Bt Research 2015, Vol.6 No.2 1-10
ISSN 1925-1939
8
appropriate glass ring was fixed with the help of
paraffin wax to each button in such a way that the ring
touched the boundary between meristematic yellow
(the exposed region after the removal of the perianth)
and non-meristematic lower greenish region of the nut.
The area (meristematic region within glass ring) of the
coconut buttons was calculated using vernier calipers.
About fifty mites (per cm
2
area on the bud within the
culture ring) were carefully introduced in the
meristematic region bordered by the glass ring using a
sterile brush. After introducing the mites, the mouth
(rim) of the glass ring was lined with a drop of water
so that the cover glass on it remains intact. This
arrangement considerably helped for observing the
behavior of individual stages of the mite through the
cover glass and their manipulation according to the
need. The culture arrangement was placed in the
centre of a plastic tray (8.5 cm diameter and 3. 5 cm
height). The tray was then filled with sterile double
distilled water up to the lower boundary of the glass
ring, so as to prevent the movement of the mites away
from the meristematic zone and also to maintain
proper humidity. The maximum aseptic conditions
were maintained throughout the experiments. Cultures
were maintained at 37
o
C in an incubator.
Btk
toxin for bioassay
Four types of samples were prepared for bioassay.
Dried crude pellets obtained from LB control (72 h)
and potato flour supplemented media (48 h) were used
for treatment. Apart from these samples, their
respective autoclaved controls were also used for
accuracy in calculation (Fig. 4).
Bioassay for
A. guerreronis
For acaricidal assay, the standardized
A. guerreronis
cultures were used. Bioassay was performed in the
culture set-up (24 h old) with about 20 active and
mature mites (with all other stages of development)
per cm
2
. Using a fine brush, fine powder (1.25, 1.88,
2.5, 3.13, or 3.73 μg/cm
2
) of the preparation as above
was carefully dusted on to the tender region of the bud
within the glass ring. Mortality rate in each treatment
was separately scored for probit analyses.
Monitoring the growth and mortality of mites
The life cycle of the mite (Fig. 3) and mortality rates
were analyzed by observing through a Magnus
compound microscope. The photographs were taken
using a digital camera attached to the microscope
(Webcam companion 2.0 MEM 1300, Japan).
Field trial
About 5- 10 years old coconut palms in a private
property near the University of Calicut campus
(11°7′N, 75°53′25″E) seriously infested with
A.
gureronis
were used for the field trial. At the onset of
South-West Monsoon (June), 10 g crude
solid-fermented obtained from potato flour fermented
medium (48 h) was deposited manually in the adaxial
side of each leaf base (leaf pocket) of all exposed
leaves with or without a bunch of nuts or
inflorescence. About 15 such deposits were made per
each coconut palm.
Statistical analysis
Lethal concentration LC
50
value of
Btk
formulation
against
A. guerreronis
was estimated from the
regression equation of
p
value from the figure,
i.e
.,
p
=
Intercept + BX (where
p
is the computer generated
table value corresponding to the concentration of the
toxin, and X is the calculated value on log
10
). Antilog
of X gives the LC
50
dose. Minimum 3 parallel studies
were conducted for each datum. SPSS (statistical
Package for the Social Sciences) version 20 was used
for the probit (p) analyses.
Conclusion
As seen in the probit values, we demonstrated that the
crude
Btk
-toxin in solid-fermented matter was 18 folds
more efficient than LB control. It indicates the
enhanced production of
δ-
endotoxin by SSF. The only
difference between the two media used was that the
latter contained 10% (w/v) potato flour extra.
Moreover, the harvest from potato flour supplemented
medium (at 48h) was 24 h ahead of the LB control
(72h), thus the gestation period for the maximum
production of
δ-
endotoxin has been reduced by 24h.
The coconut growing counties predominantly are in
the Third World, which look forward for inexpensive
methods for combating
A. guerreronis.
Our strategy
shows that one time applications of crude solid
fermented matter per palm is sufficient to control the
