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ABSTRACT
Integrated Pest Management (IPM) and bio-intensive pest management (BIPM) (where Bt biopesticide is an indispensible
component of it) are the suggested alternatives of chemical pesticides. So, a holistic approach to the isolation and detection
of potential Bt strains, production at industrial scale and administration in the field is necessary to include Bt biopesticide
in the IPM and BIPM of Bangladesh. In this connection, the bioinsecticide prepared from potential indigenous Bt strain
JSc1 was applied in cabbage, cauliflower and organic tea farming and was found to be efficient in controlling the target
lepidopteran pests such as Helicoverpa armigera, Spodoptera litura, Plutella xylostella, Hyposidra spp. etc. Results
indicated that more than 85% of the treated crops were protected from the infestation and destruction by the pests.
Obtained data analyzed using ANOVA test suggested the inclusion of Bt biopesticide in the IPM of Bangladesh as no
such differences were observed with the chemical pesticides currently in use.
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MATERIALS AND METHODS
Bacterial strains and culture conditions
Reference Bt strain kurstaki HD-73, kindly provided by Okayama University, Japan and indigenous Bt
strain JSc1 were used in this study. LB agar (per litre: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g)
and LB broth were used for culture maintenance, propagation, subculture and spore count throughout the study
and incubation temperature was maintained at 30C for all types of cultures and the liquid cultures were
incubated in an orbital shaker at 180 rpm if not otherwise stated.
Inoculum preparation
An isolated colony from overnight incubated LB agar medium plate at 30C was dispensed in 3.0 ml of LB
medium and incubated overnight at 30C. Aliquots (0.2 ml) were used to inoculate 250 ml Erlenmeyer flasks
containing 50 ml LB medium. After 6h of incubation at 30C and 200 rpm, the cell density was determined at
600 nm and was used as inoculum in amounts to start with an initial OD 600nm = 0.1.
Bio-insecticide production in 3.0 L bioreactor
Bt bioinsecticide production was carried out at 30C in a 3.0 L fully controlled bioreactor (New Brunswick
Scientific, USA) containing 2.0 L of finally optimized medium (10% soybean extract, 0.5% molasses, 20%
(v/v) sea water, 300 mg/L cystine and 4% ammonium sulfate) (Mourin et al., 2015) with continuous regulation
of pH using 2N HCl and 2N NaOH. Dissolved oxygen (dO 2 ) level in the medium was automatically controlled
and maintained at 30% by providing aeration at 1.0 standard liter per minute (SLPM) and agitation at 250 rpm.
Fermentation was carried out up to 24 hours for maximum - endotoxin yield.
Determination of spore and - endotoxin
Number of spores and - endotoxin concentration were estimated at the end of the fermentation process.
For spore count, 1.0 ml of the fermentation broth was heated at 80C for 10 min and 100 l of appropriate
dilution after serial dilution was inoculated on LB agar medium by spread plate technique. After overnight
incubation at 37C, the colonies were counted and multiplied by the dilution factor to estimate the exact
numbers. The crystal protein was partially purified following the method described by (Mourin et al., 2015)
where 1.0 ml of fermentation broth was washed twice with cold sterile distilled water, once with 1.0 M NaCl
and 5.0 mM EDTA and once with 5.0 mM EDTA alone. The washing steps were accomplished each time by
centrifugation at 10000 rpm for 10 min. Finally, the washed pellet was resuspended in 1.0 ml of 0.1 N NaOH
solution for 1 h at room temperature. Then, the partially purified crystal protein concentration in the
supernatant was estimated according to Bradford method (Bradford, 1976).
Field trial
Bt preparation containing both Cry proteins and spores was applied
in the field. The efficacy was analyzed both qualitatively and
quantitatively against the caterpillars in comparison to a positive control,
chemical pesticide and a negative control, water treatment usually. Bt
preparations were applied at an optimum concentration of 0.3 mg/ ml of
alkali soluble proteins i.e. crystal protein.
Application of Bt preparation in cabbage and cauliflower farming
The efficacy of Bt JSc1 spore-crystal protein preparation was tested
against the pests of cabbage and cauliflower in Narshingdi. Bt
preparation was applied besides control (water) and chemical pesticides
(lambda cyhalothrin, emamectin benzoate) to control the common
cabbage and cauliflower pests such as Helicoverpa armigera,
Spodoptera litura, Plutella xylostella, Hyposidra spp. etc which belong
to order Lepidoptera. Treatments were applied following randomized
complete block design (RCBD) in cabbage and cauliflower farming (Fig.
1A). Treatments were both replicated and blocked along 4 blocks, each
containing 3 replicates. Experiment was performed with 360 plants, 30
plants in each replicate and 90 plants in each block. Survival data of
plants from pest infestation and damage from each replicate was
collected and statistically analyzed.
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Hyposidra spp. twice in the year (March-April and June-July). In both cases, 60 matured tea bushes were
selected for experiments and no pesticide was applied to the bushes since trimming (Fig. 1B). Treatments were
applied weekly and the numbers of plants survived from pest infestation were recorded for individua l
treatments. Data were analyzed statistically by ANOVA test and their efficacies were compared.
RESULTS AND DISCUSSION
Efficacy of Bt preparation in cabbage and cauliflower farming
Qualitative results suggested that the efficacy of Bt biopesticide and chemical pesticide in protecting the
cabbage and cauliflower from pest infestation were equal (Fig. 2). As cabbage and cauliflower damaging
lepidopteran pests such as H. armigera, S. litura, P. xylostella, Hyposidra spp., were challenged by sporecrystal preparation from Bt JSc1 in parallel to chemical pesticide and control treatments, Bt biopesticide
preparation was found to exert almost similar bioactivity against the pests with cabbage and cauliflower as did
the chemical pesticide. At the final stage of the farming, it was observed that the untreated plants were
completely damaged whereas chemical pesticide or Bt biopesticide treated plants were protected and the
healthy cabbage and cauliflower were grown. For cabbage, more protectionwas provided by Bt biopesticide
than the chemical pesticide but for cauliflower, it was opposite with slight difference. Quantitative analysis of
the treatments revealed that both Bt biopesticide and chemical pesticide treatments protected about 90% of the
plants whereas only 25% plants survived with negative treatment i.e. untreated plants (Fig. 3A). Average
weight of cabbage was slightly higher for Bt biopesticide (1.885 kg) than that of chemical pesticides (1.855
kg) and it was opposite for cauliflower (Bt- 1.36 kg, and Chemical- 1.56 kg). The average weight of survived
untreated plants was almost 38% less in both cases (cabbage- 1.165 kg and cauliflower- 0.918 kg) (Fig. 3B).
The condition of untreated plants indicates the level of pest infestation which was very high in the
experiment as survival of untreated plants were only 25% and their weights were almost 38% less from the
treated plants. The efficiency of Bt biopesticide based on these two criteria suggests that the replacement of
chemical pesticide will provide food safety as a surplus over the food security without affecting the production
yield. Performance of the Bt biopesticide preparation was more effective on cabbage farming over cauliflower.
This might be due to the difference in their textures which facilitated more protection in cabbage than in
cauliflower.
Efficacy of Bt preparation in organic tea farming
Efficacy i.e. the mean survival percentage of tea plants with Bt biopesticide (53.8%) was found to be very
close to the other treatments i.e. garlic (51.6%) and neem formulations (44.3%), currently in use in organic tea
garden. The maximum number of survived plants (more than 75%) in the 4th week was recorded for Neem
formulation treatment and was dropped as the treatment was stopped after 4th week, followed by a resume in
the 6th week. On the other hand, relatively lower survival percentages were recorded for Bt biopesticide and
garlic formulation initially but both of them retained their impacts with time even with an interruption (Fig.
4). Both Bt biopesticide and garlic formulation protected maximum numbers of plants after 10th week of their
application but neem formulation could not regain its initial protection efficiency level.
The drastic drop in protection by neem formulation might be due to its short residual activity and thus the
larvae exposed to it might have survived. Adding to this, resistance might have evolved with them and
transferred to the offspring. But this was not evidenced for Bt biopesticide preparation and garlic formulation.
Although the variance was observed to be higher for Bt biopesticide, this does not imply random and higher
fluctuation of performance rather consistent improvement of protection over time.
Statistical Analysis of Variance
Cabbage and cauliflower farming
The statistical comparison of the efficacy of Bt biopesticide to the chemical pesticide by ANOVA (TwoWay) produced the F values which indicated that at = 0.05, significant differences among the treatments
prevailed both in cabbage (Bt biopesticide, Chemical pesticide and Control) (F= 175.658; df=11;
P-value= 0.00000473) and cauliflower (Bt biopesticide, Chemical pesticide and Control) (F= 661.151; df =
11; P-value = 0.0000000922) farming (Table 1). As significant differences among the treatments were
determined statistically (Table 1), pair wise difference analyses among the multiple treatments were calculated
by Fisher Least Significant Difference (LSD) method.
The analyzed data provides much evidences to conclude that, at = 0.05 level of significance, Bt
biopesticide and chemical pesticide did not differ significantly whereas the pairs of Bt biopesticide and control
as well as chemical pesticide and control differed significantly in cabbage farming. It means that the efficacy
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Table (1): Statistical data obtained from the ANOVA (Two- Way) test performed on the average survival of
cabbage and cauliflower plants from the pest infestation.
ANOVA for Randomized Complete Block Design (RCBD)
Source of
Variation
Cabbage
Cauliflower
Df
SS
MS
P-value
df
SS
MS
P-value
Treatment
6021
3010.6
175.658
0.00000473
6795
3398
661.151
0.0000000922
Block
89
29.6
1.724
0.261
103
34
6.676
0.0244
Residual
103
17.1
31
Total
11
6213
11
6929
Table (2): Fisher Least Significant Difference (LSD) Method for Multiple Comparison Test
Group vs Group (Contrast)
Biopesticidevs Chemical pesticide
Biopesticidevs Control
Chemical pesticide vs Control
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Table (3): Statistical data obtained from the ANOVA (One- Way) test performed on the average survival of
tea plants from the pest infestation
Summary (Organic Tea farming)Treatments
Bt biopesticide
Garlic formulation
Neem formulation
Total
Sample size
11
11
11
33
Mean
53.8181
51.6363
44.2727
49.9090
Variance
32902
30448
23221
136.5852
df
2
30
32
SS
550.3636
3820.3636
4370.7272
MS
275.1818
127.3454
F
2.1609
p- level
0.1328
F crit
3.31583
Omega Sqr.
0.06573
of Bt biopesticide and chemical pesticide in controlling the pests in cabbage existed highly in comparison to
the no treatment. But there was insignificant difference between these two efficient treatments (Table 2).
Again, in cauliflower farming, similar results were observed i.e. Bt biopesticide and chemical pesticide did not
differ significantly at = 0.05, whereas the pairs (Bt biopesticide and control and chemical pesticide and
control) differed significantly at = 0.05.
These results suggest that the substitution of chemical pesticide currently in use in cabbage and cauliflower
farming with the Bt biopesticide preparation will be feasible in terms of controlling pests keeping food safety
and without affecting the production yield.
The experiment was performed in randomized complete block design (RCBD) where treatments (Bt
biopesticide, chemical pesticide and negative control) were blocked and replicated. This design is most
effective if the patterns of non-uniformity (changing soil types, drainage patterns, fertility gradients, direction
of insect migration into the field, etc.) in a field were identified. If the extraneous variability associated with
field was not removed prior to testing for a treatment effect, it would be more difficult to detect treatment
effects via F- test. In other words, the denominator in the F- test would be larger than needed because it contains
variability associated with field. If the potential sources of variation were not identifiable, this design can still
be used keeping the blocks as square as possible which usually keeps the plots within a block most uniform.
The goal of RCBD was to maximize the differences among the blocks while minimizing the differences within
the block so that statistically sound unbiased data are produced.
Organic tea farming
Statistical data obtained from the ANOVA (One- Way) produced an F value indicating that at = 0.05,
there was insignificant difference between the mean survival of tea plants for all the treatments throughout the
field trial replicates (F= 2.1609; df= 32; P = 0.1328). This inferred that these three treatments, Bt biopesticide
formulation, garlic formulation and neem formulation were of equal effects in organic tea farming (Table 3).
Hence, Bt biopesticide preparation can be used in tea farming in parallel to other biopesticides in use and for
its longer residual activity as well as lost cost, it will be highly potential eco-friendly biological pest control
agent in organic tea farming.
ACKNOWLEDGMENT
This study was partly supported by the grant of USDA Agricultural Biotechnology research program
coordinated by Ministry of Education, Government of Bangladesh. We also acknowledge Nurjahan Akhter
Laboni, graduate student at Institute of Statistical Research and Training, University of Dhaka, for her
statistical analysis and supports.
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