Bt Research 2015, Vol.6, No.7, 1-3
2
Figure 1 Isolated Bt-like colonies from soil samples (indicated
by black arrows)
50 ml (sterile) centrifuge tubes or zip-lock bags until
isolation.
2 Isolation and identification
Vegetative cells from sporulated bacteria (Figure 1)
were isolated by homogenization of 3 g of each soil
sample in 10 ml of sterile distilled water, which was
later mixed by intense vortexing for 1 min. and
incubated at 70 ºC for 15 min., after that, samples
were vortexed and heated again as previously
described. Each sample was then subjected to five
ten-fold dilutions and 50
l (from 10
-3
to 10
-5
dilutions)
were plated using a Drigalsk spatula onto Petri dishes
containing nutrient agar (0.5% Peptone,
0.3% beef
extract, 0.5% NaCl
and 1.5% agar). Plates were
incubated at 28 ºC for at least 72 h. Bacterial colonies
exhibiting Bt-like phenotype (matte white colour, flat,
dry and uneven borders) (Figure 2) were sub-cultured
for single-colony isolation with a 5
l inoculating loop
and incubated as before. Each sporulated culture was
then heat fixed and stained (0,133 % Coomassie Blue
stain in 50 % acetic acid) in a glass microscope slide
(Ammons et al., 2002). The Bt-like cells identification
was performed by microscopic examination using a
Leyca DM 750 bright field microscope (Leyca
Microsystems). Those colonies showing Coomassie
Blue stained parasporal crystals were stored at -20 ºC
in 50 % glycerol
3 Determining the insecticidal activity of
novel Bt isolates against
Spodoptera cosmioides
.
Preliminary bioassays (Figure 3) were performed by
surface contamination of artificial diet for lepidopteran
(1.3 g benzoic acid, 128,4 g corn grits, 32.1 g wheat
germ, 34.3 g beer yeast, 4.5 g ascorbic acid, 1.1 g
methylparaben (Nipagin), 0.5 mL formaldehyde, 12 g
of Agar and 779.5 mL of distilled water), using first
Figure 2 Sporulated Bt culture of
Bacillus thuringiensis
subsp.
kurstaki
HD1 strain showing rhomboidal parasporal crystals
(C), spores (S) and vegetative cells (VC). In order to highlight
parasporal crystals, the culture has been heat fixed and stained
with Coomassie Blue (Ammons et al., 2002). Microphotograph
(1000
) was obtained with a Leica DM750 microscope and its
incorporated ICC50 digital camera (Leica Microsystems)
Figure 3. Preliminary bioassays results obtained with
S.
cosmioides larvae
. (A) Dead
S. cosmioides
larvae treated
with HD1 Bt strain; active against Lepidoptera (B) Dead
S.
cosmioides
larvae treated with HD133 Bt strain, also active
against Lepidoptera (C)
S. cosmioides
larvae exhibiting impaired
growth when treated with IPS82 Bt strain, a strain active
against Diptera (mosquitoes and black flies). (C) Negative
control (sterile water)
instar
Spodoptera cosmioides
(Walker) larvae (Figure
3). Sporulated cultures of Bt isolates (mix of spores
and crystals) were obtained from nutrient agar plates
by scrapping the surface of agar with an inoculating
loop. This material, corresponding to the entire Petri
plate surface, was then diluted in 1 ml of distilled
water and homogeneizated by vortexing vigorously
for 1 min. at room temperature (RT). After mixing,
100
l were loaded onto the surface of the solid
lepidopteran artificial diet, spread with a Drigalski
spatula and allowed to dry at RT.
Bioassays were
conducted with neonate larvae that were placed onto
the surface of contaminated artificial diet. Sterile
water was used as a negative control and the
commercial Bt strains HD1 (Bt subsp.
kurstaki
),
HD133 (Bt subsp.
aizawai
) and IPS82 (Bt subsp.
israelensis
) were used as positive controls. Ten larvae
were used for each plate and each bioassay was
