About the Editors |
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xv | |
Contributors |
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xvii | |
Preface |
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xix | |
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1 | (11) |
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1.1 Introduction: The Purpose of Sampling |
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1 | (1) |
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1 | (4) |
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1.2.1 Diagnostic sampling |
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1 | (1) |
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1.2.2 Sampling for detection |
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2 | (1) |
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1.2.3 Sampling for density estimates |
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3 | (2) |
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1.3 Sampling in Relation to Phytosanitary Requirements |
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5 | (1) |
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1.4 Soil Sampling for Endoparasitic Nematodes |
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5 | (1) |
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1.5 Soil Sampling for Ectoparasitic Nematodes |
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6 | (1) |
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1.6 Sampling for Entomopathogenic Nematodes from Soil Samples: Isolation and Baiting Techniques |
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6 | (2) |
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6 | (2) |
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1.7 Examples of Sampling Protocols for Pine Wood Nematodes and Potato Cyst Nematodes |
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8 | (1) |
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1.7.1 Sampling for B. xylophilus |
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8 | (1) |
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1.7.2 Sampling for G. rostochiensis and G. pallida |
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9 | (1) |
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9 | (1) |
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1.9 Handling and Storage of Samples |
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10 | (1) |
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10 | (2) |
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2 Methods for Nematode Extraction |
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12 | (30) |
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12 | (2) |
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2.2 Centrifugal Flotation |
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14 | (4) |
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15 | (1) |
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15 | (3) |
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18 | (9) |
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18 | (1) |
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18 | (1) |
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18 | (2) |
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20 | (1) |
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20 | (1) |
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21 | (1) |
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2.3.3 Schuiling centrifuge |
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21 | (1) |
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22 | (1) |
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22 | (1) |
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2.3.4 Seinhorst elutriator |
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23 | (1) |
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23 | (1) |
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24 | (1) |
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25 | (1) |
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26 | (1) |
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26 | (1) |
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2.4 Extraction of Vermiform Nematodes from Soil |
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27 | (11) |
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27 | (1) |
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27 | (1) |
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28 | (1) |
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2.4.2 Cobb decanting and sieving method and the Flegg-modified Cobb method |
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29 | (1) |
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29 | (1) |
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29 | (2) |
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31 | (1) |
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31 | (1) |
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32 | (1) |
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2.4.4 Oostenbrink elutriator |
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33 | (1) |
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33 | (1) |
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34 | (1) |
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2.4.5 Seinhorst elutriator |
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35 | (1) |
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36 | (1) |
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36 | (2) |
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2.5 Extraction of Nematodes from Plant Parts |
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38 | (2) |
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38 | (1) |
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39 | (1) |
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2.5.3 Baermann method and mistifier |
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39 | (1) |
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39 | (1) |
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39 | (1) |
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40 | (1) |
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40 | (2) |
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42 | (18) |
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42 | (1) |
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3.2 Estimating Nematodes in Plant Tissue |
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43 | (2) |
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3.2.1 Staining nematodes inside plant tissues with acid fuchsin |
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43 | (2) |
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3.3 Estimating Nematodes in Liquids |
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45 | (4) |
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3.3.1 Maintaining a reproducible accuracy in aliquots |
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45 | (1) |
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3.3.2 Using optical methods to estimate numbers in liquids |
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45 | (2) |
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3.3.3 Applying digital image analysis to facilitate optical methods |
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47 | (1) |
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3.3.4 Counting nematodes using light absorbance |
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48 | (1) |
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3.3.5 Counting nematodes using quantitative PCR |
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49 | (1) |
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3.4 Estimating Numbers of Cysts |
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49 | (4) |
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3.4.1 Using extraction techniques with high efficiency to separate organic debris |
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50 | (1) |
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3.4.2 Using organic solvents to separate cysts from organic float |
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50 | (1) |
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3.4.3 Mechanical separation of cysts (Globodera spp.) from organic float |
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51 | (1) |
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3.4.4 Direct visual enumeration of cysts attached to roots using transparent boxes |
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51 | (1) |
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3.4.5 Counting females using fluorescence |
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51 | (2) |
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3.5 Estimating Eggs and Juveniles Isolated from Cysts |
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53 | (1) |
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3.5.1 Estimating content of cysts using a homogenizer |
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53 | (1) |
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3.5.2 Estimating content of individual cysts by preparing a squash mount |
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54 | (1) |
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3.6 Estimating Gall Index |
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54 | (2) |
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3.7 Estimating Number of Egg Masses |
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56 | (2) |
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3.7.1 Staining egg masses with red food colour |
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56 | (1) |
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3.7.2 Estimating egg numbers isolated from egg masses |
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57 | (1) |
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58 | (2) |
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4 Screening Plants for Resistance/Susceptibility to Plant-parasitic Nematodes |
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60 | (11) |
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60 | (1) |
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4.2 Selection of Nematode Population |
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60 | (1) |
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4.3 Collection and Preparation of Inoculum |
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61 | (2) |
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4.3.1 Inoculum of cyst nematodes |
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61 | (1) |
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4.3.2 Inoculum of root-knot nematodes |
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61 | (1) |
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4.3.3 Inoculum of root-lesion and burrowing nematodes |
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62 | (1) |
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4.3.4 Inoculum of stem and bulb, and foliar nematodes |
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62 | (1) |
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4.3.5 Inoculum of semi-endoparasitic and ectoparasitic nematodes |
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63 | (1) |
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4.4 Setting up Resistance Screening Assays |
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63 | (5) |
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4.4.1 Pot type and growing medium |
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63 | (2) |
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65 | (2) |
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4.4.3 Harvest and data acquisition |
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67 | (1) |
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4.5 Marker-assisted Selection |
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68 | (1) |
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68 | (3) |
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5 Handling, Fixing, Staining and Mounting Nematodes |
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71 | (17) |
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71 | (1) |
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71 | (4) |
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71 | (2) |
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5.2.2 Nematode picking technique |
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73 | (1) |
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73 | (1) |
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73 | (1) |
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73 | (1) |
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73 | (2) |
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75 | (1) |
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5.3 Killing and Fixing Techniques |
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75 | (3) |
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76 | (1) |
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5.3.1.1 Seinhorst slow method |
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77 | (1) |
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5.3.1.2 Ryss express method |
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77 | (1) |
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5.3.1.3 Simple evaporation method |
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77 | (1) |
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5.3.2 Fixing for SEM and TEM |
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78 | (1) |
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5.4 Processing Nematodes for Temporary and Permanent Mounts |
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78 | (2) |
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78 | (1) |
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78 | (1) |
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78 | (1) |
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79 | (1) |
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5.4.2.1 Glass wool supports or Ballotini beads |
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79 | (1) |
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5.4.2.2 Fingernail polish or paint rings |
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79 | (1) |
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5.4.2.3 Wax support rings |
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79 | (1) |
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5.5 Mounting en face Views and Cross-sections |
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80 | (1) |
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5.6 Staining Nematode Structures and Secretions |
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81 | (1) |
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82 | (1) |
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5.7 Staining Nematodes in Plant Material |
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82 | (3) |
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5.7.1 Staining endoparasitic nematodes in roots |
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82 | (1) |
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5.7.2 Staining egg masses of root-knot nematodes |
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83 | (1) |
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83 | (1) |
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5.7.4 Staining techniques |
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84 | (1) |
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85 | (3) |
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88 | (44) |
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88 | (1) |
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6.2 Collecting Nematodes to Start a Culture |
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88 | (1) |
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6.3 Axenization and Gnotobiology in in Vitro Cultures |
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89 | (1) |
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6.4 Cleaning and Decontamination of Plant-parasitic Nematodes |
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90 | (6) |
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6.4.1 Cleaning of nematodes in water |
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90 | (1) |
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6.4.2 Surface sterilization |
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91 | (1) |
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6.4.2.1 Inorganic disinfectants |
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91 | (1) |
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6.4.2.2 Surface sterilization and decontamination using agar |
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92 | (1) |
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6.4.2.3 Surface sterilization assisted by glass beads and glass fibre |
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92 | (1) |
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6.4.2.4 Surface sterilization using Pluronic F127 gel |
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93 | (1) |
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6.4.3 Antiseptics and disinfectants for Tylenchida and Aphelenchida |
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93 | (1) |
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6.4.3.1 Hibitane diacetate |
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93 | (1) |
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93 | (1) |
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6.4.3.3 Hydrogen peroxide |
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93 | (1) |
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94 | (1) |
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94 | (1) |
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6.4.3.6 Disinfectants and sterilants for Dorylaimida and Diphtherophorida |
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95 | (1) |
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6.4.4 Testing for contamination by microorganisms after surface sterilization |
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96 | (1) |
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96 | (2) |
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6.5.1 Roots of whole seedlings on agar |
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96 | (1) |
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6.5.1.1 Tomato seedlings for in vitro culturing of Meloidogyne javanica |
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97 | (1) |
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6.5.1.2 Ditylenchus angustus cultured on axenic rice seedlings |
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98 | (1) |
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6.5.1.3 Xiphinema index cultured on tomato and fig seedlings |
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98 | (1) |
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98 | (3) |
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6.6.1 Tomato root explants |
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99 | (1) |
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6.6.2 Maize root explants |
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100 | (1) |
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6.6.3 Cucumber excised roots |
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100 | (1) |
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6.6.4 Cyst nematodes on excised roots |
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100 | (1) |
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100 | (1) |
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6.6.6 Potato tuber, potato plug and storage root cultures |
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101 | (1) |
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6.7 Callus Tissue Culture |
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101 | (4) |
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103 | (1) |
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6.7.2 Banana fruit callus |
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103 | (1) |
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6.7.3 Rice and wheat callus |
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104 | (1) |
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104 | (1) |
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105 | (1) |
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105 | (1) |
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6.9 In Vitro Banana Plantlets |
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106 | (1) |
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6.10 Gnotobiotic Culture of Whole Plants |
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107 | (2) |
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108 | (1) |
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6.11 Genetically Engineered Plants |
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109 | (2) |
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111 | (2) |
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6.12.1 Arabidopsis thaliana |
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111 | (1) |
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112 | (1) |
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113 | (3) |
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6.13.1 Pot cultures of Meloidogyne and Nacobbus |
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113 | (2) |
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6.13.2 Pot cultures of cyst nematodes |
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115 | (1) |
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6.13.3 Storage of viable cysts of Heterodera spp. |
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116 | (1) |
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6.14 Production of Entomopathogenic Nematodes |
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116 | (5) |
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116 | (1) |
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6.14.1.1 Production of the host insect, Galleria mellonella |
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116 | (1) |
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6.14.1.2 Counting dauer juveniles |
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117 | (1) |
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6.14.2 In vitro culturing |
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117 | (1) |
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6.14.2.1 Isolation of symbiotic bacteria and production of bacterial stock cultures |
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118 | (1) |
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6.14.2.2 Identification of contaminants and phase variants of symbiotic bacteria |
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118 | (1) |
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6.14.2.3 Production of bacteria-free nematodes |
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118 | (2) |
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6.14.3 Establishment of monoxenic cultures |
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120 | (1) |
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6.14.3.1 Monoxenic cultures for mass production |
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120 | (1) |
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6.14.4 Culture of single nematodes or pairs in hanging drops |
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120 | (1) |
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6.15 Cryopreservation of Nematodes |
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121 | (1) |
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6.15.1 Protocol for cryopreservation and revival of nematodes |
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121 | (1) |
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122 | (1) |
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122 | (10) |
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7 Measuring Nematodes and Preparation of Figures |
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132 | (20) |
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132 | (1) |
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133 | (1) |
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134 | (7) |
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134 | (1) |
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134 | (1) |
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7.3.3 Drawing nematodes for measurement |
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135 | (1) |
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7.3.4 Nematode measurement ruler |
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136 | (1) |
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7.3.5 Measurement criteria |
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137 | (4) |
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7.4 Processing Measurement Data and Preparing for Publication |
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141 | (9) |
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144 | (2) |
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7.4.2 Preparing figures for publication |
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146 | (1) |
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146 | (1) |
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147 | (1) |
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147 | (1) |
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7.4.2.4 Combination artwork |
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147 | (1) |
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147 | (1) |
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7.4.2.6 Encapsulated PostScript (EPS) files |
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148 | (1) |
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148 | (2) |
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7.4.2.8 Resolution and apparent image quality |
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150 | (1) |
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150 | (1) |
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151 | (1) |
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8 Electron Microscopy Techniques |
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152 | (25) |
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8.1 Transmission Electron Microscopy |
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152 | (16) |
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153 | (1) |
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154 | (1) |
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154 | (1) |
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8.1.4 Infiltration and embedding: epoxy resins |
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155 | (1) |
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8.1.5 Specimen preparation: acrylic resins |
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155 | (1) |
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8.1.6 Cryopreparation methods for TEM |
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156 | (3) |
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8.1.7 High-pressure freezing (HPF) |
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159 | (3) |
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8.1.8 Self-pressurized rapid freezing |
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162 | (1) |
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8.1.9 Freeze substitution |
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163 | (1) |
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8.1.10 Preparation of sections for TEM observations |
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164 | (3) |
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8.1.11 Transmission electron microscopy |
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167 | (1) |
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8.1.12 Concluding remark on TEM applications |
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168 | (1) |
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8.2 Scanning Electron Microscopy |
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168 | (4) |
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8.2.1 Preparation of nematodes for SEM |
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168 | (1) |
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169 | (1) |
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170 | (1) |
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8.2.3.1 Critical-point drying |
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170 | (1) |
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170 | (1) |
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171 | (1) |
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171 | (1) |
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8.2.3.5 Freeze substitution |
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171 | (1) |
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8.2.3.6 Resin infiltration |
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171 | (1) |
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8.2.4 Glycerin embedded specimens |
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171 | (1) |
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172 | (1) |
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8.2.6 Mounting and coating |
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172 | (1) |
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172 | (1) |
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172 | (5) |
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9 Behavioural and Physiological Assays |
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177 | (18) |
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177 | (1) |
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9.2 Attraction/Repulsion Plate Assays |
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177 | (2) |
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179 | (1) |
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179 | (2) |
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9.4.1 Response to non-volatiles |
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179 | (1) |
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9.4.2 Response to volatiles |
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180 | (1) |
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181 | (1) |
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181 | (1) |
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9.7 Water Content Changes |
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181 | (1) |
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9.8 Oxygen Consumption Assays |
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182 | (1) |
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9.9 Collecting Female Sex Pheromone, Virgin Females and Males |
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183 | (1) |
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183 | (6) |
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183 | (1) |
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9.10.1.1 Vital/non-vital stains |
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183 | (1) |
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184 | (1) |
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185 | (1) |
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9.10.2.1 Standard hatching assays |
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185 | (2) |
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9.10.2.2 Root diffusates and other hatching agents |
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187 | (1) |
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9.10.2.3 Fluorescence assay |
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188 | (1) |
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9.10.2.4 Adenosine triphosphate assay |
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188 | (1) |
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189 | (1) |
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189 | (1) |
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189 | (6) |
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195 | (11) |
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195 | (1) |
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10.2 Staining Chromosomes of Root-knot Females |
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195 | (6) |
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10.2.1 Propionic-orcein staining of root-knot females |
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195 | (1) |
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10.2.1.1 Preparation of propionic-orcein stain |
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195 | (1) |
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10.2.1.2 Selecting and obtaining nematode material for cytological study |
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196 | (1) |
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10.2.2 Preparation of smears to study oogonial divisions and early stages of maturation of oocytes |
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197 | (1) |
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198 | (1) |
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198 | (1) |
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198 | (1) |
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199 | (1) |
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10.2.2.5 Examination of temporary preparations |
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199 | (2) |
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10.2.3 Summary of procedures for staining Meloidogyne chromosomes with propionic-orcein |
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201 | (1) |
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10.3 Staining Chromosomes of Free-living Nematodes |
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201 | (1) |
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10.3.1 Propionic-orcein staining (Hechler, 1970) |
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201 | (1) |
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10.3.2 DAPI florescent nuclear staining (Hasegawa et al, 2006) |
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202 | (1) |
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10.4 Staining Chromosomes of Giant Cells |
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202 | (2) |
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10.4.1 Seed germination pouch cultures |
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202 | (1) |
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10.4.2 Glasshouse cultures |
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202 | (1) |
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203 | (1) |
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10.4.4 Observation of stained material |
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203 | (1) |
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204 | (2) |
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11 Isoelectric Focusing of Proteins |
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206 | (12) |
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206 | (1) |
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11.2 Isoelectric Focusing of Proteins for Diagnostics of Cyst Nematodes |
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207 | (2) |
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11.2.1 Sample preparation and loading |
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207 | (1) |
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11.2.2 Isoelectric focusing run |
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207 | (1) |
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208 | (1) |
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11.2.4 Interpretation of results |
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208 | (1) |
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11.3 Isoelectric Focusing of Isozymes for Diagnostics of Root-knot Nematodes |
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209 | (7) |
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11.3.1 Preparation of egg-laying females for isozyme extraction |
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209 | (1) |
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210 | (1) |
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11.3.2.1 Resolving gel (8%); reagents and preparation |
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210 | (1) |
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11.3.2.2 Stacking gel (4%); reagents and preparation |
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211 | (1) |
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11.3.2.3 Electrode (running) buffer |
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211 | (1) |
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211 | (1) |
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11.3.4 Isozyme extraction and gel loading |
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212 | (1) |
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212 | (1) |
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212 | (1) |
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11.3.6.1 Staining solution for esterase |
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212 | (4) |
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11.3.6.2 Staining solution for malate dehydrogenase |
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216 | (1) |
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11.3.6.3 Staining solution for glutamate-oxaloacetate transaminase |
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216 | (1) |
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11.3.6.4 Staining solution for superoxide dismutase |
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216 | (1) |
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11.3.6.5 Fixative solution (20% ethanol and 10% glycerin) |
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216 | (1) |
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216 | (2) |
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12 Molecular Identification of Nematodes using Polymerase Chain Reaction (PCR) |
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218 | (22) |
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218 | (2) |
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12.2 Preservation of Nematodes for Molecular Studies |
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220 | (1) |
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220 | (1) |
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12.2.2 Ethanol (ethyl alcohol) preservation |
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220 | (1) |
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220 | (1) |
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12.2.3.1 Protocol for preparing DESS solution |
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220 | (1) |
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221 | (1) |
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12.3.1 Protocols for DNA extraction from nematodes |
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221 | (1) |
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222 | (9) |
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230 | (1) |
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12.4.2 Gel electrophoresis |
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231 | (1) |
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12.5 PCR-Restriction Fragment Length Polymorphism |
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231 | (1) |
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232 | (2) |
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12.7 Problems with PCR-based Methods |
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234 | (1) |
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234 | (1) |
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12.9 Cloning of PCR Products |
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235 | (1) |
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235 | (1) |
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235 | (5) |
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13 Isolation and Characterization of Tandem Repeats in Nematode Genomes |
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240 | (10) |
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Philippe Castagnone-Sereno |
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240 | (1) |
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13.2 Bench Strategies for the Identification of Tandem Repeats |
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241 | (3) |
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13.2.1 The case of satellite DNA |
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241 | (1) |
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13.2.2 The case of microsatellites |
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242 | (2) |
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13.3 In silico Identification of Tandem Repeats |
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244 | (1) |
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13.4 The Contribution of Tandem Repeats as Molecular Markers in Nematodes |
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245 | (1) |
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13.4.1 Satellite DNA-based species-specific diagnostic tools |
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245 | (1) |
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13.4.2 Microsatellites and population genetic studies in plant-parasitic nematodes |
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245 | (1) |
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246 | (1) |
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246 | (4) |
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14 Characterization of Nematode Mitochondrial Genomes |
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250 | (15) |
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Danny A. Humphreys-Pereira |
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250 | (2) |
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14.2 PCR Amplification of Mitochondrial Genomes |
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252 | (1) |
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252 | (3) |
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14.3.1 Amplification of partial mt genes/short DNA fragments (< 3 kb) |
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252 | (1) |
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252 | (3) |
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14.4 Cloning and Sequencing |
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255 | (1) |
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14.5 Mitochondrial Genome Annotation and Gene Identification |
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255 | (6) |
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14.5.1 Protocols for mitochondrial genome annotation |
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256 | (5) |
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14.5.2 Important information after automated annotation |
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261 | (1) |
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261 | (4) |
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15 Phylogenetic Analysis of DNA Sequence Data |
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265 | (18) |
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265 | (2) |
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15.2 Visualization and Editing of Raw Sequence Data |
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267 | (1) |
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268 | (1) |
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15.4 Similarity Search in a Sequence Database |
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269 | (1) |
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15.5 Sequence Retrieval from the Database |
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270 | (3) |
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15.5.1 Multiple alignment |
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271 | (1) |
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15.5.2 Sequence alignment editing |
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271 | (2) |
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15.6 File Format Converting |
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|
273 | (1) |
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15.7 Selection of Model of Sequence Evolution |
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274 | (1) |
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15.7.1 jModelTest: A tool to select the best-fit model of nucleotide substitution |
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274 | (1) |
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15.8 Phylogenetic Analysis with PAUP* |
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275 | (3) |
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276 | (1) |
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276 | (1) |
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15.8.3 Maximum likelihood |
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277 | (1) |
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15.9 Phylogenetic Analysis with MrBayes |
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278 | (1) |
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15.10 Visualization of Phylogenetic Trees |
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279 | (2) |
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15.11 Sequence Submission |
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|
281 | (1) |
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|
281 | (2) |
Index |
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283 | |