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Tables of Contents for Practical Plant Virology
Chapter/Section Title
Page #
Page Count
Part I Virus Inoculation
1
34
Introduction I Virus Inoculation
3
2
Protocol 1 Mechanical Inoculation of Plants
5
9
Protocol 2 Inoculation of Protoplasts Isolated from Leaves
14
15
2.1 Inoculation of Cowpea Protoplasts with Cowpea Mosaic Comovirus in PEG
15
6
2.2 Inoculation of Barley Protoplasts with Brome Mosaic Bromovirus-RNA by Electroporation
21
8
Protocol 3 Differential Temperature Treatment of Infected Plants
29
3
Exercise 1 Infectivity of Tobacco Mosaic Tobamovirus
32
3
Part II Symptomatology
35
30
Introduction II Symptomatology
37
5
Protocol 4 Description of External Symptoms
42
6
Protocol 5 Determination of Internal Symptoms
48
9
5.1 Staining with Trypan Blue
50
1
5.2 Staining with Phloxine-Methylene Blue
51
1
5.3 Staining with Methyl Green-Pyronin
52
1
5.4 Staining with Azure A
53
1
5.5 Staining with Orange-Green (O-G)
54
3
Exercise 2 Systemic Acquired Resistance (SAR)
57
3
Exercise 3 Demonstration of Phloem Necrosis in Plants Affected by Potato Leafroll
60
3
Exercise 4 Inclusions in Hair Cells of Leaves of Tobacco Infected with Tobacco Mosaic Tobamovirus
63
2
Part III Infectivity Assays
65
32
Protocol 6 Infectivity Assay on Local-Lesion Hosts
67
11
6.1 Infectivity Assay on Detached Leaves
68
6
6.2 Infectivity Assay on Nondetached Leaves
74
4
Protocol 7 Infectivity Assay on Hosts Producing Starch Lesions
78
4
Protocol 8 Infectivity Assay on Hosts Producing Systemic Symptoms Only
82
4
Exercise 5 Effect to Temperature on the Susceptibility of Plants
86
2
Exercise 6 Effect of Abrasives on the Infection
88
2
Exercise 7 Effect of Inhibitors on the Infection
90
2
Exercise 8 Effect of Light on the Susceptibility of Plants
92
2
Exercise 9 Effect of Time of the Day on the Susceptibility of Plants
94
3
Part IV Infectivity of Virus in Crude Sap
97
16
Introduction IV Infectivity of Virus in Crude Sap
99
1
Protocol 9 Determination of the Dilution End-Point
100
2
Protocol 10 Determination of the Thermal Inactivation Point
102
3
Protocol 11 Determination of the Longevity In Vitro
105
3
Exercise 10 Determination of the Dilution End-Point
108
2
Exercise 11 Determination of the Thermal Inactivation Point
110
1
Exercise 12 Determination of the Longevity In Vitro
111
2
Part V Transmission of Viruses
113
76
Introduction V Transmission of Viruses
115
1
Protocol 12 Virus Transmission by Grafting
116
9
12.1 Bud Grafting
117
3
12.2 Wedge Grafting
120
3
12.3 Tuber Grafting
123
2
Protocol 13 Virus Transmission Through Soil
125
3
13.1 Testing of Roots for the Presence of Virus
125
1
13.2 Use of Bait Plants to Reveal the Presence of Viruliferous Vectors
126
2
Protocol 14 Virus Transmission by Nematodes
128
15
Protocol 15 Virus Transmission by Fungi
143
5
Protocol 16 Virus Transmission by Aphids
148
11
16.1 Nonpersistent Transmission
151
1
16.2 Persistent Transmission of a Circulative Virus
152
2
16.3 Acquisition of a Circulative Virus by Aphids Feeding on Artificial Diets Through a Membrane
154
5
Protocol 17 Transmission by Leafhoppers (Cicadellidae)
159
10
17.1 Transmission of Semipersistent Viruses
162
1
17.2 Nontransovarial Transmission of a Circulative-Propagative Virus
163
1
17.3 Transovarial Transmission of a Circulative-Propagative Virus
164
1
17.4 Glasshouse Mass Screening for Resistance to Rice Tungro Disease
165
4
Protocol 18 Transmission by Whiteflies (Aleyrodidae)
169
5
Protocol 19 Transmission by Thrips (Thripidae)
174
4
Exercise 13 Wedge-Grafting of Tobacco
178
2
Exercise 14 Separation of a Nonpersistently and a Persistently Transmitted Virus by Differential Transmission
180
4
Exercise 15 Visualisation of Salivary Sheaths Formed by Aphids
184
2
Exercise 16 Separation of a Semipersistently Transmitted and a Circulative-Propagative Virus by Differential Transmission
186
3
Part VI Maintenance of Virus Isolates
189
14
Introduction VI Maintenance of Virus Isolates
191
1
Protocol 20 Freezing
192
2
Protocol 21 Dehydration
194
4
Protocol 22 Freeze-Drying
198
2
Protocol 23 Short-Term Storage
200
3
Part VII Production of Virus-Free Material from Infected Plants
203
16
Introduction VII Production of Virus-Free Material from Infected Plants
205
2
Protocol 24 Heat Treatment
207
2
Protocol 25 Meristem-Tip Culture
209
7
Protocol 26 Heat Treatment Combined with Meristem-Tip Culture
216
3
Part VIII Virus Isolation and Purification
219
56
Introduction VIII Virus Isolation and Purification
221
11
Protocol 27 Density-Gradient Centrifugation
232
6
Protocol 28 Virus Purification Protocols
238
37
28.1 Alfalfa Mosaic Alfamovirus
240
3
28.2 Bean Yellow Mosaic Potyvirus
243
2
28.3 Beet Necrotic Yellow Vein Furovirus
245
2
28.4 Carnation Ringspot Dianthovirus
247
1
28.5 Cherry Leaf Roll Nepovirus
248
2
28.6 Cowpea Mosaic Comovirus
250
2
28.7 Cucumber Mosaic Cucumovirus
252
2
28.8 Elderberry Carlavirus
254
2
28.9 Potato Leafroll Luteovirus
256
2
28.10 Potato X Potexvirus
258
3
28.11 Sunn-Hemp Mosaic Tobamovirus
261
1
28.12 Tobacco Mosaic Tobamovirus
262
2
28.13 Tobacco Necrosis Necrovirus
264
2
28.14 Tobacco Rattle Tobravirus
266
1
28.15 Tobacco Streak Ilarvirus
267
2
28.16 Tomato Spotted Wilt Tospovirus
269
6
Part IX Physicochemical Properties of Virus Particle
275
24
Protocol 29 UV Light Absorption
277
7
Protocol 30 Buoyant Density in Caesium Chloride
284
7
Protocol 31 Particle Morphology and Size as Determined with the Electron Microscope
291
8
31.1 Leaf-Chopping or Leaf-Dip Method
291
2
31.2 Particle Size Determination
293
6
Part X Properties of Viral Components
299
18
Introduction X Properties of Viral Components
301
1
Protocol 32 Determination of the Molecular Mass of Coat Proteins by Gel Electrophoresis
302
6
Protocol 33 RNA Extraction from Purified Virus Particles
308
4
Protocol 34 Determination of the Molecular Mass of Ribonucleic Acids by Gel Electrophoresis
312
5
Part XI Serological Techniques for Detection and Identification of Viruses and Viral Products
317
60
Introduction XI Serological Techniques for Detection and Identification of Viruses and Viral Products
319
3
Protocol 35 Production of Antiserum
322
7
35.1 Immunisation of Rabbits and Acquisition of Serum
322
5
35.2 Purification of Immunoglobulins by Ammonium Sulphate Precipitation
327
2
Protocol 36 Absorption of Antiserum
329
4
36.1 Absorption with Acetone-Extracted Plant Powder
329
2
36.2 Absorption with Sap from Virus-Free Plants
331
2
Protocol 37 Chloroplast Agglutination Test
333
3
Protocol 38 Microprecipitin Test
336
5
Protocol 39 Gel Double-Diffusion Test in Plates
341
7
Protocol 40 Enzyme-Linked Immunosorbent Assay
348
15
40.1 Standard (Direct) DAS-ELISA
349
5
40.2 Two-Step DAS-ELISA (Cocktail ELISA) (Modification 1)
354
1
40.3 Penicillin-DAS-ELISA (Modification 2)
354
4
40.4 Amplified ELISA (Modification 3)
358
1
40.5 Indirect ELISA
359
4
Protocol 41 Immunoblotting Assays
363
10
41.1 Electroblot Immunoassay
363
4
41.2 Dot-Blot Immunoassay
367
4
41.3 Tissue-Blot Immunoassay
371
2
Exercise 17 Standard DAS-ELISA
373
4
Part XII Electron Microscope and Light Microscope Serology
377
24
Introduction XII Electron Microscope and Light Microscope Serology
379
1
Protocol 42 Electron Microscope Serology
380
12
42.1 Immunosorbent Electron Microscopy
380
2
42.2 Decoration
382
2
42.3 Gold Labelling
384
8
Protocol 43 Light Microscope Serology
392
9
43.1 Gold-Silver Labelling of Viral Antigen
392
3
43.2 Immunostaining
395
3
43.3 Immunofluorescence Microscopy
398
3
Part XIII Detection of Viral RNA
401
26
Introduction XIII Detection of Viral RNA
403
1
Protocol 44 Northern Blotting
404
4
Protocol 45 Northern Hybridisation
408
5
Protocol 46 Dot-Blot Hybridisation (Nucleic Acid Spot Hybridisation)
413
2
Protocol 47 Polymerase Chain Reaction
415
2
Part XIV Diagnosis of a Disease and Identification of the Causal Virus(es)
427
8
Introduction XIV Diagnosis of a Disease and Identification of the Causal Virus(es)
429
6
Appendix
435
16
Index
451