ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC ...

necessary serial dilutions under phase contrast microscope. From the stock solution, further
dilutions were made to obtain the required concentrations for further studies.
3.3 INSECTICIDAL ACTIVITY OF Metarhizium anisopliae AND
Verticillium lecanii AGAINST DIFFERENT INSECT PESTS
The fungal spores from SMAY plates were mixed with distilled water and 0.2 per cent
Tween-80 to get the spores suspension. The number of conidia were determined using a
Neubauer haemocytometer. Finally, the spore suspension containing 1 x 10 8 conidia/ml were
obtained for the seven individual isolates (Metarhizium anisopliae (4) and Verticillium lecanii
(3) isolates). The spore suspensions of 1 x 10 8 conidia ml -1 of all isolates were topically
applied on early instar of pest with a chromatography sprayer. Three replications were
maintained and in each replication 20 larvae/nymph were tested. Another set was kept
without addition of spores as negative control. The number of dead larvae was noted down
from fifth day of inoculation. Finally, the per cent mortality of each isolate was computed. The
data were subjected to statistical analysis by DMRT (Duncan’s Multiple Range Test).
3.4 EFFECT OF NUTRITION ON THE TOTAL DRY MYCELIAL
PRODUCTION OF ENTOMPATHOGENIC FUNGI
The effect of different carbon and nitrogen sources on the development of biomass
of fungi were evaluated by varying different carbon sources in the basic Czapeks medium
with lactose, starch, fructose and dextrose at 1, 2, 3 and 4 per cent. Sources at recommended
dose (3%) in the basal media served as the check. Similarly, different nitrogenous sources
viz., KNO3, NaNO3, (NH4)2SO4, NH4NO3 were evaluated at 0.1, 0.15, 2.0 and 2.5 per cent.
Initially, the fungi were cultured on SMAY plates for two weeks. The conidial
suspension was prepared by shaking conidia from a 5 mm diameter agar plug into a test tube
containing in 10 ml sterile water blank mixed with 0.05 per cent Tween 80. The conidial
suspension was mixed thoroughly by shaking at 80 rpm for 10 min and the concentration of
conidia was determined using haemocytometer. Two hundred micro tube (0.2 ml) aliquot of
the final suspension was inoculated into 250 ml broth containing different sources and levels
of carbon and nitrogen. The flasks were aerated on a shaker at 90 rpm under room
temperature for 8-9 days. After complete mycelial growth, the fungal mat was taken out and
filtered through Whatman No. 1 filter paper later dried under oven at 60°C for two days and
dry weight was recorded.
3.5 COMPATIBLITY OF THE FUNGI WITH PESTICIDE
In vitro studies were under taken to assess compatibility of selected insecticides,
fungicides and weedicides by following the poisoned food technique (Nene and Thapliyal,
1997) and the details of the different pesticides and the concentration used are given below
(Table 2).
The pesticides at required concentration were added to the sterilized SMAY agar and
poured into the petriplates after proper agitation and allowed to solidify. The conidial
suspension of the fungi were prepared by shaking conidial from a 5 mm diameter agar plug
(from a mat of the fungi) into a test tube containing in 10 ml sterile water blank mixed with
0.05 per cent Tween 80. The conidial suspension was mixed thoroughly shaking at 80 rpm for
10 min. Hundred microlitre (0.1 ml) of the suspension was spread on SMAY plates containing
different pesticides and control (no pesticide added). All the plates were incubated at 28±1°C
and a 12:12 light dark regime.