatellite-Primed (MP) Polymerase Chain
Reaction for DNA Fingerprinting (Ma and Michailides
2005) .................................................... 209
Appendix 2: Amplified Fragment Length Polymorphism (AFLP)
Analysis of Pythium spp. (Garzon et al. 2005) ............ 210
References .................................................... 211
Glossary ...................................................... 227
Index ......................................................... 245
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