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.
INTRODUCTION
Pigeon pea is an important pulse/grain legume and various constrains of pigeonpea production, the
commonly known as red gram or arhar. It is the rich insect pest are of prime important. Pigeon pea is
source of protein, iron, iodine and essential amino crop of intermediate plant type with long duration
acids likewise methionine, lysine, tyrosine (Alykroid hence serves as an ideal host for many insect pests.
and Doughty 1964) and also supply vitamins-B, Disease and insect pest are estimated to cause yield
minerals and fats. The presence of different type of losses from 470 to 988 kg ha-1 (FAO STAT, 2008).
proteins and their smaller molecules including More than 200 insect species have been reported
alkaloids, is flavones, polyphenols and a variety of infesting this crop. Major pest of pigeon pea are pod
oligosaccharides make legume seed unique in fly and pod borer. In India 3 species of Helicoverpa
providing nutraceuticals. Asia produces 3.00 million are found viz. H. armigera, Helicoverpa assulta and
tones grains from 3.81 million hectare area with 789 H. peltigera .The larvae of H. armigera destroys the
kg/ha productivity. India accounts for 78% of global buds, flowers and pods. It bores inside the pod of
output with current production of 2.45 million tones pigeon pea during vegetative state and damages
from 3.80 million hectare recording average yield of inside the pod. In the absence of pod larvae feeds on
686 kg/ha. In UP it is grown on 0.33 million hectare foliage. The world wide annual losses caused by
area producing 0.30 million tones of grain with H.armigera in pigeon pea are $ 317 million
average yield of 910 kg/ha (FAO, 2006). Among (Shanower et al 1999). In India yield losses of
pigeon pea due to insect and disease ranged from
NA Khan ( ), Raja Husain, Anamika Kaushal, Pratibha Singh 470 to 988 kg ha-1, which accounts for 29-85% of
Department of Plant Molecular Biology & Genetic Engineering,
Faizabad grain yield in different cultivars. The non-judicious
Email-nakhan0110@gmail.com use of chemical insecticides has led to the
K N Singh development of resistance to one or more multiple
Department of Biochemistry, NDUA&T Kumarganj, Faizabad insecticides in over 500 species of insects (Akhurst
(U.P.) 224229
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2002). Both bacteria and Fungi can be pathogenic 500 l TCA (Tri chloro acetic acid) and left it for 3-
and killed Helociverpa armigera. Bacillus 4 hours on ice. Again centrifuged it, at 5000 rpm for
thuringeniesis (Bt) produces large insecticidal 10 minutes and dissolved pellet in 250 l phosphate
protein crystals (ICPs) during sporulation (Khalique buffer and 50 l of sample was electrophoresed on
and Ahmad 2002). In that the proteins are isolated, SDS-PAGE.
purified and studied for their efficacy to kill insect
larval. Determination of the composition and Purification of bacterial protein by ammonium
toxicity of the parasporal crystals, by means of SDS- sulphate fractionation: The bacterial crude protein
PAGE analysis and bioassay, is useful complement were precipitated using ammonium sulfate at
for gene identification. different saturation level such as 30%, 60%.and
70%. For the preparation of 30% saturation 1.15g
MATERIALS AND METHODS ammonium sulfate was dissolved in 10 ml of crude,
stirred 30 minute by magnetic strirrer at 40C. After
Screening of different varieties of Pigeonpea 30 minutes, the sample was centrifuged at 10,000
against H. armigera at different time interval (0, 6, rpm for 20 minute at 40C. The supernatant was
12, 18 hrs.): Larvae were collected from Pigeon pea collected for 60% ammonium sulfate fractionation
field and screened all ten varieties of Pigeon pea in and pellet was dissolved in small amount of same
artificial/ lab condition. Two larvae were left on the phosphate buffer the dissolved pellet was 30%
pods of each variety with three replications per ammonium sulfate fractionated protein. For 60%
variety for feeding the pods. Initially the larvae were fractionation, 1.22g ammonium sulfate was
weighted under control condition than after 6, 12 dissolved in 10 ml of 30% supernatant then again it
and 18 hrs, respectively. The reduction percentage in was stirred 30 minute by magnetic stirrer at 40C.
each variety of pods was calculated by the mean of Then sample was centrifuged at 10, 000 rpm for 20
R1, R2 & R3. minutes at 40C. Supernatant was collected for 70%
ammonium sulfate fractionation and pellet was
Collection of naturally killed larvae (Helicoverpa dissolved in small amount of same phosphate buffer.
armigera) for identification of micro- organism: The dissolved pellet was 60% ammonium sulfate
The naturally killed larvae of pod borer fractionated protein.
(Helicoverpa armigera) were collected from the
experimental site of Genetics and Plant Breeding Dialysis of 30%, 60% and 70% ammonium sulfate
Department and Student Instructional Farm. The fractionated protein of bacteria: The purified
dead larvae reared in 40C for identification of protein of bacteria (30%, 60% and 70% ammonium
microorganism. Dead larvae (Helicoverpa armigera) sulfate fractionated protein) was filled inside the
were collected from pigeon pea field and each larva dialysis tube and sealed both side with plastic plug.
was crushed in 0.1 M sodium phosphate buffer, and It was put in the same phosphate buffer (sodium
stored it at 40C for streaking on L.B agar media, phosphate buffer 0.1 M, pH = 7.0) and dialyzed for
growth occurred on L.B agar plate indicates the 4-8 hours. These salt free purified proteins were
bacterial growth. used for mortality test. The single larvae were put on
the surface of petridish above whatmann filter paper
Isolation of protein from bacteria and characterizat moistened with dipped pod and leaves of purified
ion of protein (SDS-PAGE): In 500 ml liquid L.B protein. Newly molted 3rd instars larvae was dipped
media, the colony of strain of bacteria was in protein fraction for continuously 3 days.
inoculated and kept the media in shaker at 370C for Thereafter, they were fed with fresh pigeon pea pods
overnight and centrifuged at 5000 rpm for 10 min till they died or pupate.
and dissolved pellet in 5 ml phosphate buffer and
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larvae was dead and at this stage rate of mortality 2(2.40%), NDA-94-2(4.64%), MAL-13 (6.84%),
was 75% and after 72 hour the rate of mortality was BAHAR (8.14%), NDA-3 (12.81%). While
also 75% the larvae which was treated with 80% maximum percentage increment in weight of Larvae
fraction two larvae dead after 12 hour and rate of was observed in NDA-14-6(25.89) followed by
mortality was 50%, 3 larvae dead after 24 hour rate UPAS-120 (24.46 %), NDA-13-6 (21.30%), NDA-1
of mortality was 75% and all larvae dead after 60 (16.64 %). Approximately the same result was
hour and rate of mortality was 100% maximum rate observed at 12 hr while after 18 hr. percentage
of mortality was observed in case of 70% protein reduction was found in all varieties. Maximum
fraction because this fraction was much pure as reduction was found in UPAS-120 (18.03%) and
compared to 30% and 60% minimum rate of minimum reduction was found MAL-6 (3.01 %).
mortality was observed in case of 20% and crude
protein. No mortality was observed in case of SDS- PAGE analysis of larval protein: Preparation
control condition. Thus, 70% fraction can be useful of L.B. Broth media and inoculated with Bt strain.
to identify specific protein to control pod borer. The protein was isolated by crushing the sample in
100% mortality was observed when larvae phosphate buffer (pH 7). The SDS-PAGE analysis
(Helicoverpa armigera) treated with 70% fraction, it was done of Bt Strain. The subunit molecular weight
was done to identify the correct protein by SDS of Larval protein and Cry protein of Bt strain was
PAGE. achieved by running the fraction of these protein
along with standard molecular weight marker 6000
According to table 1 Minimum Percentage Da - 43000 Da (Bangalore Genei Private Limited)
increment in weight of Larvae in MAL-6 after 6 Hr on 12% SDS-PAGE.
(1.36 %) after 6 Hr (1.36 %) followed by NDA-
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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The molecular mass of cry protein of Bt strain along toxic strain under mortality test of larvae. So the B-
with the molecular marker was determined on 12 % strain was sent to MMTC, Chandigarh for
SDS-PAGE gel. Approximately 43 KDa protein was identification of bacterial strain.
band was observed (Figure 2).
The comparative study between BT protein and
There were six bacterial strain (A, B, C, D, E & F) Bacterial protein (H.armigera) for mortality
were isolated, in which B-strain was found highly 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.
REFERENCES
Table 2 Determination of Bt. Protein of mortality of
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