2. Permit to mate and lay eggs.
3. Take out the adults after a 48 h oviposition period.
4. Preserve those plants within the greenhouse at 25 Æ 1
C, 65%
RH for 16:8h (L:D).
5. Retain the plants in the greenhouse for a further 12 days until
the presence of the second nymphal instar [253].
6. Choose the second nymphal instar since the second nymphal
stages are the most vulnerable to nematode infection.
7. Pick randomly 5–6 leaves from each plant (cucumber or pepper) infested with T. vaporariorum second nymphal instars.
8. Relocate each leaf into the petri dish filled with a thin layer of
1.5% agar.
9. Amend the nematode suspensions to yield the concentrations
0-control, 25, 50, 100, 150, 200, and 250 IJ/cm
2 by volumetric dilutions in distilled water to an absolute volume of 1 ml.
10. Mix Triton X-100 to the EPN concentrations and the control
comprising only distilled water with a handheld sprayer.
11. Spray each suspension onto the surface of each petri dish till
runoff by means of a handheld sprayer. Have four replicates for
each treatment, and repeat the whole experiment twice, with
diverse batches of fresh nematodes, insects, and plants.
12. Execute the spraying at greenhouse in the afternoon.
13. Conceal the petri dish after inoculation of nematodes.
14. Maintain the plants at 20 Æ 1
C, 85% RH, and a 12:12 h L:D
period for 72 h [254].
15. Check the infection of S. feltiae and H. bacteriophora after 72 h
treatment.
16. Have three replicates for each treatment, and repeat the whole
experiment twice.
17. Dissect the cadavers using a stereomicroscope to check that
mortality ensued from EPN infection.
2.2 Diaspora of
Infective Juveniles
from Greenhouse
Cadavers [255]
To demonstrate the efficacy of formulated IJs against Hoplia philanthus in greenhouse.
2.2.1 Materials Required
l
Hoplia philanthus grub.
l
Greenhouse crop ryegrass.
l
Cadaver (HbK15-5).
l
PVC pipe (3.6 cm inner diameter).
l
Plastic pot.
156
Application Efficacy Against Insects in Other Than Soil Habitats
3. Take out the adults after a 48 h oviposition period.
4. Preserve those plants within the greenhouse at 25 Æ 1
C, 65%
RH for 16:8h (L:D).
5. Retain the plants in the greenhouse for a further 12 days until
the presence of the second nymphal instar [253].
6. Choose the second nymphal instar since the second nymphal
stages are the most vulnerable to nematode infection.
7. Pick randomly 5–6 leaves from each plant (cucumber or pepper) infested with T. vaporariorum second nymphal instars.
8. Relocate each leaf into the petri dish filled with a thin layer of
1.5% agar.
9. Amend the nematode suspensions to yield the concentrations
0-control, 25, 50, 100, 150, 200, and 250 IJ/cm
2 by volumetric dilutions in distilled water to an absolute volume of 1 ml.
10. Mix Triton X-100 to the EPN concentrations and the control
comprising only distilled water with a handheld sprayer.
11. Spray each suspension onto the surface of each petri dish till
runoff by means of a handheld sprayer. Have four replicates for
each treatment, and repeat the whole experiment twice, with
diverse batches of fresh nematodes, insects, and plants.
12. Execute the spraying at greenhouse in the afternoon.
13. Conceal the petri dish after inoculation of nematodes.
14. Maintain the plants at 20 Æ 1
C, 85% RH, and a 12:12 h L:D
period for 72 h [254].
15. Check the infection of S. feltiae and H. bacteriophora after 72 h
treatment.
16. Have three replicates for each treatment, and repeat the whole
experiment twice.
17. Dissect the cadavers using a stereomicroscope to check that
mortality ensued from EPN infection.
2.2 Diaspora of
Infective Juveniles
from Greenhouse
Cadavers [255]
To demonstrate the efficacy of formulated IJs against Hoplia philanthus in greenhouse.
2.2.1 Materials Required
l
Hoplia philanthus grub.
l
Greenhouse crop ryegrass.
l
Cadaver (HbK15-5).
l
PVC pipe (3.6 cm inner diameter).
l
Plastic pot.
156
Application Efficacy Against Insects in Other Than Soil Habitats