From Soil to Lab: Sampling and Extracting Nematodes Like a Pro


Updated: 4 Feb 2024

32


Hello, lovely readers!

Are you searching for the ideal method of nematode sampling? Unsure about how the nematodes are extracted?

No worries! You’re in the right place. We’re here to walk you through easy step-by-step methods that’ll simplify your life. Our goal is to make things practical and straightforward for you, so go ahead and take a look.

Let’s dive in and get started together!

Sampling of Nematodes

The main purposes are:

Objectives

  • Identification of nematode species.
  • Diagnosis of nematode diseases.
  • Population estimation for research, advisory, regulatory, and predictive programs.
  • Making management decisions.
  • Determination of crop losses and the degree of damage to crops.
  • Determination of incidence, prevalence, and population densities of nematodes in surveyed areas.
  • Studying the relationship between nematodes and other pathogens.
  • Studying the influence of environmental factors (e.g., climate, moisture, soil texture, age of plant, pH, and resistance) on population changes of nematodes.

Time of sampling

  • The time of sampling is very important as nematode populations vary significantly with the season and age of the host plants.
  • For advisory services, samples can be collected right after land preparation to get the initial population during the maximum vegetative growth, as well as during the flowering stage, either before or after harvest.
  • It mainly depends on the objectives.

Tools required

  • Spade
  • Soil auger
  • Shovel
  • Trowel
  • Plastic bags
  • Permanent ink marker
  • Bucket

Sample Material

For isolation of endoparasitic and semi-endoparasitic nematodes, infected plant parts are collected e.g., roots (Meloidogyne), bulbs, tubers, rhizome (Ditylenchus), leaves (Aphelenchoides), trunk (Bursaphelenchus), seeds (Anguina), and fruit (Schistonchus caprifici).

Soil samples are collected for ectoparasites and free-living nematodes.

Sample Collection

Root Sampling

For root samples, carefully collect the entire root system up to a depth of 30 cm along with soil. When dealing with perennial plants, take samples approximately 30-40 cm away from the base of the plant.

Soil Sampling

When collecting soil samples, remove the top 5-10 cm of soil and gather samples up to 30 cm in depth along with roots. Avoid sampling in excessively dry or wet soil; instead, prefer sampling in moist soil conditions for accurate results.

Handling Nematode Samples

Here are some steps to take:

  • Handle with Care: Nematodes are living animals; handle the samples gently.
  • Use Plastic Bags: Place samples in plastic bags to prevent soil drying.
  • Temperature Control: Protect samples from temperature extremes.
  • Separate Shoots and Roots: Since shoots decompose faster than roots, keep them in separate polythene bags.
  • Labeling: Clearly label each sample with the required information.
  • Delivery to Lab: Bring the sample to the lab without delay. If samples won’t be processed immediately, store them at 4°C for preservation until extraction

Sampling Pattern

There are different patterns of soil sampling which depend on the purpose and requirement. It is essential to obtain reliable data on a field or an area.

The nematode population usually occurs in patches, and if such patches are missed, the average population may be too low. In a reverse situation, the population will be too high. 

To minimize such variations and to have a reliable average nematode population, different patterns of soil collection are used depending on the objective of sampling area and type of vegetation.

Extraction of Nematodes

Plant parasitic nematodes can be extracted from soil and plants in many different ways but the most commonly used methods are listed below:

  • Cobb’s Decanting and Sieving Method
  • Baermann Funnel Technique
  • Baermann trays (modified Baermann funnel)

Cobb’s Decanting and Sieving Method

Materials required

  • Sieves of different mesh sizes
  • Plastic buckets
  • 250 ml beakers
  • 600 ml beakers
  • Wash bottles
  • Plastic cups
  • Stirring rod / stick about 45 cm long and 2.5 cm wide

10 Easy Steps

  1. Place the soil sample (500 to 1000 cc) in the container and then add 2-3 liters of water.
  2. Until all clods are broken up, stir the mixture with a stick. The main goal is to separate soil particles from nematodes. The nematodes get suspended in the water.
  3. Allow the mixture to stand for 30 seconds to 1 minute to let heavy soil particles settle at the bottom of the bucket.
  4. Into the second container, pour the water through a 20-mesh sieve leaving the heavy soil particles behind. The coarse sieve will easily allow nematodes to pass through.
  5. Repeat steps 2 and 3 after adding approximately 1 liter of water. The second bucket will now include most of the nematodes.
  6. The residue present on the coarse sieve should be washed with additional water, ensuring no nematodes remain. Let the water flow into the 2nd bucket.
  7. The leftover present in the 1st bucket should be discarded, and both buckets are to be washed, and cleaned.
  8. Stir the amalgam in the 2nd container and wait for it to settle down for 30-40 seconds. Use a 200-mesh sieve and pour through it into the 1st bucket.
  9. Wash the leftover retained on the fine 200-mesh sieve with a gentle water stream.  Into a 250 ml beaker, backwash the material with big nematodes.
  10. Repeat steps 8 and 9 for the material in the first bucket, using a 350-mesh sieve.  Backwash the contents into a 250 ml container/beaker with small to medium nematodes.
Advantages
  • This method is not dependent on nematode movement; even sluggish nematodes can be effectively recovered.
  • It removes most of the nematodes from big soil samples
  • Nematodes become ready for direct proper examination within 30 minutes.
Disadvantages
  • The method demands expensive sieves and skilled workers.
  • Nematodes may be challenging to observe due to the presence of fine soil particles.

Baermann Funnel Technique

Principle

The extraction depends on the nematodes’ active movement.

Materials Required

  • Funnel stand
  • Beaker 250 ml
  • Funnel
  • Plastic drink cup cover
  • Wire mesh
  • Tissue paper
  • Clamp
  • Rubber or plastic tube

11 Easy Steps

  1. A tube of 10 cm in length is to be attached to the funnel opening. The tube should strongly clamp the tubing.
  2. The glass funnel is then to be mounted on the stand.
  3. Then fill two-thirds of the funnel with with water.
  4. To support tissue on the funnel opening, place a basket of wire mesh.
  5. Pass mixed soil sub-sample from a coarse sieve to separate the roots and stones, etc.
  6. Spread 50 cc soil sub-samples properly on the tissue paper and then fold in the sides of the tissue.
  7. Add water to the funnel while the level of the water should be about 5 mm above from the wire mesh.
  8. At room temperature, keep the setup undisturbed for 2-3 days.
  9. Daily add fresh water to the funnel in compensation for the evaporation loss. Pour water through the sides, not directly onto the sample. Cover the funnel with a petri dish.
  10. Nematodes will wriggle out through the filter paper into the water and settle at the bottom of the tubing.
  11. After 48 hours, collect 10-20 ml of water containing nematodes and examine under stereomicroscope for identification.
Advantages
  • Clean and live nematode samples can be easily extracted.
  • Sieves are not needed.
  • Inexpensive to construct.
Disadvantages
  • This technique is not effective for inactive nematodes.
  • Recovery percentages may be low.

Baermann Trays

Baermann trays are a modification of Baermann’s funnel and nematodes can be extracted from relatively larger samples. No special racks, tubing, or clamps are needed.

Principle

The extraction depends on the nematodes’ active migration (motility-dependent method).

Materials Required

  • Pan or tray
  • Wire mesh
  • Tissue paper
  • Squeeze bottle
  • 500 mesh sieve

8 Easy Steps

  1. Add water to the dish.
  2. Place soil on tissue paper.
  3. Put the tissue on the wire mesh or plastic tray with holes.
  4. Fold the tissue over the soil and add water as needed.
  5. Incubate the tray at room temperature for 3 days.
  6. After incubation, remove the wire mesh from the dish.
  7. Pour water containing nematodes onto the sieve.
  8. Backwash nematodes into the beaker.

Conclusion

The discussion on nematode sampling and extraction methods has been comprehensive. Each extraction method has been briefly outlined with easy-to-follow steps. Practice these steps with care, using the provided information on materials and tools.

Remember, we’re here for you. Should you encounter any challenges or have questions along the way, our “Contact Us” section is your resource. Don’t hesitate to seek guidance if needed. Happy extracting!

FAQs

Here are some frequently asked questions for your convenience.

When should we collect samples?

Samples can be collected right after land preparation to get the initial population during the maximum vegetative growth, as well as during the flowering stage, either before or after harvest.

How are root samples collected?

For root samples, carefully collect the entire root system up to a depth of 30 cm along with soil. When dealing with perennial plants, take samples approximately 30-40 cm away from the base of the plant.

How are soil samples collected?

When collecting soil samples, remove the top 5-10 cm of soil and gather samples up to 30 cm in depth along with roots. Avoid sampling in excessively dry or wet soil; instead, prefer sampling in moist soil conditions for accurate results.

How the nematodes are extracted? Name them.

Plant parasitic nematodes can be extracted from soil and plants in many different ways but the most commonly used methods are listed below:

  • Cobb’s Decanting and Sieving Method
  • Baermann Funnel Technique
  • Baermann trays (modified Baermann funnel)
What are the advantages of Cobb’s Decanting and Sieving method?
  • This method is not dependent on nematode movement; even sluggish nematodes can be effectively recovered.
  • It separates most of the nematodes from big soil samples.
  • Nematodes become ready for proper examination in under 30 minutes.
What are the disadvantages of Cobb’s Decanting and Sieving method?
  • The method demands expensive sieves and skilled workers.
  • Nematodes may be challenging to observe due to the presence of fine soil particles.
What is the principle of the Baermann Funnel Technique?

The extraction depends on the nematodes’ active movement.

What are the advantages of the Baermann Funnel Technique?
  • Clean and live nematode samples can be easily extracted.
  • Sieves are not needed.
  • Inexpensive to construct.
What are the disadvantages of the Baermann Funnel Technique?
  • This technique is not effective for inactive nematodes.
  • Recovery percentages may be low.
What is the principle of the Baermann Trays method?

The extraction depends on the nematodes’ active migration (motility-dependent method).


Dr. M Awais

Dr. M Awais

This is Awais. I am a phyto doctor. I have studied plants my whole life. Plants are my best friends. I have gathered detailed information about plants. My brilliant team is always ready to accept the challenges. Together, we find the solutions for our clients.

Please Write Your Comments