Vol.5, No.11, 1139-1144 (2013) Natural Science
http://dx.doi.org/10.4236/ns.2013.511140
Production of low cost Bacillus thuringiensis
based-biopesticide for management of chickpea
pod-borer Helicoverpa armigera (Huebn) in Pakistan
Abida Bibi1*, Khalique Ahmed2, Najma Ayub1, Sadia Alam3*
1Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan; *Corresponding Author:
2KarakuramUniversity, Gilgit, Pakistan
3Departmentof Microbiology, University of Haripur, Haripur, Pakistan; *Corresponding Author:
Received 30 April 2013; revised 30 May 2013; accepted 7 June 2013
Copyright © 2013 Abida Bibi et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The biopesticide was prepared from locally
available low cost ingredients: dried beef blood,
molasses and mineral salts (ZnCl2, MgCl2, MnCl2,
CaCl2CaCl2, and FeCl3) which were used as me-
dium for the laboratory scale production of Ba-
cillus thuringiensis (B.t.) bio-pesticide by shake
flask technique. Indigenous B.t. isolate PA-Sb-
46.3 which produced two crystals—bi-pyramidal
and cuboidal was found 73.6 times toxic against
H. armigera than reference strain Bacillus thur-
ingiensis var. kurstaki (HD-I-S-1980) used. Me-
dium was fermented for 72 hours at 30˚C ± 2˚C
and 160 rpm. 72 h fermented medium showed
95% - 99% sprulation, with spore yield of 3.97 ×
109 spores/ml, and LC50 value to 1st instar larvae
of H. arm igera was 0.53 μg/ml diet. Preservatives
and diluents used in the biopesticide were found
to be effective to store at room temperature over
a period of 30 months. These observations sug-
gested that the biopesticide produced was ef-
fective and highly economical for the industrial
scale production to manage H. armigera in Paki-
stan.
Keyw ords: Beef Blood; Molasses; Indigenous B.t.
Isolate PA-Sb-46.3; Sporulation; Entomotoxicity;
Biopesticide
1. INTRODUCTION
Among the insect pests, chickpea pod-borer, Heli-
coverpaarmigera occupies a prominent position and has
attained a status of topmost agricultural pest in Pakistan,
China and India [1]. The most destructive pest H. ar-
migera is polyphagous in nature and has been recorded by
Iqbal, and Mohyuddin, 1990 [2] on 62 host plants, and on
more than 100 plant hosts by different researchers [3-6].
Synthetic chemical insecticides for the control of in-
sect pests are commonly used in Pakistan which are not
only expensive but also have created a number of prob-
lems like water pollution, biological and environmental
hazards, and development of resistant in pests [6]. H.
armigera has also shown resistance to various groups of
chemicals in Pakistan. These problems have increased the
importance for the development and the use of safe and
target specific bio-insecticides, e.g., Bacillus thuringien-
sis (B.t.).
The entomopathogenic bacterium (Bacillus thurin-
giensis (B.t.)) during sporulation produces crystalline
proteins which are associated with the insecticidal activity
of B.t. against different insects larvae of Lepidopteran,
Dipteran, Coleopteran and some other pests. These crys-
talline proteins are harmless to humans, vertebrates and
plants, are completely biodegradable and cause no toxic
residual products to accumulate in the environment. That
is why this bacterium is widely used as microbial insec-
ticide in agriculture and forestry for the control of pests,
and in human health sector for the elimination of disease
vectors [7].
Hence due to the safety associated with B.t., tremen-
dous interest has been developed towards the production
of a new commercial B.t. products. B.t. products are being
used successfully on large scales in India, China, US,
Australia and many other countries of the world whereas
in Pakistan no attention has been given towards their
production and use. The main objective of this diligent
effort is the production of low cost and effective B.t. bio-
pesticide by a simple and effective process (shake flask/
fermentation technology) to manage H. armigera in-
festing chickpea in Pakistan.

A. Bibi et al. / Natural Science 5 (2013) 1139-1144
1140
2. MATERIALS AND METHODS
2.1. Laboratory Scale Production of
B.t.-Biopesticide against
Helicoverpa armigera
Biopesticide was prepared in liquid form with spore
counts ×109/ml by using shake flask/fermentation tech-
nology [8]. Indigenous B.t. isolate, PA-Sb-46.3 that had
bipyramidal and cuboidal crystals and was 73.6 times
toxic against H. armigera than reference standard strain
B.t. var. kurstaki (HD-I-S-1980) used in this study Toxic-
ity was evaluated against 1st instar larvae of the test in-
sect chickpea pod-borer, H. armigera mass reared on
artificial diet according to the method reported by scien-
tists [9,10].
2.1.1. Composition of Medium
Local available inexpensive raw material was selected
as culture medium for the laboratory scale production of
B.t. biopesticide which comprised, dried beef blood 30.0
g, molasses 15.0 ml, CaCl2 0.03 g and salt solution 1.0
ml (ZnCl2, MgCl2, MnCl2, CaCl2, FeCl3 and 5 drops of
HCl) in1000 ml distilled water.
2.1.2. Preparation of Medium
Beef blood obtained from slaughterhouse was dried in
an oven at 90˚C for 24 hour. Dried blood crystals were
ground and sieved through muslin cloth in order to ob-
tain a very fine powder. Thirty gram of dried beef blood
powder was added into flask containing 350 ml of dis-
tilled water. Flask was tightly plugged with cotton wool
covered with polythene paper to avoid absorbance of
suspension in the cotton plug during shaking. This flask
was placed in a shaking water bath for proper mixing of
blood in distilled water at 160 rpm for 03 h at 30˚C ±
2˚C.
The blood-water mixture was transferred into flask of
one litre capacity and other contents of medium i.e., mo-
lasses 15 ml and CaCl2 0.03 g were added and volume
was made up to one litre with distilled water. Flask was
placed into water bath for an hour at 80˚C with continu-
ous stirring for proper mixing of medium ingredients.
This mixture was then sieved through six-fold of muslin
cloth in order to remove the traces of solid beef blood.
This process was repeated twice to obtain suspension
free of solid blood traces. The volume was again made
up to one litre by adding some distilled water, which was
evaporated during heating. The medium was then equally
distributed into ten flasks (500 ml capacity) in such a
way that each flask contained 100 ml of the medium. All
the flasks were tightly plugged and autoclaved at 121˚C
for 20 minutes at 15 lbs/inc2.
2.1.3. Inoculation of Medium
When medium was cool down approximately to 60˚C
then each flask was inoculated with 100 µl of inoculum
which was prepared by adding loop full from 72 h old,
pure 95% - 98%. sporulated B.t. culture (examined in a
phase contrast microscope (PCM) at 100×) into 5.0 ml of
sterile salt solution and vortexed. Smear was prepared
from this suspension and examined in PCM to check the
purity of B.t. culture.
2.1.4. Fermentation of Medium
Inoculated flasks were placed in a shaking water bath
for 72 hours at 30˚C ± 2˚C and 160 rpm. After 72 h
flasks were removed from shaking water bath, smear was
prepared from each flask and examined in a PCM in or-
der to check the purity and growth stage of fermented
culture. Flasks that contained pure and 95% - 98% spo-
rulated culture was pooled into flask of 2-litre capacity.
2.1.5. Suitability of Medium for the Growth of B.t.
Medium giving high yield of spore and crystal were
considered suitable for the production of biopesticide and
was further subjected for toxicity evaluation through
bioassay.
Bioassay of fermented culture Diet reported by scien-
tists was used for all bioassays experiments performed
during this study [10]. Six different concentrations i.e.,
0.5%, 1%, 2%, 4%, 8% and 16% of fermented culture of
biopesticide were prepared in total volume of 400 ml
artificial diet (Table 1).
Four hundred milliliter diet of each concentration was
prepared by mixing fermented culture with diet as given
in Table 1 and poured with the help of pouring bottles in
glass vials and allowed to cool and solidify. Separate
pouring bottles were used for each concentration and
control. Four replicates were maintained for each con-
centration and each replicate comprise 25 vials. Similarly
four replicates of control without fermented cultures
were also carried out in parallel.
Each vial containing intoxicated diet and that of con-
trol was aseptically infested with the single 1st in star
larvae with sterilized soft camel hair brush. Vials were
tightly plugged with sterilized cotton wool and incubated
Table 1. Concentration of fermented culture used in diet for
bioassay.
S. NosConcentrationFermented
culture “A”
Diet
“B”
Total volume
“A” & “B”
1 0.5% 2.0 ml 398 ml 400 ml
2 1% 4.0 ml 396 ml 400 ml
3 2% 8.0 ml 392 ml 400 ml
4 4% 16.0 ml 384 ml 400 ml
5 8% 32.0 ml 368 ml 400 ml
6 16% 64.0 ml 336 ml 400 ml

A. Bibi et al. / Natural Science 5 (2013) 1139-1144 1141
in an inverted position at 26˚C ± 2˚C for 7 days. At the
end of incubation period, experiment was terminated and
mortality was recorded by counting dead and alive larvae
in each vial The LC50 values of the toxins were worked
out through probit analysis computer programme [11].
2.1.6. Preparation of Biopesticide Formulation
Storage of biopesticide for a long period at room tem-
perature diluents and preservatives i.e., Glycerol 10.0 ml,
Boric acid 10.0 g, Corn starch 35 g, Methyl-Para-hy-
droxybenzoate 10.0 g were added into one litre fresh
fermented B.t. culture and mixed well by shaking. The
pH was noted and placed in a dark room.
2.2. Bioassay of Biopesticide Formulation
Toxicity of biopesticide formulation was evaluated
against larvae of the H. armigera using the same method
and concentrations in 400 ml of diet as used for fer-
mented culture.
2.3. Influence of Diluents and Preservatives
Influence of preservatives and diluents on toxicity of
biopesticide was also noted by comparing values: pH,
CFU × 109/ml and LC50 μg/ml diet of the fermented cul-
ture and biopesticide formulation formed after adding
diluents and preservatives. Diluents and preservatives
showing no effect on toxicity was considered suitable for
biopesticide preservation and shelf life.
2.4. Storage and Shelf Life Evaluation of
Biopesticide
Five more batches (each batch of one litre) of biopes-
ticide were prepared by the same procedure as used for
the production of first batch. The six batches were com-
bined and toxicity was evaluated; pH was checked and
stored at room temperature in a dark room. Bioassay
experiments were also conducted against H. armigera at
three month’s time interval over a period of 30 months in
order to measure the stability period of toxin and effec-
tiveness period of preservatives and diluents in the bio-
pesticide placed at room temperature.
Concentrations 4%, 8% and 16% of pooled batch of
biopesticide showed 100% mortality (Figure 1). There-
fore shelf-life was evaluated with reduced concentrations
i.e., 0.125%, 0.25%, 0.5%, 1.0%, 2.0%, and 4.0%, (same
for all experiment i.e., 1st to 10th) of the biopesticide pre-
pared in total volume of 400 ml of artificial diet (Table
2).
All bioassay steps and method of determination of
LC50 values were same as used for the toxicity evaluation
of fermented culture.
Viable spore counts colony forming units (CFU) in
1.0 ml of biopesticide was calculated for each batch of
0
20
40
60
80
100
120
1234567
Concentration of toxin
Mortality %
Figure 1. Pooled batch of biopesticide.
Tab le 2. Preparation of different concentrations of biopesticide
in artificial diet.
S.Nos Concentration
(biopesticide)
Biopesticide
“A”
Diet
“B”
Total volume
“A” & “B”
1 0.125% 0.5 ml 399.5 ml 400 ml
2 0.25% 1.0 ml 399 ml 400 ml
3 0.5% 2.0 ml 398 ml 400 ml
4 1.0% 4.0 ml 396 ml 400 ml
5 2.0% 8.0 ml 392 ml 400 ml
6 4.0% 16 ml 384 ml 400 ml
one litre, pooled batch of six litres and after every three
months during shelf-life evaluation (Table 3). For this
purpose 1.0 ml of biopesticide was added into 9.0 ml of
sterile distilled water in sterilized test tube and vortexed.
Ten fold dilutions of this sample was prepared and 100
µl of dilution 10−7 was spread-plated (four replicates) on
the surface of solid BGM after heat shock at 80˚C for
10.0 min and incubated at 30˚C for 24 h. After 24 h,
colonies showing B.t. like morphology were counted
under magnifying lens and confirmed as B.t. by ran-
domly selecting 5 - 6 colonies per plate and observing in
phase contrast microscope. Mathematical average of four
replicates was calculated. Number of CFU/mg was cal-
culated by the following formula:
Colony Forming Unitsmg
Number of colony forming units
mlDilution factor


3. RESULTS
3.1. Suitability of Medium Selected for the
Preparation of Biopesticide
Results of fermented media as indicated in Table 4
revealed that the selected media was suitable for the
growth of B.t. and the production of biopesticide as B.t.
showed 95% - 99% sporulation and produced high yield
of crystal after 72 h at adjusted conditions i.e., rpm,
temperature time and size of flask. It also showed high

A. Bibi et al. / Natural Science 5 (2013) 1139-1144
1142
Table 3. Effect of preservatives and diluents on the toxicity of
biopesticide.
Culture pH spores × 109/ml LC50 μg/ml diet
Fermented culture 7 3.97 0.53
Biopesticide formulation 7 4.25 0.50
Table 4. Toxicity of different batches and combined batch of
biopesticide against H. armigera.
Batches of
biopesticide
Biopesticide
pH
No. of spores/ml
(×109) LC50 μg/ml Diet
1 7 4.25 0.50
2 7 4.54 0.39
3 7 3.61 0.91
4 7 3.89 0.69
5 7 4.37 0.93
6 7 3.81 0.65
Pooled batch 7 4.01 0.59
toxicity i.e. LC50 value against 1st instar larvae of H. ar-
migera 0.53 μg/ml diet.
3.1.1. Influence of Preservatives and Diluents on
the Toxicity of Biopesticide
Toxicity results of fermented culture and biopesticide
formulation against 1st instar larvae of H. armigera as
shown in Table 5 indicated no effect of preservatives and
diluents on pH, number of spores ×109/ml and toxicity as
they did not reduce or enhance toxicity because there is
no difference between the LC50 value of fermented cul-
ture and LC50 value of biopesticide formulation prepared
from the same fermented culture.
3.1.2. Toxicity Evaluation of Biopesticide
The LC50 value of six batches and pooled batch of
biopesticide as in Ta b l e 6 showed that biopesticide was
effective against H. armigera. Their pH value and spores
×109/ml were also given. These values also indicated that
no correlation exists between number of spores and tox-
icity of biopesticides as the toxicity value did not in-
crease or decrease with increase or decrease of number
of spores.
3.2. Mortality Increase with the Increase of
Toxin Concentration
Mortality % observed in six batches and pooled batch
of biopesticides given in Figures 1 and 2 indicated that
with the increase of toxin concentration in diet, mortality
of larvae also increased.
Figure 2. Mortality % of different concentrations of H. ar-
migera.
3.2.1. Shelf Life Evaluation of Biopesticide
Bioassay experiments performed as results indicated
in Table 3 at three month’s time interval over a period of
30 months showed that there was gradual decrease in the
toxicity of biopesticide after nine months but no change
in the number of spores and no contamination was ob-
served during this time period. Biopesticide was still
considered effective because its LC50 μg/ml diet is 4.95
which is slightly higher than LC50 value that is 4.54
μg/ml diet 4.54 of the same isolate PA-Sb-46.3.
3.2.2. Effectiveness of Preservatives and
Diluents in Biopesticide
Results of shelf-life evaluation of biopesticide (Table 3)
indicated that preservatives and diluents used were effec-
tive because no: 1) change in pH value 2) significant
change in number of spores 3) growth of other microor-
ganism (bacteria and fungi) was observed during spore
counting over a period of thirty months
4. DISCUSSION
Due to the safety associated with B.t. production sci-
entists all over the world are trying to produce effective
B.t. biopesticide by using inexpensive and locally avail-
able raw material as medium in order to lower the pro-
duction cost and compete with the commercial B.t.
products. Obeta and Okafor (1984) formulated five dif-
ferent media from the seeds of legumes dried beef blood
and mineral salts and assessed growth and production of
insecticidal toxins of B.t. which were effective against A.
aegypti, C. quinquefasciatus, A. gambiae [12]. Bioor-
ganic wastes (chicken feathers) as medium for the pro-
duction B.t. biopesticide were also used. A similar at-
tempt has been made here for the production of effective
B.t. biopesticide against H. armigera by selecting local,
cheap and easily available medium ingredients consisting
of e.g., molasses as a carbohydrate source, beef blood as
protein source and salt solution as inorganic ions for the
enhancement of sporulation. Similar requirements are
reported by Prasertphon (1996) that the sporulation and
rystal production depends upon a source of carbon, ni- c

A. Bibi et al. / Natural Science 5 (2013) 1139-1144 114 3
Table 5. Characteristics of fermented medium of biopesticide.
Fermented medium
B.t. isolate Medium Composition
Time pH Sporulation CFU/mL (×109) LC50 mg/mL diet
PA-Sb-46.3 Blood, Molases, CaCl, Distilled water 72 h 7 95% - 99% 3.97 0.53
Table 6. Shelf life evaluation of biopesticide.
S.Nos Interval
(in months)
Time period
(in months) pH No. of spores/ml (×109)
LC50 μg/ml
diet
1 00 (Fresh) 00 7 3.89 0.59
2 3 03 7 3.71 0.23
3 3 06 7 4.40 0.30
4 3 09 7 3.55 1.22
5 3 12 7 4.27 1.83
6 3 15 7 3.21 2.08
7 3 18 7 3.70 2.34
8 3 21 7 3.45 3.23
9 3 24 7 4.30 3.80
10 3 27 7 3.65 4.14
11 3 30 7 3.93 4.95
trogen, minor elements and growth factor [8]. Bernhard
and Utz (1993) stated that sporulation was stimulated by
the inorganic ions particularly Ca+2 and Mg+2 [13].
Medium contained entire nutrient in proper ratio nec-
essary for the optimal growth of B.t. and for the produc-
tion of crystal in good quality and quantity. During proc-
ess of bacterial growth no smell of ammonia was ob-
served indicating an appropriate amount of protein in the
medium These results were also in agreement with the
result reported by Prasertphon (1996) who stated that
good quality and quantity of crystal production can be
achieved by carefully balancing the nutrition ratio i.e.,
carbohydrate and protein in the medium [8]. If the nutri-
tion ratio is not properly balanced it will prevent the B.t.
to sporulate. Insufficient protein in the medium results
poor quantity of crystal and too rich of protein results in
bacterial lysis giving odor of ammonia.
The neutral pH of fermented culture and biopesticide
formulations was observed that indicated appropriate
amount of carbohydrates and protein in the media. Simi-
lar pH was also reported by Morris et al., 1998 [14].
Conditions for shake flask technique such as temperature,
rpm, time size of flask was suitable for the growth of B.t.
Temperature, rpm and time were in close agreement with
the values reported by Yusoff et al., 2003 [15].
Insect bioassay is the best way of finding the toxicity
of B.t. preparation. All bioassays results showed that the
fermented culture and biopesticide formulation were
toxic against the H. armigera and was suitable to control
them different concentrations used showed that with the
increase of toxin, mortality also increased. Ahmed et al.,
(1996) have also reported similar results of B.t. var
kurstaki against H. armigera. They found with the in-
crease of toxin concentration mortality was also increa-
sed [16].
All bioassay results also indicated that there was no
correlation between the number of spores and toxicity
value of biopesticide which are in agreement with the
results that there is no relationship between the number
of spores in preparation and its insect killing power
which is true for lepidopterous active isolate [17].
Toxicity test of biopesticide stored at room tempera-
ture against H. armigera at three month’s time interval
over a period of 30 months indicated that B.t. biopesti-
cide can be stored at room temperature for more than 30
months without losing its effectiveness by using pre-
servatives and diluents. These results were in agreement
with Prasertphon (1996) results who stated that fresh
liquid B.t. culture could be stored at room temperature
fore more than 24 months without losing its effectiveness
by using preservatives and diluents [8]. Some character-
istic s such as no change in pH value, number of spores
and no growth of other microorganism (bacteria and
fungi) also supported these results.
5. CONCLUSION
Cost analysis of raw material used as culture medium
indicated that it is highly economical (7 US Dollars for
the production of 10 litre biopesticide formulation), and
bioassay result indicated that production of indigenous
B.t. biopesticide on commercial scale can serve as a sub-
stitute of chemical pesticides which will be a remarkable
achievement in the field of pest management in Pakistan.
It will reduce the use of highly toxic chemical insecticides,
save foreign exchange used in their import, help to control
insects that have developed resistance to chemical insec-
ticides and will save our environment from pollution
problems which are the major concerns at present.
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