Nematode Pests of Horticultural Crops and their Management (1+1)

Practical - 3

Exercise: Extraction of Nematodes from Soil Samples.
Objectives:

• To conduct faunistic studies of nematodes belonging to different groups present in soil and their qualitative and quantitative estimation.

Material required:

• Beakers, plastic pans/enamelled metallic pans, Petri-plates, sieves, soil sample, facial tissue papers, wire nets, glass funnels, funnel stand, rubber tube, glass vials.

Method:

• Cobb’s decanting and sieving technique and Modified Baermann’s funnel method (Schindler’s modification)

i. Cobb’s Sieving and Decanting Technique
Principles

• Differential sedimentation rate i.e. the soil particles and nematodes settle at different rates due to differences in their specific gravity.(specific gravity of nematodes is 1.05)
• Different sized nematodes are retained on sieves of different pore sizes.

Procedure (Fig. 6)

• Take out the composite sample in a pan, mix it thoroughly and take 200 cc for processing. Store the remaining sample in a refrigerator.
• Transfer 200 cc soil to Pan A and add about one litre water, mix well with hands, breaking clods and clumps.
• Wait for a few seconds and pass this soil suspension through a 20-mesh sieve (pore size 840µm), collecting the filtrate in pan B. Wash the Pan A.
• Collect roots present on the 20-mesh sieve in a beaker and discard the remaining material.
• Stir the suspension of pan B gently, wait for a few seconds and pour it through a 60-mesh sieve (pore size 250µm) in pan A. Collect the residue left over 60-mesh sieve in a beaker and label it as 60. This residue will contain cyst nematodes in case of their infestation and can be viewed directly under a stereomicroscope.
• Pass the contents of pan A through a 300-mesh sieve (pore size 53 µm). Discard the suspension passed through the sieve.
• Collect the residue left over 300-mesh sieve in a beaker and label as 300.
• Further process 300-mesh residue by either of the following techniques.

Fig. 3.1 Cobb’s Decanting and Sieving Technique

ii. Baermann’s Funnel Technique
Principle:

• The active and motile nematode pass through the tissue paper and get collected at the base of rubber tube/specimen vial due to movement and gravitational force whereas inert soil particles/debris remain on the tissue paper.

Procedure(Fig.7)

• Take the residue of 300 mesh sieve collected by Cobb’s decanting and sieving technique.
• Fix a glass funnel with a piece of rubber tube (10-12’’long) bearing a glass vial (5 ml capacity) attached to its distal end on a stand.
• Fill the funnel assembly with water and press the rubber tube gently to remove the air bubbles.
• Transfer the sieved nematode suspensions (labelled as 300) to a moulded piece of wire net covered with a double layered tissue paper and place it over the funnel.
• Add water till it touches the lower surface of the wire net.
• After 24-48 hours remove the glass vial and observe the nematodes under a stereoscopic binocular microscope.

Advantages

• Clear nematode suspension, free of dirt and debris is obtained.
• The nematodes are collected in a small amount of water, thus making further working with them easy.

Disadvantages

• It is a time consuming.
• Nematodes may lose their activity/viability due to lack of oxygen.
• Sedentary and slow moving nematodes cannot be extracted

Fig. 3.2 Baermann’s Funnel Technique

iii. Modified Baermann’s funnel Technique (Schindler’s modification) (Fig. 8):

• In this technique Petri-plate is used in place of funnel. Other steps are same as described above for Baermann’s funnel method and the nematodes get collected in Petri-plate

Advantage:

• Maximum recovery of active nematode is achieved by this method because the nematodes do not lose their activity/viability since the oxygen is always available.

Precautions:

• Thorough mixing of the soil and breaking of clods is important.
• While pouring nematode suspension on the tissue paper, a thumb should be placed in between to avoid tearing of the tissue paper.