l
Silicone adhesive.
l
Sterilized sandy soil.
l
24-Well plates.
l
Tap water.
l
Stereomicroscope.
2.2.2 Procedure
1. Attach the PVC pipe to a hole (4 cm diameter) on the lateral
surface of the plastic pot (8.2 Â 6.7 Â 9 cm; surface area
52.8 cm
2 ) and fix by means of a silicone adhesive.
2. Close the left-out end of the pipe by a detachable cap.
3. Fill the pot and pipe with humid sanitized sandy soil.
4. Seed the pots consequently with perennial ryegrass.
5. Permit the plant to rise for 4 weeks earlier to infestation with
H. philanthus grubs.
6. Keep the grubs separately in moist sand in 24-well plates at
15 ºC for 1 week.
7. Remove the dead grubs during this period.
8. Add six vigorous grubs on the surface of every pot 4 h before
placement of one recently prepared cadaver (HbK15-5) at the
opposite end of the pipe.
9. In the control treatment, place in every pot a single prepared
cadaver in a hole made to a depth of 5 cm in the center of the
pot and conceal with soil.
10. Remove the grubs that did not pass into the soil within 2 h and
replace with healthy ones.
11. Follow five experimental treatments with five replications of
each treatment.
12. Add 50 ml tap water on alternate days to every pot to sustain
soil moisture.
13. Maintain the average air temperature within the greenhouse at
26 ºC (range 20–30 ºC).
14. After a fortnight, sample the pots detrimentally.
15. Count the live and deceased grubs.
16. Dissect the dead larvae under the stereomicroscope to check
that mortality resulted from EPN infection.
17. Duplicate the experiment.
2.3 Greenhouse Pot
Trial [256]
To study the efficacy and dispersal of EPN for control of black vine
weevil (BVW).
Greenhouses
157
Silicone adhesive.
l
Sterilized sandy soil.
l
24-Well plates.
l
Tap water.
l
Stereomicroscope.
2.2.2 Procedure
1. Attach the PVC pipe to a hole (4 cm diameter) on the lateral
surface of the plastic pot (8.2 Â 6.7 Â 9 cm; surface area
52.8 cm
2 ) and fix by means of a silicone adhesive.
2. Close the left-out end of the pipe by a detachable cap.
3. Fill the pot and pipe with humid sanitized sandy soil.
4. Seed the pots consequently with perennial ryegrass.
5. Permit the plant to rise for 4 weeks earlier to infestation with
H. philanthus grubs.
6. Keep the grubs separately in moist sand in 24-well plates at
15 ºC for 1 week.
7. Remove the dead grubs during this period.
8. Add six vigorous grubs on the surface of every pot 4 h before
placement of one recently prepared cadaver (HbK15-5) at the
opposite end of the pipe.
9. In the control treatment, place in every pot a single prepared
cadaver in a hole made to a depth of 5 cm in the center of the
pot and conceal with soil.
10. Remove the grubs that did not pass into the soil within 2 h and
replace with healthy ones.
11. Follow five experimental treatments with five replications of
each treatment.
12. Add 50 ml tap water on alternate days to every pot to sustain
soil moisture.
13. Maintain the average air temperature within the greenhouse at
26 ºC (range 20–30 ºC).
14. After a fortnight, sample the pots detrimentally.
15. Count the live and deceased grubs.
16. Dissect the dead larvae under the stereomicroscope to check
that mortality resulted from EPN infection.
17. Duplicate the experiment.
2.3 Greenhouse Pot
Trial [256]
To study the efficacy and dispersal of EPN for control of black vine
weevil (BVW).
Greenhouses
157