Bt Research 2015, Vol.6 No.2 1-10
ISSN 1925-1939
4
intermediate stages (Fig. 5). In all other treatments, all
mites (with all stages) were dead within 24 h of
treatment (Fig. 5). It clearly shows that the crude
Btk
toxin is not only lethal to the nymphal stages, but the
adults as well.
Figure 4
Btk
toxin used for treatment. A. crude pellet (48 h) of
solid-fermented matter containing a mixture of
Btk
spore,
crystal, debris of cells and potato flour (SEM view),
i.e
., LB
was supplemented with 10% (w/v) of potato flour and
incubated in a shaker (Scigenics Biotech, India) at 125 rpm and
37
o
C for 12 h; the resultant semi-solid viscous fermented
matter with embedded
Btk
was centrifuged (1000 ×
g
for 10
min), and the wet solid pellet with no free solution was
further incubated (37
o
C, static); and B. crude pellet of
Btk
containing a mixture of spore obtained after submerged
fermentation in LB (72 h), vegetative cells, spores, crystal and
cell debris are seen (SEM view).
Probit analysis
Figures 6A and B give a vivid picture of probit
analyses. Figure 6A is the probit for the dried and
powdered pellet obtained form 72 h old
Btk
culture in
LB (control),
i.e.,
after subtracting the effect of its
autoclaved control. At 5 % significance level, the
probit value (a concentration required to kill 50% of
the target organism) of pellet from LB control was
2.026 µg/cm
2
, which was 19% less effective than the
corresponding treatment of potato flour supplemented
pellet (whose probit was 1.639 µg/cm
2
). For the
treatment, only unpurified crude pellet was used. It is
advantageous that no further purification of the
crystals in the crude fermented matter was required.
It has to be noted that the dry weight of pellet obtained
from LB control (6.32 mg/mL) was different from that
of potato flour supplemented medium (91.25
mg/mL
eqv
),
i.e
., the weight of 10% starchy potato flour
added to LB must have enhanced the dry weight.
Figure 5 Bioassay for
Btk
with
A. gurreronis
. Images were
taken at 48 h of treatment. Various concentrations (1.25μg,
1.88 μg, 2.5 μg, 3.13 μg and 3.73 μg, all per cm
2
area) of
Btk
toxin (dry and powdered crude fermented matter from potato
flour supplemented medium, harvested at 48h incubation) was
carefully dusted on the tender region culture set-up without
disturbing the mites (after initial 24 h observation for their
vitality). A is the image of control mites fed with pellet from
LB control, and B-F are that of treated mites fed with pellet
from potato flour supplemented medium): A. image of mites
treated with autoclaved control of potato flour supplemented
medium (3.73 μg/cm
2
) showing most of them are alive and
active (above 90%) with both nymphal stages (two adult mites
at inset); B. mites treated with 1.25 μg/cm
2
fermented matter,
here some mites (about 61%) were alive but less active (1
active with 2 dead mites at inset); C. mites treated with 1.88
μg/cm
2
fermented matter, here all mites were dead (3 dead
mites at inset); D. mites treated with 2.5 μg/cm
2
fermented
matter, here also all mites were dead (2 dead mites at inset); E.
mites treated with 3.13 μg/cm
2
fermented matter, all mites were
dead (5 dead mites at inset); F. mites treated with 3.73 μg/cm
2
fermented matter, all mites were dead (3 dead mites at inset).
