2. Apply various concentrations of nematodes (1000, 5000, and
10,000 IJs) in 10 ml distilled water per kilogram of soil.
3. Release ten larvae (fourth instar) of test insect into each jar.
4. Add food for insect larvae in the jars.
5. Repeat three replicates for each treatment.
6. Cover the glass jars with muslin cloth.
7. Keep the jars at room temperature.
8. Check the insect mortalities after 3, 5, and 7 days of
inoculation.
9. Tip out the soil and gently search the larvae in soil for data
recording.
10. Count the healthy and dead larvae.
11. Fill the same jars again with the soil along with the healthy
larvae.
12. Correct the mortality data using Abbot’s formula [200].
13. Use the corrected per cent mortality data for further analysis
which is done using completely randomized design.
9.1.3 Leaf Disc Bioassay
Materials Required
l
Petri dish.
l
Leaves (depend upon the plant host).
l
EPN (IJs).
l
Distilled water.
l
Test insects (larvae).
Procedure
1. Insect mortality bioassay is conducted with species of entomopathogenic nematodes.
2. Wash the host leaves with sterile distilled water.
3. Cut the leaves into 9-cm diameter discs.
4. Place the leaves inside the petri dishes which act as a source of
nourishment for the developing larvae.
5. Inoculate 2 ml of nematode suspension containing 200 IJ/ml
on the leaf disc.
6. Place ten healthy larvae (third instar) of test insect on the
leaf disc.
7. Incubate the plate at room temperature for 24–72 h.
8. Arrange the treatments in a completely randomized design
with replicates.
9. Maintain control experiment by adding 0.5 ml distilled water
alone to the larvae on leaf discs in the petri dishes.
110
Laboratory Techniques for Entomopathogenic Nematodes
10,000 IJs) in 10 ml distilled water per kilogram of soil.
3. Release ten larvae (fourth instar) of test insect into each jar.
4. Add food for insect larvae in the jars.
5. Repeat three replicates for each treatment.
6. Cover the glass jars with muslin cloth.
7. Keep the jars at room temperature.
8. Check the insect mortalities after 3, 5, and 7 days of
inoculation.
9. Tip out the soil and gently search the larvae in soil for data
recording.
10. Count the healthy and dead larvae.
11. Fill the same jars again with the soil along with the healthy
larvae.
12. Correct the mortality data using Abbot’s formula [200].
13. Use the corrected per cent mortality data for further analysis
which is done using completely randomized design.
9.1.3 Leaf Disc Bioassay
Materials Required
l
Petri dish.
l
Leaves (depend upon the plant host).
l
EPN (IJs).
l
Distilled water.
l
Test insects (larvae).
Procedure
1. Insect mortality bioassay is conducted with species of entomopathogenic nematodes.
2. Wash the host leaves with sterile distilled water.
3. Cut the leaves into 9-cm diameter discs.
4. Place the leaves inside the petri dishes which act as a source of
nourishment for the developing larvae.
5. Inoculate 2 ml of nematode suspension containing 200 IJ/ml
on the leaf disc.
6. Place ten healthy larvae (third instar) of test insect on the
leaf disc.
7. Incubate the plate at room temperature for 24–72 h.
8. Arrange the treatments in a completely randomized design
with replicates.
9. Maintain control experiment by adding 0.5 ml distilled water
alone to the larvae on leaf discs in the petri dishes.
110
Laboratory Techniques for Entomopathogenic Nematodes