Laboratory and Field Manual for Plant Nematology/ P. Parvatha Reddy

1.NEMATOLOGYEQUIPMENT 1
1.1. LaboratoryEquipment 1
1.1.1. Furniture and Fittings 1
1.1.2. Microscopesand Equipment 3
1.2. Field Equipment 5
1.3. Library 5
1.3.1. Books 5
1.3.2. Journals 7
1.3.3. Internet Websites 8
2.MICROSCOPE 9
2.1. Stereoscopic Microscope 9
2.1.1. Illumination ofDissecting Microscope 9
2.1.2. Use 10
2.2. Compound Microscope 11
2.2.1. Components 11
2.2.2. Use 13
2.2.3. Alignment and Care 13
2.2.4. Drawing and MeasuringofNematodes 14
2.3. scanning electron Microscope(SEM) 18
2.3.1. AdvantagesofSEM 18
2.3.2. Use 19
3.SAMPLING NEMATODEPOPULATIONS 21
3.1. SamplingProcedures' 21
3.1.1. Detection and Survey 21
3.1.2. Diagnosis or Detection 21
3.1.3. Advisory 22
3.1.4. Research 22
3.2. Collection of samples 22
3.2.1. sampling equipment 22
3.2.2. Time of sampling 23
3.2.3. Sample Size 23
3.2.4. SamplingPatterns 23
3.3. Care and Conditioning ofSoil and plant tissue Samples 25
3.3.1. Handling of samples during Collection and Transit 25
3.3.2. Storage 25
3.3.3. Storage Temperature and Moisture 25
3.3.4. Storage Time 2$
3.4. Fact Sheet 26
3.4.1. Nematode Survey 2uid Collection 26w
^ COTltCFltS \f.
4.EXTRACTION OF NEMATODESFROMSOIL 29
4.1. Cotton Wool Filter Technique 30
4.2. Decanting and Sieving Technique 31
4.3. Simple Extraction Method for Large Nematodes like 33
Xiphinema and Longidorus
4.4. Two-Flask Technique
4.5. Elutriation Techniques
4.5.1. Gostenbrink Elutriation System
4.5.2. Seinhorst Elutriation System
4.6. Centrifugal Flotation Technique
4.7. Estimating Nematodesin Aqueous Suspensions
4.8. British Standard Sieves
5.EXTRACTION OFCYSTNEMATODESFROMSOIL
5.1. A Simple Extraction Method
5.2. Acetone or Acetone: Carbon Tetrachloride Method
5.3. Paper Strip Method
5.4. Kirchner's Funnel Method
5.5. Modified Fenwick Can Method
5.6. DPI Cyst Separation Technique
5.7. Gostenbrink's Flotation Apparatus
5.8. Steinhorst's Extraction Apparatus for Cystsfrom Moist Soil
5.9. Reinmuth's Centrifuging Method
5.10. Centrifugal Flotation Technique
5.11. Separation ofCystsfrom Admixtures
5.12. Estimation ofEgg contentofCysts
5.13. HatchingTests on Heterodera/Globodera Cysts
5.14. Perspex Slides for Mounting Cyst Nematodes
6.EXTRACTIONOFNEMATODESFROMPLANTMATERIAL
6.1. DissectingPlant Materialien Water
6.2. Modified Baermann FunnelTechnique
6.3. MacerationyFiltration Technique
6.4. RootIncubation Technique
6.5. Mistifier Technique
6.6. A Centrifugal Flotation Method for Recovering Nematodes'
and Eggs from Plant Tissue
6.7. Modified Centrifugal Flotation Method
6.8. Maceration-sedimentation Technique for Recovery of
Rhadinaphelenchuscocophilusfrom CoconutPalm Tissue
6.9. Extraction ofSedentary Nematodes and Eggs
6.10.Mechanical Maceration Technique
6.11.Enzjrme and Chemical Maceration Technique
6.12.Fact Sheet
6.12.1. ProcessingofRootand Soil SamplesLaboratory&Field ManualforPlantNematology xi
7.EXTRACTION OF EGGS 73
7.1. From Roots 73
7.1.1. With Few Exposed Eggs 73
7.1.2. With ManyExposed Eggs ' 73
7.2. From Soil 75
7.2.1. Root-knot Nematode Eggs 75
7.2.2. Free-living Nematode Eggs 76
7.3. Hatching £ind Collection of Meloidogyne spp.Juveniles 77
7.4. Procedure for Collection,Extraction and Calibration of 77
Root-knot Nematode Eggsfor Inoculum
8.HANDLING NEMATODES 79
8.1. Pickingand TransferringIndividualNematodes 79
8.2. Preparing SharplyPointed Metal Needles 79
8.3. Anaesthetization 80
8.4. Killingand Fixing 80
8.4.1. Fixatives 82
8.4.2. Properties ofMostImportant Fixatives 83
8.5. Preparation of nematode Moments 84
8.5.1. Mounting Nematodes in Water 84
8.5.2. Temporary Mounts 85
8.5.3. Semi-permanent mounts 85
8.5.4. Permanent Mounts 86
8.6. Glycerol Mounts for Free-living Nematodes 89
8.7. Aluminium Double Cover Slip Slide 90
8.8. Fixation and Preservation of plant Material 90
8.9. Preparation ofPosterior Cuticular Pattern of root-knot 91
Nematodes
8.10. Preparation ofVulval Cone of cyst Nematodes 93
8.11. Preparation of Face View ofNematodes 94
8.12. Preparation ofDissected Specimens 95
8.12.1. Dissecting Stylets ofMeloidogyne Females 95
8.12.2. Dissecting Stylets ofMe/oido^yne Males and 97
Second-stage juveniles
8.12.3. Dissecting Stylets of other Nematode Genera 97
8.12.4. Dissecting Spicules 97
8.13. Prep£iration of issues and Cells 98
8.13.1. Dissection of sperm from Seminal Vesicle ofMale 98
8.13.2. Dissection of Sperm from Inseminated Females 98
9.STAINING 101
9.1. Staining ofRoots 101
9.1.1. Staining by Fleming's Reagent 101
9.-1.2. Staining with Picric acid and Aniline Blue for 102
Hardwood Roots
9.1.3. Staining with Bromophenol or Bromothymol Blue 102i
xii Contents |
5;
9.1.4. Staining in Aceto-osmium 103 |
9.1.5. Staining in Lugol's Solution ' 103
9.1.6. Sodium Hypochlorite(NaOCl)-Acid Fuchsin Method|103
9.1.7. Differential StainingofEndo-parasitic nematodes 104
9.2. Staining Shoots 104
9.2.1. Staining with Cotton Blue or Acid Fuchsine in Lacto- 104
phenol
9.2.2. Staining with Acid Fuchsin-Glycerol-Lactic Acid 105
9.2.3. Staining with Fleming's Reagent 105
9.3. Stains for Distinguishing Live from Dead Nematodes 106
9.3.1. Staining with New BlueR 106
9.3.2. Staining with Chrysoidin 106
9.3.3. Staining with PhloxineB 106
9.3.4. StguningwithEosin-Y 106
9.3.5. Staining with Acridine Orange 106
9.4. Specialized Staining 107
9.4.1. Staining with Acetic Orcein 107
9.4.2. Staining with Gold Chloride 107
9.4.3. Staining with Silver Nitrate 107
9.4.4. Silver Staining for Faint Surface Structures of 107
Nematodes
9.4.5. Staining of eggs and Egg Sacs with Phloxine B 108
10.MICROTOMESECTIONING 109
10.1. Killing and Fixation 110
10.2. Dehydration HI
10.2.1. Dehydration by Non-solventsofParaffin 111
10.2.2. Dehydration by Solvents ofParaffin 111
10.3. Infiltration and Embedding 112
10.4. Microtome Sectioning 112
10.5. Staining 113
11.CULTURINGNEMATODES 117
11.1. SterilizingNematodes 117
11.2. SterilizingEggMasses 119
11.3. SterilizingSeeds 120
11.4. Mass Culturing of mushroom Nematode{Ditylenchus 121
mycetophagous)
11.5. Mass Culturing oiAphelenchoidesritzemabosi 122
11.6. Mass Culturing ofDitylenchus destructor 122
11.7. Mass Culturing ofGlobodera rostochiensis and G.pallida 123
11.8. Mass Culturing of Meloidogyne spp.on Excised Tomato Roots 124
11.9. Mass Multiplication of root-knot Nematodes on Tomato Plants 131
11.10.CultxiringofRadopholussimilis andPratylenchusspp.on Carrot 132
Discs
11.11.A Culturing Method for Nematodes on an Aerated Nutrient 137
AgarMixtureLaboratory&Field ManualforPlantNematology xiii
12.SCREENING OF GERMPLASM 141
12.1. Screening ofBanana Germplasm against Migratory
endoparasitic Nematodes(Pratylenchus coffee and
Radopholussimilis)under Greenhouse Conditions 141
12.1.1. Equipment and Materials 141
12.1.2. Preparation of Plant Material 142
12.1.3. Extraction and Inoculation with Nematodes 142
12.1.4. Data Collection 143
12.2. Screening ofBanana Germplasm against root-knot 147
Nematodes{Meloidogyne spp.)under Greenhouse
Conditions
12.2.1. Equipment and Materials 147
12.2.2. Preparation of plans Material 147
12.2.3. Extraction and Inoculation with Nematodes 148
12.2.4. Data Collection 148
12.3. Screening ofBanana Germplasm againstNematodes 151
imderField Conditions
12.3.1. Equipment and Materials 151
12.3.2. Preparation of Plant Material 152
12.3.3. Site Selection and Preparation 152
12.3.4. Transfer to Field 152
12.3.5. Data Collection 153
13.MASSPRODUCTIONOFBIOLOGICALCONTROLAGENTS 157
13.1. Bacteria 157
13.1.1. Pasteuriapenetrans 157
13.1.2. Pseudomonasfluorescens 160
13.1.3. Rhizobacteria(Psewdo/nonasspp.,BaciZZus spp., 161
Serratia spp.,etc.)
13.1.4. EndophyticBacteria 162
13.1.5. Streptomyces avermitilis(Avermectins) 164
13.2. Fungi 168
13.2.1. Nematode Trapping or Predatory Fungi 168
13.2.2. Trichoderma viride and T.harzianum 172
13.2.3. Paecilomyceslilacinus 177
13.2.4. Arbuscular mycorrhizal fungi(AMF) 178
13.2.5. EnrichmentofFYMwithBioagentsCTrZcAocZerma 182
harzianum^Paecilomyceslilacinus)
13.2.6. Seed Biopriming/Solid MatrixPriming 184
13.2.7. Formulation ofAlginated Pellets ofBiocontrol Agents 184
13.2.8. Preservation ofBiocontrol Agents 184
13.2.9. Production ofBioagent Colonized Seedlings 187
13.3. Entomopathogenic Nematodes(EPN) 187
13.3.1.In vivo Production 188
13.3.2. In vitro Production 191
13.3.3. Formulation 193XIV
14.roENTIFICATIONOFCOMMON GENERA OF
PLANTPARASITICANDENTOMOPATHOGENIC
NEMATODES
14.1. Plant Parasitic Nematodes
14.1.1. Tylenchorhynchus spp.(StuntNematodes)
14.1.2. Ditylenchus spp.(Stem and Bulb Nematodes)
14.1.3. Belonolaimus spp.(Sting Nematodes)
14.1.4. Hoplolaimusspp.(Lance Nematodes)
14.1.5. Helicotylenchus spp.(Spiral Nematodes)
14.1.6. Rotylenchulus spp.(Reniform Nematodes)
14.1.7. Pratylenchus spp.(Lesion Nematodes)
14.1.8. Hirschmanniella spp.(Rice-rootNematodes)
14.1.9. Ra4opholus spp.(Burrowing Nematodes)
14.1.10. Meloidogyne spp.(Root-knot Nematodes)
14.1.11. Heterodera and Globodera spp.(CystNematodes)
14.1.12. Criconemoidesspp.(Ring Nematodes)
14.1.13. HemicriconeTnoidBs spp.(Sheathoid Nematodes)
14.1.14. Hemicycliophora spp.(Sheath Nematodes)
14.1.15. Tylenchulus spp.(Citrus RootNematodes)
14.1.16. Paratylenchus spp.(Pin Nematodes)
14.1.17. Anguina spp.(Seed Gall Nematodes)
14.1.18. Aphelenchoides spp.(Bud and LeafNematodes)
14.1.19. Ipanema spp.(Dagger Nematodes)
14.1.20. Longidorus spp.(Needle Nematodes)
14.1.21. Trichodorus spp.(Stubby-root nematodes)
14.2. Entomopathogenic Nematodes
14.2.1. Steinernema
14.2.2. Heterorhabditis
15.TECHNIQUESUSEDTOCONTROLPLANTNEMATODES
15.1. Soil Solarization
15.2. Soil Fumigation with Methyl Bromide
15.2.1. Nursery-Beds
15.2.2. Potting Soil
15.2.3. Phjrtotoxicity Test