Journal of Biotechnology

and Crop Science

6(8): 34-39, 2017

Controling pod borer (Heliocoverpa armigera) by natural toxin(s) in pigeon pea

NA Khan, Raja Husain, Anamika Kaushal, Pratibha Singh, KN Singh

Received: 09 February 2017 Revised Accepted: 13 April 2017

ABSTRACT

Among various constraints pigeon pea production, the insect pest has one of the prime importance. Helicoverpa armigera
is one of the most devastating pests of this crop. Excessive use of chemical pesticides has resulted in environmental
degradation, adverse effects on human health and other organism. For eradication of this insect, the researchers have
shifted from chemical to biological control. The naturally killed larvae of Helicoverpa were crushed and growth was
tested on LB agar plate, which got the Helicoverpa larvae dead due to bacterial micro-organism and growth on PDA
media indicated the presence of fungi. The protein profiling of bacterial protein on (SDS-PAGE) 12%, indicated very low
molecular weight of protein which was responsible for death of Helicoverpa armigera. The ammonium sulphate fraction
of bacterial protein was applied @ 30%, 60% and 70% on live larvae. The rate of mortality was maximum in case of
70% ammonium sulphate fractionated protein.

Key Words: Ammonium Sulphate, PDA media, SDS-PAGE, Sephadex G-75

INTRODUCTION

Pigeon pea is an important pulse/grain legume and
commonly known as red gram or arhar. It is the rich
source of protein, iron, iodine and essential amino
acids likewise methionine, lysine, tyrosine (Alykroid
and Doughty 1964) and also supply vitamins-B,
minerals and fats. The presence of different type of
proteins and their smaller molecules including
alkaloids, is flavones, polyphenols and a variety of
oligosaccharides make legume seed unique in
providing nutraceuticals. Asia produces 3.00 million
tones grains from 3.81 million hectare area with 789
kg/ha productivity. India accounts for 78% of global
output with current production of 2.45 million tones
from 3.80 million hectare recording average yield of
686 kg/ha. In UP it is grown on 0.33 million hectare
area producing 0.30 million tones of grain with
average yield of 910 kg/ha (FAO, 2006). Among
NA Khan ( ), Raja Husain, Anamika Kaushal, Pratibha Singh
Department of Plant Molecular Biology & Genetic Engineering,
Faizabad
Email-nakhan0110@gmail.com
K N Singh
Department of Biochemistry, NDUA&T Kumarganj, Faizabad
(U.P.) 224229
various constrains of pigeonpea production, the
insect pest are of prime important. Pigeon pea is
crop of intermediate plant type with long duration
hence serves as an ideal host for many insect pests.
Disease and insect pest are estimated to cause yield
losses from 470 to 988 kg ha-1 (FAO STAT, 2008).
More than 200 insect species have been reported
infesting this crop. Major pest of pigeon pea are pod
fly and pod borer. In India 3 species of Helicoverpa
are found viz. H. armigera, Helicoverpa assulta and
H. peltigera .The larvae of H. armigera destroys the
buds, flowers and pods. It bores inside the pod of
pigeon pea during vegetative state and damages
inside the pod. In the absence of pod larvae feeds on
foliage. The world wide annual losses caused by
H.armigera in pigeon pea are $ 317 million
(Shanower et al 1999). In India yield losses of
pigeon pea due to insect and disease ranged from
470 to 988 kg ha-1, which accounts for 29-85% of
grain yield in different cultivars. The non-judicious
use of chemical insecticides has led to the
development of resistance to one or more multiple
insecticides in over 500 species of insects (Akhurst

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2002). Both bacteria and Fungi can be pathogenic
and killed Helociverpa armigera. Bacillus
thuringeniesis (Bt) produces large insecticidal
protein crystals (ICPs) during sporulation (Khalique
and Ahmad 2002). In that the proteins are isolated,
purified and studied for their efficacy to kill insect
larval. Determination of the composition and
toxicity of the parasporal crystals, by means of SDS-
PAGE analysis and bioassay, is useful complement
for gene identification.
MATERIALS AND METHODS
Screening of different varieties of Pigeonpea
against H. armigera at different time interval (0, 6,
12, 18 hrs.): Larvae were collected from Pigeon pea
field and screened all ten varieties of Pigeon pea in
artificial/ lab condition. Two larvae were left on the
pods of each variety with three replications per
variety for feeding the pods. Initially the larvae were
weighted under control condition than after 6, 12
and 18 hrs, respectively. The reduction percentage in
each variety of pods was calculated by the mean of
R1, R2 & R3.
Collection of naturally killed larvae (Helicoverpa
armigera) for identification of micro- organism:
The naturally killed larvae of pod borer
(Helicoverpa armigera) were collected from the
experimental site of Genetics and Plant Breeding
Department and Student Instructional Farm. The
dead larvae reared in 40C for identification of
microorganism. Dead larvae (Helicoverpa armigera)
were collected from pigeon pea field and each larva
was crushed in 0.1 M sodium phosphate buffer, and
stored it at 40C for streaking on L.B agar media,
growth occurred on L.B agar plate indicates the
bacterial growth.
Isolation of protein from bacteria and characterizat
ion of protein (SDS-PAGE): In 500 ml liquid L.B
media, the colony of strain of bacteria was
inoculated and kept the media in shaker at 370C for
overnight and centrifuged at 5000 rpm for 10 min
and dissolved pellet in 5 ml phosphate buffer and
J of Biotech & Crop Sci (2017) 6(8): 34-39
500 l TCA (Tri chloro acetic acid) and left it for 3-
4 hours on ice. Again centrifuged it, at 5000 rpm for
10 minutes and dissolved pellet in 250 l phosphate
buffer and 50 l of sample was electrophoresed on
SDS-PAGE.
Purification of bacterial protein by ammonium
sulphate fractionation: The bacterial crude protein
were precipitated using ammonium sulfate at
different saturation level such as 30%, 60%.and
70%. For the preparation of 30% saturation 1.15g
ammonium sulfate was dissolved in 10 ml of crude,
stirred 30 minute by magnetic strirrer at 40C. After
30 minutes, the sample was centrifuged at 10,000
rpm for 20 minute at 40C. The supernatant was
collected for 60% ammonium sulfate fractionation
and pellet was dissolved in small amount of same
phosphate buffer the dissolved pellet was 30%
ammonium sulfate fractionated protein. For 60%
fractionation, 1.22g ammonium sulfate was
dissolved in 10 ml of 30% supernatant then again it
was stirred 30 minute by magnetic stirrer at 40C.
Then sample was centrifuged at 10, 000 rpm for 20
minutes at 40C. Supernatant was collected for 70%
ammonium sulfate fractionation and pellet was
dissolved in small amount of same phosphate buffer.
The dissolved pellet was 60% ammonium sulfate
fractionated protein.
Dialysis of 30%, 60% and 70% ammonium sulfate
fractionated protein of bacteria: The purified
protein of bacteria (30%, 60% and 70% ammonium
sulfate fractionated protein) was filled inside the
dialysis tube and sealed both side with plastic plug.
It was put in the same phosphate buffer (sodium
phosphate buffer 0.1 M, pH = 7.0) and dialyzed for
4-8 hours. These salt free purified proteins were
used for mortality test. The single larvae were put on
the surface of petridish above whatmann filter paper
moistened with dipped pod and leaves of purified
protein. Newly molted 3rd instars larvae was dipped
in protein fraction for continuously 3 days.
Thereafter, they were fed with fresh pigeon pea pods
till they died or pupate.
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Dialysis of Crude Protein: Preparation of Dialysis
tube in 0.1 mM EDTA and 5% Na2CO3 solution by
Boiling at 100 C in water bath. Fractionated
Protein sample of different ammonium sulphate
conc. were filled in dialysis tubes, each sample was
filled in separate dialysis tube. Closed the dialysis
tube at both end tightly so that sample could not
come out of tube. Filled 1 litre conical flask with 0.1
M phosphate buffer (pH 7) with approximately 500
ml to 800 ml. The conical flask was kept on
Magnetic stirrer for 8 hrs to 10 hrs. Finally dialyzed
sample was used for further analysis.
Identification of the correct fraction related to
mortality and gel electrophoresis of the identified
fraction: 100 l of 30%, 60% and 70% ammonium
sulfate fraction protein and live protein taken in
eppendorf tube and mixed with equal amount of
loading dye, heat it for 5-10 minutes for
denaturation. The denatured sample was loaded in
the well of gel and run for 8-10 hours. The complete
running, the gel was stained for six hours and
destained for gel documentation reading.
Purification of appropriate fraction on sephedex
G-100 column: Firstly the matrix (Sephadex G-100)
was suspended in large volume of water and left it
over night. Until, the gel was fully swollen after this
the bottom of column was plugged by the glass wool
and stands up right the column. After making a good
slurry of the matrix (stationary phases) in the
phosphate buffer, the matrix was poured in the
column with the help of same buffer. After this
fractionated protein sample was loaded in the
column, then the column was subsequently eluted by
sodium phosphate buffer. The flow rate was
controlled at 1 ml/minute. After collecting 12
fractions, each fraction was observed at 280 nm to
monitor the protein content in the test tube. After
this, the maximum peaks fractionation samples were
used as the source of protein.
RESULTS AND DISCUSSION
The larvae (Helicoverpa armigera) were responsible
for maximum damage of pod in pigeonpea. The
micro-organism (bacteria and fungi) when attacked
on Helicoverpa armigera secrete the toxin which
cause the death of Helicoverpa armigera. It is an
alternative option to Bacillus thuringienesis
endotoxin for controlling the insect pest. Cannan
(1996) had reported that the endotoxin crystal (Cry)
proteins produced in the bacteria are specific in their
insecticidal activity, viz., Cry 1/(Lepidoptera), Cry 2
(Lepidoptera and Diptera, Cry 3 (coleoptera), Cry 4
(Diptera) and Cry 5 (Lepidoptera and Coleoptera).
Now it was aimed to obtain the molecular weight of
specific protein which caused death of (Helicoverpa)
larvae. For this, we isolated crude protein of bacteria
and used this crude protein sample for gel
electrophoresis. After an electrophoresis, a very high
molecular weight protein band was observed, which
was easily identified. Determination of the
composition and toxicity of parasporal crystals, by
means of SDS-PAGE analysis and bioassay, is a
useful method for gene identification. The crude
protein isolated from bacteria was not pure so there
was need of purification of crude protein. For
purification crude protein precipitated with
ammonium sulfate with 30%, 60% and 70% fraction
of protein. The fractionated protein of the given
sample was purified by the column chromatography.
The ammonium sulfate fractionated by the sephadex
G-100 and 12 samples were collected. The
Maximum fraction OD was recorded in no. 9 (1.011)
and fraction no. 10 (1.22) and the lowest fraction
OD was obtained in fraction no. 5 (0.344). Pod and
leaves dipped in each fraction separately and treated
the (3rd Instars) larvae with, control crude protein,
30% 60% and 70% protein fraction. In case of 30%
treated larvae mortality observed after 36 hour and
single larvae dead in this case the rate of mortality
after 72 hour was 25%. The larvae which was treated
with 60% fraction single larvae dead after 12 at this
stage the rate of mortality was 25%. After 36 hour 3
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larvae was dead and at this stage rate of mortality
was 75% and after 72 hour the rate of mortality was
also 75% the larvae which was treated with 80%
fraction two larvae dead after 12 hour and rate of
mortality was 50%, 3 larvae dead after 24 hour rate
of mortality was 75% and all larvae dead after 60
hour and rate of mortality was 100% maximum rate
of mortality was observed in case of 70% protein
fraction because this fraction was much pure as
compared to 30% and 60% minimum rate of
mortality was observed in case of 20% and crude
protein. No mortality was observed in case of
control condition. Thus, 70% fraction can be useful
to identify specific protein to control pod borer.
100% mortality was observed when larvae
(Helicoverpa armigera) treated with 70% fraction, it
was done to identify the correct protein by SDS
PAGE.
According to table 1 Minimum Percentage
increment in weight of Larvae in MAL-6 after 6 Hr
(1.36 %) after 6 Hr (1.36 %) followed by NDA-
2(2.40%), NDA-94-2(4.64%), MAL-13 (6.84%),
BAHAR (8.14%), NDA-3 (12.81%). While
maximum percentage increment in weight of Larvae
was observed in NDA-14-6(25.89) followed by
UPAS-120 (24.46 %), NDA-13-6 (21.30%), NDA-1
(16.64 %). Approximately the same result was
observed at 12 hr while after 18 hr. percentage
reduction was found in all varieties. Maximum
reduction was found in UPAS-120 (18.03%) and
minimum reduction was found MAL-6 (3.01 %).
SDS- PAGE analysis of larval protein: Preparation
of L.B. Broth media and inoculated with Bt strain.
The protein was isolated by crushing the sample in
phosphate buffer (pH 7). The SDS-PAGE analysis
was done of Bt Strain. The subunit molecular weight
of Larval protein and Cry protein of Bt strain was
achieved by running the fraction of these protein
along with standard molecular weight marker 6000
Da - 43000 Da (Bangalore Genei Private Limited)
on 12% SDS-PAGE.

Figure 1 Collected Larvae of Helicoverpa armigera.

Table 1 Screening showing the percentage of increase / decrease weight of Larvae after feeding the pods at
different time interval.
Name of varieties Percent difference b/w 0 Percent difference b/w 6 Percent difference b/w Overall percentage
to 6 hr to 12 hr 12 to 18 hr
MAL-13 6.84 2.40 -6.09 2.74
NDA-2 2.40 4.49 -3.18 3.60
NDA-1 16.64 6.18 -6.42 15.90
NDA-13-6 21.30 1.68 -12.31 8.15
BAHAR 8.14 2.75 -6.48 3.91
NDA-94-2 4.64 1.21 -7.37 -1.90
NDA-14-6 25.89 13.24 -10.23 27.98
NDA-3 12.81 6.18 -3.93 15.07
UPAS-120 24.46 1.54 -18.03 3.60
MAL-6 1.36 1.72 -3.01 0.00

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Figure 2 SDS-PAGE Analysis of Bt. Protein.

Figure 3 SDS-PAGE Analysis of Bacterial proteins.

Figure 4 SDS-PAGE Analysis of Bacterial proteins.

The molecular mass of cry protein of Bt strain along
with the molecular marker was determined on 12 %
SDS-PAGE gel. Approximately 43 KDa protein was
band was observed (Figure 2).
There were six bacterial strain (A, B, C, D, E & F)
were isolated, in which B-strain was found highly
toxic strain under mortality test of larvae. So the B-
strain was sent to MMTC, Chandigarh for
identification of bacterial strain.
The comparative study between BT protein and
Bacterial protein (H.armigera) for mortality
against Pod borer (H.armigera) were observed at

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different concentration: The comparative study Table 3 Determination of Bacterial Protein against
between Bt. protein and Bacterial protein the mortality of Pod borer (H. armigera).
(H.armigera) for mortality against pod borer Bacterial Protein in g/ g % Mortality
(H.armigera) were observed at different 0.00 0.00
0.50 0.00
concentration. In case of Bt. Protein, eight different
1.00 8.33
doses of Bt. protein (00.0 g/ g, 0.20 g/ g, 0.50 g/ 2.00 12.50
g, 1.00 g/ g, 2 g/ g, 4 g/ g, 6 g/ g, 8 g/ g) were 5.00 20.83
observed on live larvae. Maximum mortality % 10.00 33.33
15.00 41.66
(91%) was found at 8 g/ g while the minimum 20.00 45.83
mortality % (4.16 %) was found at 8 g/ g. 30.00 58.33
40.00 70.83
Effect of different dosages of Bacterial protein on 50.00 83.30
larvae of Pod borer (H.armigera): In case of 60.00 95.83
Bacterial protein, Twelve different doses of Bt.
protein (00.0 g/ g, 0.50 g/ g, 1.00 g/ g, 2 g/ g, 5 ACKNOWLEDGEMENT
g/ g, 10 g/ g, 15 g/ g, 20 g/ g, 30 g/ g, 40 g/
g, 50 g/ g, 60 g/ g) were observed on live larvae. The author is highly thankful to DG, UPCAR Dr.
Rajendra Kumar for providing funds for this project
Maximum mortality % (91%) was found at 60 g/ g
work.
while the minimum mortality % (4.16 %) was found
at 0.50 g/ g.
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1.00 20.80 Alykroid WR, Doughty J (1964) Legumes in human
2.00 41.66
4.00 62.50 nutrition. FAO Nutritional Studies 19: 45-
6.00 83.33 47.
8.00 91.66 Cannon RJC (1996) Bacillus Thuringiensis use in
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thuringiensis Berliner strains on
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behaviors of H. armigera (Lepidoptera:
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841.

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