"This up-to-date, comprehensive book covers the practicalities of working with and studying soil and plant nematodes. Written by an international team of experts, it includes traditional and new methodologies, as well as sections on behavioural and physiological assays, and cytogenetic, biochemical and molecular biology techniques"--.

• Cover
• Techniques for Work with Plant and Soil Nematodes
• Copyright
• Contents
• About the Editors
  • Roland N. Perry
  • David J. Hunt
  • Sergei A. Subbotin
• Contributors
• Preface
• 1 Sampling
  • 1.1 Introduction: The Purpose of Sampling
  • 1.2 Sampling Strategies
    • 1.2.1 Diagnostic sampling
      • 1.2.2 Sampling for detection
      • 1.2.3 Sampling for density estimates
  • 1.3 Sampling in Relation to Phytosanitary Requirements
  • 1.4 Soil Sampling for Endoparasitic Nematodes
  • 1.5 Soil Sampling for Ectoparasitic Nematodes
  • 1.6 Sampling for Entomopathogenic Nematodes from Soil Samples: Isolation and Baiting Techniques
    • 1.6.1 Baiting method
  • 1.7 Examples of Sampling Protocols for Pine Wood Nematodes and Potato Cyst Nematodes
    • 1.7.1 Sampling for B. xylophilus
    • 1.7.2 Sampling for G. rostochiensis and G. pallida
  • 1.8 Sampling Tools
  • 1.9 Handling and Storage of Samples
  • 1.10 References
• 2 Methods for Nematode Extraction
  • 2.1 Introduction
  • 2.2 Centrifugal Flotation
    • 2.2.1 Materials
    • 2.2.2 Procedure
  • 2.3 Extraction of Cysts
    • 2.3.1 Baunacke method
      • 2.3.1.1 Materials
      • 2.3.1.2 Procedure (Fig. 2.3)
    • 2.3.2 Fenwick can
      • 2.3.2.1 Materials
      • 2.3.2.2 Procedure
    • 2.3.3 Schuiling centrifuge
      • 2.3.3.1 Materials
      • 2.3.3.2 Procedure
    • 2.3.4 Seinhorst elutriator
      • 2.3.4.1 Materials
      • 2.3.4.2 Procedure
    • 2.3.5 Wye washer
      • 2.3.5.1 Materials
      • 2.3.5.2 Procedure
  • 2.4 Extraction of Vermiform Nematodes from Soil
    • 2.4.1 Baermann method
      • 2.4.1.1 Materials
      • 2.4.1.2 Procedure
    • 2.4.2 Cobb decanting and sieving method and the Flegg-modified Cobb method
      • 2.4.2.1 Materials
      • 2.4.2.2 Procedure
    • 2.4.3 Two-flask method
      • 2.4.3.1 Materials
      • 2.4.3.2 Procedure
    • 2.4.4 Oostenbrink elutriator
      • 2.4.4.1 Materials
      • 2.4.4.2 Procedure
    • 2.4.5 Seinhorst elutriator
      • 2.4.5.1 Materials
      • 2.4.5.2 Procedure
  • 2.5 Extraction of Nematodes from Plant Parts
    • 2.5.1 Direct observation
    • 2.5.2 Incubation
    • 2.5.3 Baermann method and mistifier
    • 2.5.4 Maceration
    • 2.5.5 Eggs
  • 2.6 Acknowledgement
  • 2.7 References
• 3 Estimating Numbers
  • 3.1 Introduction
  • 3.2 Estimating Nematodes in Plant Tissue
    • 3.2.1 Staining nematodes inside plant tissues with acid fuchsin
  • 3.3 Estimating Nematodes in Liquids
    • 3.3.1 Maintaining a reproducible accuracy in aliquots
    • 3.3.2 Using optical methods to estimate numbers in liquids
    • 3.3.3 Applying digital image analysis to facilitate optical methods
    • 3.3.4 Counting nematodes using light absorbance
    • 3.3.5 Counting nematodes using quantitative PCR
  • 3.4 Estimating Numbers of Cysts
    • 3.4.1 Using extraction techniques with high efficiency to separate organic debris
    • 3.4.2 Using organic solvents to separate cysts from organic float
    • 3.4.3 Mechanical separation of cysts (Globodera spp.) from organic float
    • 3.4.4 Direct visual enumeration of cysts attached to roots using transparent boxes
    • 3.4.5 Counting females using fluorescence
  • 3.5 Estimating Eggs and Juveniles Isolated from Cysts
    • 3.5.1 Estimating content of cysts using a homogenizer
    • 3.5.2 Estimating content of individual cysts by preparing a squash mount
  • 3.6 Estimating Gall Index
  • 3.7 Estimating Number of Egg Masses
    • 3.7.1 Staining egg masses with red food colour
    • 3.7.2 Estimating egg numbers isolated from egg masses
  • 3.8 References
• 4 Screening Plants for Resistance/Susceptibility to Plant-parasitic Nematodes
  • 4.1 Introduction
  • 4.2 Selection of Nematode Population
  • 4.3 Collection and Preparation of Inoculum
    • 4.3.1 Inoculum of cyst nematodes
    • 4.3.2 Inoculum of root-knot nematodes
    • 4.3.3 Inoculum of root-lesion and burrowing nematodes
    • 4.3.4 Inoculum of stem and bulb, and foliar nematodes
    • 4.3.5 Inoculum of semi-endoparasitic and ectoparasitic nematodes
  • 4.4 Setting up Resistance Screening Assays
    • 4.4.1 Pot type and growing medium
    • 4.4.2 Inoculation
    • 4.4.3 Harvest and data acquisition
  • 4.5 Marker-assisted Selection
  • 4.6 References
• 5 Handling, Fixing, Staining and Mounting Nematodes
  • 5.1 Introduction
  • 5.2 Handling Nematodes
    • 5.2.1 Nematode picks
    • 5.2.2 Nematode picking technique
    • 5.2.3 Pipettes
    • 5.2.4 Forceps
    • 5.2.5 Aspirator
    • 5.2.6 Micro-chambers
    • 5.2.7 Useful dishes
  • 5.3 Killing and Fixing Techniques
    • 5.3.1 Permanent mounts
      • 5.3.1.1 Seinhorst slow method
      • 5.3.1.2 Ryss express method
      • 5.3.1.3 Simple evaporation method
    • 5.3.2 Fixing for SEM and TEM
  • 5.4 Processing Nematodes for Temporary and Permanent Mounts
    • 5.4.1 Temporary mounts
      • 5.4.1.1 Support rings
      • 5.4.1.2 Agar pads
    • 5.4.2 Slide making
      • 5.4.2.1 Glass wool supports or Ballotini beads
      • 5.4.2.2 Fingernail polish or paint rings
      • 5.4.2.3 Wax support rings
  • 5.5 Mounting en face Views and Cross-sections
  • 5.6 Staining Nematode Structures and Secretions
    • 5.6.1 Vital stains
  • 5.7 Staining Nematodes in Plant Material
    • 5.7.1 Staining endoparasitic nematodes in roots
    • 5.7.2 Staining egg masses of root-knot nematodes
    • 5.7.3 Histology staining
    • 5.7.4 Staining techniques
  • 5.8 References
• 6 Culturing Techniques
  • 6.1 Introduction
  • 6.2 Collecting Nematodes to Start a Culture
  • 6.3 Axenization and Gnotobiology in in Vitro Cultures
  • 6.4 Cleaning and Decontamination of Plant-parasitic Nematodes
    • 6.4.1 Cleaning of nematodes in water
    • 6.4.2 Surface sterilization
      • 6.4.2.1 Inorganic disinfectants
      • 6.4.2.2 Surface sterilization and decontamination using agar
      • 6.4.2.3 Surface sterilization assisted by glass beads and glass fibre
      • 6.4.2.4 Surface sterilization using Pluronic F127 gel
    • 6.4.3 Antiseptics and disinfectants for Tylenchida and Aphelenchida
      • 6.4.3.1 Hibitane diacetate
      • 6.4.3.2 Cetavlon
      • 6.4.3.3 Hydrogen peroxide
      • 6.4.3.4 Antibiotics
      • 6.4.3.5 Mercury chloride
      • 6.4.3.6 Disinfectants and sterilants for Dorylaimida and Diphtherophorida
    • 6.4.4 Testing for contamination by microorganisms after surface sterilization
  • 6.5 Plant Tissue Culture
    • 6.5.1 Roots of whole seedlings on agar
      • 6.5.1.1 Tomato seedlings for in vitro culturing of Meloidogyne javanica
      • 6.5.1.2 Ditylenchus angustus cultured on axenic rice seedlings
      • 6.5.1.3 Xiphinema index cultured on tomato and fig seedlings
  • 6.6 Explant Culture
    • 6.6.1 Tomato root explants
    • 6.6.2 Maize root explants
    • 6.6.3 Cucumber excised roots
    • 6.6.4 Cyst nematodes on excised roots
    • 6.6.5 Leaf cultures
    • 6.6.6 Potato tuber, potato plug and storage root cultures
  • 6.7 Callus Tissue Culture
    • 6.7.1 Alfalfa callus
    • 6.7.2 Banana fruit callus
    • 6.7.3 Rice and wheat callus
    • 6.7.4 Groundnut callus
    • 6.7.5 Carrot callus
  • 6.8 Carrot Disc Culture
  • 6.9 In Vitro Banana Plantlets
  • 6.10 Gnotobiotic Culture of Whole Plants
    • 6.10.1 Seed pouches
  • 6.11 Genetically Engineered Plants
  • 6.12 Model Plants
    • 6.12.1 Arabidopsis thaliana
    • 6.12.2 Lotus japonicus
  • 6.13 Pot Cultures
    • 6.13.1 Pot cultures of Meloidogyne and Nacobbus
    • 6.13.2 Pot cultures of cyst nematodes
    • 6.13.3 Storage of viable cysts of Heterodera spp.
  • 6.14 Production of Entomopathogenic Nematodes
    • 6.14.1 In vivo culturing
      • 6.14.1.1 Production of the host insect, Galleria mellonella
      • 6.14.1.2 Counting dauer juveniles
    • 6.14.2 In vitro culturing
      • 6.14.2.1 Isolation of symbiotic bacteria and production of bacterial stock cultures
      • 6.14.2.2 Identification of contaminants and phase variants of symbiotic bacteria
      • 6.14.2.3 Production of bacteria-free nematodes
    • 6.14.3 Establishment of monoxenic cultures
      • 6.14.3.1 Monoxenic cultures for mass production
  • 6.14.4 Culture of single nematodes or pairs in hanging drops
  • 6.15 Cryopreservation of Nematodes
    • 6.15.1 Protocol for cryopreservation and revival of nematodes
  • 6.16 Acknowledgements
  • 6.17 References
• 7 Measuring Nematodes and Preparation of Figures
  • 7.1 Introduction
  • 7.2 Calibration
  • 7.3 Direct Measurement
    • 7.3.1 Computer measuring
    • 7.3.2 Drawing apparatus
    • 7.3.3 Drawing nematodes for measurement
    • 7.3.4 Nematode measurement ruler
    • 7.3.5 Measurement criteria
  • 7.4 Processing Measurement Data and Preparing for Publication
    • 7.4.1 Cobb formula
    • 7.4.2 Preparing figures for publication
      • 7.4.2.1 Line drawings
      • 7.4.2.2 Greyscale
      • 7.4.2.3 Colour
      • 7.4.2.4 Combination artwork
      • 7.4.2.5 Labelling
      • 7.4.2.6 Encapsulated PostScript (EPS) files
      • 7.4.2.7 Layers
      • 7.4.2.8 Resolution and apparent image quality
  • 7.5 Acknowledgement
  • 7.6 References
• 8 Electron Microscopy Techniques
  • 8.1 Transmission Electron Microscopy
    • 8.1.2 Primary fixation
    • 8.1.3 Chemical fixation
    • 8.1.4 Infiltration and embedding: epoxy resins
    • 8.1.5 Specimen preparation: acrylic resins
    • 8.1.6 Cryopreparation methods for TEM
    • 8.1.7 High-pressure freezing (HPF)
    • 8.1.8 Self-pressurized rapid freezing
    • 8.1.9 Freeze substitution
    • 8.1.10 Preparation of sections for TEM observations
    • 8.1.11 Transmission electron microscopy
    • 8.1.12 Concluding remark on TEM applications
    • 8.1.1 Before fixation
  • 8.2 Scanning Electron Microscopy
    • 8.2.1 Preparation of nematodes for SEM
    • 8.2.2 Fixation
    • 8.2.3 Drying
      • 8.2.3.1 Critical-point drying
      • 8.2.3.2 Air drying
      • 8.2.3.3 Acetone drying
      • 8.2.3.4 Freeze drying
      • 8.2.3.5 Freeze substitution
      • 8.2.3.6 Resin infiltration
    • 8.2.4 Glycerin embedded specimens
    • 8.2.5 Cryofracture
    • 8.2.6 Mounting and coating
  • 8.3 Acknowledgements
  • 8.4 References
• 9 Behavioural and Physiological Assays
  • 9.1 Introduction
  • 9.2 Attraction/Repulsion Plate Assays
  • 9.3 Pluronic Gel Assays
  • 9.4 Movement Assays
    • 9.4.1 Response to non-volatiles
    • 9.4.2 Response to volatiles
  • 9.5 Electrophysiology
  • 9.6 Stylet Activity
  • 9.7 Water Content Changes
  • 9.8 Oxygen Consumption Assays
  • 9.9 Collecting Female Sex Pheromone, Virgin Females and Males
  • 9.10 Viability Tests
    • 9.10.1 Staining
      • 9.10.1.1 Vital/non-vital stains
      • 9.10.1.2 Lipid reserves
    • 9.10.2 Hatching
      • 9.10.2.1 Standard hatching assays
      • 9.10.2.2 Root diffusates and other hatching agents
      • 9.10.2.3 Fluorescence assay
      • 9.10.2.4 Adenosine triphosphate assay
      • 9.10.2.5 Trehalose assay
    • 9.10.3 Other methods
  • 9.11 References
• 10 Staining Chromosomes
  • 10.1 Introduction
  • 10.2 Staining Chromosomes of Root-knot Females
    • 10.2.1 Propionic-orcein staining of root-knot females
      • 10.2.1.1 Preparation of propionic-orcein stain
      • 10.2.1.2 Selecting and obtaining nematode material for cytological study
    • 10.2.2 Preparation of smears to study oogonial divisions and early stages of maturation of oocytes
      • 10.2.2.1 Hydrolysis
      • 10.2.2.2 Fixation
      • 10.2.2.3 Staining
      • 10.2.2.4 Mounting
      • 10.2.2.5 Examination of temporary preparations
    • 10.2.3 Summary of procedures for staining Meloidogyne chromosomes with propionic-orcein
  • 10.3 Staining Chromosomes of Free-living Nematodes
    • 10.3.1 Propionic-orcein staining (Hechler, 1970)
    • 10.3.2 DAPI fluorescent nuclear staining (Hasegawa et al., 2006)
  • 10.4 Staining Chromosomes of Giant Cells
    • 10.4.1 Seed germination pouch cultures
    • 10.4.2 Glasshouse cultures
    • 10.4.3 Feulgen staining
    • 10.4.4 Observation of stained material
  • 10.5 References
• 11 Isoelectric Focusing of Proteins
  • 11.1 Introduction
  • 11.2 Isoelectric Focusing of Proteins for Diagnostics of Cyst Nematodes
    • 11.2.1 Sample preparation and loading
    • 11.2.2 Isoelectric focusing run
    • 11.2.3 Gel staining
    • 11.2.4 Interpretation of results
  • 11.3 Isoelectric Focusing of Isozymes for Diagnostics of Root-knot Nematodes
    • 11.3.1 Preparation of egg-laying females for isozyme extraction
    • 11.3.2 Gel preparation
      • 11.3.2.1 Resolving gel (8%); reagents and preparation
      • 11.3.2.2 Stacking gel (4%); reagents and preparation
      • 11.3.2.3 Electrode (running) buffer
    • 11.3.3 Running the gel
    • 11.3.4 Isozyme extraction and gel loading
    • 11.3.5 Electrophoresis
    • 11.3.6 Staining
      • 11.3.6.1 Staining solution for esterase
      • 11.3.6.2 Staining solution for malate dehydrogenase
      • 11.3.6.3 Staining solution for glutamate-oxaloacetate transaminase (Harris and Hopkinson, 1976)
      • 11.3.6.4 Staining solution for superoxide dismutase (Ravindranath and Fridovich,1975 cited by Esbenshade and Triantaphyllou (1985b))
      • 11.3.6.5 Fixative solution (20% ethanol and 10% glycerin)
  • 11.4 References
• 12 Molecular Identification of Nematodes Using PolymeraseChain Reaction (PCR)
  • 12.1 Introduction
  • 12.2 Preservation of Nematodes for Molecular Studies
    • 12.2.1 Dry preservation
    • 12.2.2 Ethanol (ethyl alcohol) preservation
    • 12.2.3 DESS preservation
    • 12.2.3.1 Protocol for preparing DESS solution
  • 12.3 DNA Extraction
    • 12.3.1 Protocols for DNA extraction from nematodes
  • 12.4 PCR Technique
    • 12.4.1 Protocols for PCR
    • 12.4.2 Gel electrophoresis
  • 12.5 PCR-Restriction Fragment Length Polymorphism
  • 12.6 Real-time PCR
  • 12.7 Problems with PCR-based Methods
  • 12.8 PCR Purification
  • 12.9 Cloning of PCR Products
  • 12.10 DNA Sequencing
  • 12.11 References
• 13 Isolation and Characterizationof Tandem Repeats in Nematode Genomes
  • 13.1 Introduction
  • 13.2 Bench Strategies for the Identification of Tandem Repeats
    • 13.2.1 The case of satellite DNA
    • 13.2.2 The case of microsatellites
  • 13.3 In silico Identification of Tandem Repeats
  • 13.4 The Contribution of Tandem Repeats as Molecular Markers in Nematodes
    • 13.4.1 Satellite DNA-based species-specific diagnostic tools
    • 13.4.2 Microsatellites and population genetic studies in plant-parasitic nematodes
  • 13.5 Concluding Remarks
  • 13.6 References
• 14 Characterization of Nematode Mitochondrial Genomes
  • 14.1 Introduction
  • 14.2 PCR Amplification of Mitochondrial Genomes
  • 14.3 Protocols for PCR
    • 14.3.1 Amplification of partial mt genes/short DNA fragments (< 3 kb)
    • 14.3.2 Long-range PCR
  • 14.4 Cloning and Sequencing
  • 14.5 Mitochondrial Genome Annotation and Gene Identification
    • 14.5.1 Protocols for mitochondrial genome annotation
    • 14.5.2 Important information after automated annotation
  • 14.6 References
• 15 Phylogenetic Analysis of DNA Sequence Data
  • 15.1 Introduction
  • 15.2 Visualization and Editing of Raw Sequence Data
  • 15.3 Sequence Assembling
  • 15.4 Similarity Search in a Sequence Database
  • 15.5 Sequence Retrieval from the Database
    • 15.5.1 Multiple alignment
    • 15.5.2 Sequence alignment editing
  • 15.6 File Format Converting
  • 15.7 Selection of Model of Sequence Evolution
    • 15.7.1 jModelTest: A tool to select the best-fit model of nucleotide substitution
  • 15.8 Phylogenetic Analysis with PAUP*
    • 15.8.1 Distance method
    • 15.8.2 Maximum parsimony
    • 15.8.3 Maximum likelihood
  • 15.9 Phylogenetic Analysis with MrBayes
  • 15.10 Visualization of Phylogenetic Trees
  • 15.11 Sequence Submission
  • 15.12 References
• Index
• Back Cover