Production of low cost <i>Bacillus thuringiensis</i> based-biopesticide for management of chickpea pod-borer <i>Helicoverpa armigera</i> (Huebn) in Pakistan

doi:10.4236/ns.2013.511140

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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: [email protected]

2KarakuramUniversity, Gilgit, Pakistan

3Departmentof Microbiology, University of Haripur, Haripur, Pakistan; *Corresponding Author: [email protected]

Received 30 April 2013; revised 30 May 2013; accepted 7 June 2013

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

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

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

OPEN ACCESS

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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