NEMATOLOGY 100

Laboratory Exercise 4

Rev. 01/01/2020

Purpose:  Quantitative population assessment; effects of plant residues and defense compounds on nematode survival; nematode morphology and anatomy.

1.   Powerpoint presentation and slides of nematode damage to plants.

2.  Experiment:  Effect of substances of plant origin on nematode survival and activity.  The following materials are some examples:

• cellulose powder, high C:N ratio, not considered a source of toxic metabolites.
• milled mustard (Brassica juncea), source of the glucosinolate sinigrin which is metabolized to allyl isothiocyanate.
• yellow mustard seed, Sinapis hirta, source of the glucosinolate sinalbin which is metabolized to 4-hydroxybenzyl isothiocyanate
• garlic powder, a source of allicin which is oxidized to S-allylcysteine and other sulfur-containing compounds.

some of the compounds, for example the isothiocyanates, are unstable, some with a half-life of only a few hours.

• Work in groups of two.  Use different plant sources provided by students.  We will consider this a preliminary screening test of different materials, so we will maximize the number of plant sources at the cost of replication.
• Each group select one of the plant sources
• You are provided with 50 ml beakers containing 30 ml of sterilized sand.  The weight of the sand in each beaker is approximately 40g.
• Place the 30 ml sand from two beakers into a plastic tub and blend in 0.8g (dry weight) of the milled residue (1% w/w) or macerated material.
• Place 30ml of the blends back into each of the two 50 ml beakers and use 30g unamended sand in another two 50 ml beakers as controls. Each group should have a total of 4 beakers, 2 amended and 2 unamended.
• Label your beakers with the treatment and group name.
• Add 4 ml water to the beakers of amended sand, 3 ml to the unamended sand.  The differences in water added are to compensate for water absorbed by the organic amendment.
• Calibrate the nematode inoculum, root-knot nematode (Meloidogyne incognita).
•  Pipette a suspension containing 500 second stage juveniles into each beaker in 2 ml water.
• Separate amended and control beakers into different covered plastic boxes to minimize drying.
• Leave exposed for 48 hours.
• After 48 hours, lab #5,  place soil from each beaker onto Baermann funnels on the side bench to allow nematodes to move through tissue.  After 60 hours (Sunday evening), instructor will tap 20 ml of nematode suspension from the funnels for you to assay during lab #6.
• Determine numbers of surviving nematodes during lab #6.   Calculate mortality rates relative to the unamended control treatment.
• Consider possibel errors and problems with the experimental design.

3.  Note differences in the nematode communities extracted from soil samples in the last lab period and in other samples provided.. 

4.  Practice basic skills:  hand-picking nematodes, heat-killing with flame or slide-warmer device, and making temporary mounts.  You need to be able to see internal structures clearly.

5.  Use the  keys provided in Nemaplex to identify the nematodes to the level of family (in Nemapex Select Biology and Taxonomy then Nematode Characteristics and Taxonomic Keys then Families of Plant and Soil Nematodes.

6.  Complete observations on stoma and esophagus structures of nematodes with different feeding habits:
      plant parasites, bacteria feeders, fungus feeders, predators.

7.  Complete observations of reproductive systems.

8.  Extract nematodes from your own samples as assigned

9.  Homework Assignment:

     Review areas of NEMAPLEX dealing with nematodes as plant parasites and nematode damage to plants.

     Main Menu:

      Select...General Nematology
       Select...Introduction to Nematodes
                 Select...Nematodes as Plant Parasites
                            Select...General Concepts of Disease
                                     How Nematodes Damage Plants
                                     Taxa Containing Plant Parasites

                            Back to previous menu

                 Select...Characteristics of Nematodes
                            Select...Reproductive Strategies of Nematodes

    Main Menu:

      Select...In the News - review current and recent news issues regarding nematodes.  Note issues regarding pesticides and volatile organic compounds (VOCs).

Return to Nematology 100 Menu