STUDY OF GENETIC VARIATION ON BASIS OF

FEEDING BEHAVIOR OF BOLLWORM (Helicoverpa

armigera) ATTRIBUTE MOLECULAR MARKER.

by
PAKHARE PALLAVI EKNATH
B.Sc. (Agril. Biotechnology)

DISSERTATION

Submitted to the

Vasantrao Naik Marathwada Krishi Vidyapeeth,

Parbhani in partial fulfillment of the requirements
for the Degree of

MASTER OF SCIENCE

(Agriculture)

in

AGRICULTURAL BIOTECHNOLOGY

VILASRAO DESHMUKH
COLLEGE OF AGRICULTURAL BIOTECHNOLOGY,
LATUR

VASANTRAO NAIK MARATHWADA KRISHI VIDYAPEETH

PARBHANI - 431 402 (M.S.) INDIA.

2020
 CANDIDATE’S DECLARATION

I hereby declare that this dissertation or

part Thereof has not been previously
Submitted by me for a degree of any other
Institution

Or
University.

Place : LATUR

Date : / /20 (Pallavi. E. Pakhare)

Dr. A.A.Bharose
Associate Professor
Department of Plant Biotechnology,
Vilasrao Deshmukh College of Agricultural Biotechnology, Latur

C E R T I F I C A T E– I

This is to certify that Miss. Pallavi Eknath Pakhare has satisfactorily

prosecuted his course and research for period of not less than four
semesters and that the dissertation entitled- “Study of genetic variation on
basis of feeding behavior of bollworm (Helicoverpa armigera) attribute
molecular marker” submitted by him is the result of original research work
and is of sufficiently high standard that warrants its presentation to the
examination.

I also certify that the dissertation or part thereof has not been
previously submitted by his for a degree of any university.

Place : Latur (A.A.Bharose)

Date : / / Research Guide
 C E R T I F I C A T E– I I

This is to certify that the dissertation entitled “Study of genetic variation

on basis of feeding behavior of bollworm (Helicoverpa armigera) attribute
molecular marker by VNMKV, Parbhani.” submitted by Miss. Pallavi Eknath
Pakhare to the Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani in partial
fulfillment of the requirement for the degree of MASTER OF SCIENCE
(Agriculture) in the subject of AGRICULTURAL BIOTECHNOLOGY has
been approved by the Student’s Advisory Committee after oral examination in
collaboration with the External Examiner.

( ) (A.A.Bharose)
External Examiner Research Guide and Chairman
Advisory Committee

Members of Advisory Committee:

(S. R. Bhalerao)

(M.S. Dudhare)

(D.G.More)
 PLAGIARISM CLEARANCE CERTIFICATE

This is to certify that thesis entitled “Study of genetic variation on basis of

feeding behavior of bollworm (Helicoverpa armigera) attribute molecular

marker” submitted by Pallavi Eknath Pakhare has been properly examined by

using URKUND: Anti Plagiarism Software.

No sentence, equation, diagram, table, paragraph or section has been copied

verbatim from previous work unless it is placed under quotation marks duly

referenced. The work presented is original and own work of the researcher (i.e. there

is no plagiarism). No ideas, process, result or words of other have been presented as

researcher’s own work.

Signature and Name of the Signature and Name of the

Research Student Research Guide

URCUND ANALYSIS REPORT

XVI
 Acknowledgment

Emotions cannot be adequately expressed in words, because then emotions are

transformed into mere formalities. My acknowledgments are many more than I am expressing
here.

I avail this opportunity to acknowledge my sincere, humble indebtedness and deepest

sense of gratitude to my honorable guide Prof . A..A..Bharose, Associate Professor, Department
of Plant biotechnology, Vilasrao Deshmukh College of Agricultural Biotechnology, Latur, whose
insight, unfailing interest, inspiring guidance, infinite patience was an asset throughout the
course of investigation, providing necessary facilities and valuable help in conducting the
studies. I would like to thank Dr. M. S. Dudhare, Associate Professor (Department of Plant
Biotechnology) for his valuable suggestion throughout the research programe.

I record my sense of gratitude to Dr. H.B.Patil, Associate Dean and Principal, V.D.
College of Agricultural Biotechnology, Latur, V.N.M.K.V. Parbhani for their kind co-operation
and providing facilities and resources during my P.G. Programme. I take upon this opportunity
to express my sincere gratitude towards my Advisory Committee members. Dr. A.A. Bharose,
(Dept. of Plant Biotechnology) Dr. S. R. Bhalerao, ( Dept. of Biochemistry and Mol Bio.). Dr. M. S.
Dudhare ( Department of Biotechnology and Microbiology) , Dr. D.G. More, (Department of
Entomology), for their inspirative guidance and providing basic facilities and support required
for completion of this study.

I wish to accord my sincere thanks to Hon’ble Faculty Prof. H.B. Patil, Dr. V.D. Surve, , Dr.
Y.S. Bhagat, Dr. V.R. Hinge, Prof. B.N. Aglave, Prof. K.M. Sharma, Vilasrao Deshmukh College of
Agril. Biotechnology, Latur. I wish to accord my warmest thanks to Mr. Raut, Mr. Ugile, Mrs.
Sorekar, and Mrs. Ambatwad, VDCOAB, Latur, for their kind co-operation during the entire
tenure of my study in this V.D. College of Agricultural Biotechnology, Latur.

A friend in need is a friend indeed. This project would not have been completed without
the continual support and help from my friend. I wish to accord my gratitude to Thanu,
Yeshavant, Ashwini, Archana, Shamkar, Shubhangi and omkar sir for their constant
encouragement, support and valuable suggestions throughout the project work.

I would like to express my heartiest special thanks to Dr. Mahesh, RA, DBT-BIF project,
Mr. Abhijeet, JRF-DNF, for their kind co-operation, help and valuable suggestion as and when
needed.

Parents teach us to dream, to try, with our feet on the ground and sights on the sky. My
Father Shri. Eknath Pakhare has always enlightened me to believe, in the beauty of dreams, I
express deep sence of gratitude to my Mother, Mrs. Vanita Pakhare for her inexhaustible source
of inspiration throughout my life, their blessings love and affection has brought the cherished
dream to reality. I take this opportunity to express my affection and obligation to my sister,
Shubhangi for continuous motivation and support.

I am also deeply obliged to all the Authors of past and present whose literature has been
cited in this dissertation.

Place : Latur
Date: / /20 (Pallavi E.Pakhare)
 CONTENTS

Chapter Title Page No.

No.
I INTRODUCTION 1-4

II REVIEW OF LITERATURE 5-18

III MATERIAL AND METHODS 19-27

IV RESULTS AND DISCUSSION 28-37

V SUMMARY AND CONCLUSION 38-40

LITERATURE CITED i-Xii

ABSTRACT I-II
 LIST OF TABLES

Table
Title of tables Page No.
No.

3.1 Composition of 2% extraction buffer 22

3.2 List of SSR primer for molecular characterization 24-25

3.3 Reagent and stock solution used for SSR primer 26

3.4 PCR programme used for SSR analyasis 26

The mean incubation period, percent egg hatch, larval duration,

4.1 percent pupation and growth index of H. armigera on different 29
host plant.

The mean larval instars duration of H.armigera on different

4.2 30-31
host plants

The mean pre pupal and pupal duration, percent adult

4.3 emergence and total development period of H. armigera on 31-32
different host plants

4.4 Larval body weight gained by H. armigera on different host 33

plants (Experiment I)

4.5 Larval body weight gained by H. armigera on different host 34- 36

plants (Experiment II)

4.6 List of H. armigera’s host plant for cluster analysis with 35

DNA based SSR marker

4.7 Similarity matrix of 5 H.armigera on different host plants 35

based on NTSYS-pc UPGMA clustering method

4.8 Polymorphic SSR primer used for analyasis of H.armigera 36

on different host plants
 LIST OF PLATES

Plate Title Between

No. pages

4.1 Larval instars of H.armigera(Hubner) 32-33

4.2 A SSR profile of H.armigera on different host plant with 35-36

primer HaSSR1 , HaSSR 2, HaSSR3 compared with 1 Kb DNA
ladder.

4.3 A SSR profile of H.armigera on different host plant with 35-36

primer HaSSR4 , HaSSR5, HaSSR6 compared with 1 Kb DNA
ladder.

4.4 A SSR profile of H.armigera on different host plant with 35-36

primer HaSSR7 , HaSSR8, HaSSR9 compared with 1 Kb DNA
ladder.

4.5 A SSR profile of H.armigera on different host plant with 35-36

primer HaSSR10 compared with 1 Kb DNA ladder.
 LIST OF FIGURES

Figure Page
No. Title no.

Dendrogram of H.armigera on different host plants 35-36

4.1 by SSR Markers
 ABBREVIATIONS

A : Adenine
Bp : Base pair
C : Cytosine
dATP : Deoxyadenosin-5 '-triphosphat
dCTP : Deoxycytidin-5' –triphosphate
ddH2O : Double distilled water
dGTP : Deoxyguanine-5' –triphosphate
DNA : Desoxyribonucleic acid
DNase : Desoxyribonuclease
dNTP : Desoxynucleotide
dTTP : Desoxythymine-5 '-triphosphat
EDTA : Ethylendiamintetra-acetat
et al. : et alii, and others
EtBr : Ethidium bromide
EtOH : Ethanol
G : Guanine
Hr : Hour
Kb : kilobase pair
L : Liter
M : Molar
mA : Milliampere
Mg : Milligram
MgCl2 : Magnesium Chloride
min : Minute
ml : Milliliter
mM : Millimolar
NaCl2 : Sodium Chloride
ng : Nanogramm
nt : Nucleotide
OD : Optical density
PCR : Polymerase chain reaction
RAPD : Random amplified polymorphic DNA
RNase : Ribonuclease
Rpm : Revolution per minute
RT : Room temperature
S : Seconds
T : Thymine
TAE : Tris acetate EDTA
Taq : Thermophilusaquaticus
TE : Tris-EDTA-buffer
Tris : Tris-(hydroxymethyl)-amino-methane
UV : Ultraviolet light
W : Weight
%(v/v) : Volume percentage
%(w/v) : Weight percentage
°C : Grade Celsius
µg : Microgram
µl : Microliter
INTRODUCTION
 CHAPTER - I
INTRODUCTION

Helicoverpa armigera(Hubner) is one of the most important agricultural pests

throughout the world which classified as follows Kingdom: Animalia, Phylum:
Arthropoda, Class: Insecta, Order: Lepidoptera, Familty: Noctuidae. In Asia, Europe,
Africa, Australia and other country, H. armigera causes economical losses at a couple of
billion US dollars annually, not including the socio-economic and environmental costs
associated with chemical control and the introduction of GM crops (CABI/EPPO 1990,
Tay, et al., 2013).

H. armigera is commonly known as old world bollworm which considered as

omnivorous, with the larvae attacking at least 60 cultivated and 67 wild host plants from
different families including Asteraceae, Fabaceae, Malvaceae, Poaceae and Solanaceae
(Fitt, 1989, Pogue, 2004) among these cotton, tomato, maize, chickpea, pigeon pea,
sorghum, sunflower, soybean and groundnut (Fitt, 1989) crops having agricultural
importance.

Tang, et al., (2012) observed more than 180 species of plants as hosts distributed
in nearly 45 families. The feeding larvae can be seen on the surfaces of plants, but
sometimes they are hidden within plant organs (i.e. flowers or fruits), in which case bore
holes may be visible. If bore holes are not visible, then it requires dissection of the plant
for finding larvae (CABI & EPPO 1990). The larvae feed inside the host plant (fruit)
therefore control measures of Helicoverpa spp. are difficult and also difficult to kill with
insecticides because they have gained resistance to variety of insecticides (Kranthi, et al.,
2001).

After emergence, females of the cotton bollworm start laying eggs within 2-6
days. They can lay between 300 and 400 eggs, which hatch in two days after oviposition.
A characteristic color pattern develops in different stage instars that can be variable and is
formed from shades of green, straw-yellow and pinkish to reddish-brown or yellow-

1
black. The 1st and 2nd instars larvae are yellowish white to reddish-brown-black in color
and lack prominent markings; their head, prothoracic shield, supra-anal shield,
prothoracic legs, as well as the spiracles and the tuberculate bases of setae are dark-
brown to black and give the larva a spotted appearance. (CABI, EPPO 1990).

Young larvae (third and fourth instars) can cause up to 67% loss to cotton yields
(Ting, 1986). The larvae make round holes which are visible at the base of the flower
buds, flowers, and the bolls. Leaves and shoots damaged. Large larvae bore into maturing
cotton green bolls. In pigeon pea, an important grain legume in south Asia, east Africa,
and Latin America, this pest causes yield losses of up to 100% in some years and per
year, worldwide losses to pigeon pea of more than $300 million (Thomas, et al.,
1997).On corn, consume grains by the Helicoverpa armigera larvae. On tomatoes, larvae
bore fruits, it also causing preventing fruit development and the fruit to fall (CABI,
2007).

Versatility of H. armigera which is evolving resistance to almost all classes of

insecticides and its ability to survive on variable hosts help it in making serious pest on
several crops. There have been variations in morphological parameters that observed
specific separation within (Bhatnagar and Davies, 1978) and between populations of H.
armigera collected from different host plants (Bhattacharjee and Gupta, 1972).

The host selection behavior of H. armigera is variable which arises through both
non-genetic and genetic factors. Important factors such as surface attributes of the host
plant, host age and host abundance helps to determine the feeding behavior in this
polyphagous insect. The expression of insect preferences depends on the spatial
availability of hosts at the suited stage of development. Preference of H. armiger is
flowering/fruiting parts of high value crops including cotton, tomato, corn and pulses,
which give a high socio economic cost to its depredations in subsistence agriculture in the
tropics (Parihar and Singh, 1992).

Feeding behavior of H. armigera on different host is effect on growth and

development of H.armigera. Control and management of H. armigera is very difficult

2
because its having high mobility, high fecundity, high survival rate, short life span and
ability to develop resistance against pesticides (Drake, 1991). Therefore, to prepare the
best integrated pest management strategy of H. armigera, it is necessary to know how
insects grow and develop (life history, behavior, feeding habits and their susceptibility
and resistance to chemical and biological pesticides).

Molecular markers have been used to evaluate genetic similarity and estimate
gene flow among insect populations. (Figueroa et al., 2002, Martinelli et al., 2006).

SSR markers are better in measuring the genetic structure in H. armigera because
of their characteristics such as coverage of multiple loci, co- dominance and high
polymorphisum (Scott, et al., 2003) than the RAPD markers. Non-availability of the
DNA sequence information hampered use of SSR markers for H. armigera. But, many
SSR markers specific for H. armigera have been identified (Tan et al., 2001; Ji et al.,
2003; Scott et al., 2004; Ji et al., 2005).

Microsatellites consist of short, tandemly repeated sequences of 1–6 base pairs

within the nucleus of the cell (Ashley, 1999). They have an elevated rate of mutation due
to “slipped-strand mispairing” (Levinson and Gutman, 1987; Eisen, 1999), resulting in a
high proportion of polymorphism even between closely related lines (Semagn et al.,
2006). Resulting variations (alleles) are scored through differing banding patterns. This
marker is neutral to selection and is inherited co-dominantly as a standard Mendelian trait
(Meglecz and Solignac 1998; Ashley 1999; Luikart and England 1999).

H. armigera attack on more than 181 species of plants as hosts distributed in

nearly 45 families. The crop losses in India due to this polyphagous pest are estimated to
be US $350 million annually. Therefore in the present investigation, study on the feeding
behavior of H.armigera was carried out. It will help to prepare the best integrated pest
management strategy based on the timing and preferences of these larvae at the age when
they are most vulnerable. Mass rearing of H. armigera on different host plants will be
ultimately beneficial for making better management practices of this noxious pest.
Integrated pest management has historically placed great hopes on host plant resistance.

3
 Understanding the genetic variation among the H. armigera populations occurring
on different host plants has become important to understand the veriability in their
susceptibility to different insecticides, including Bacillus thuringiensis.

Considering the above point, the present investigation were carried out to the
feeding behavior of Helicoverpa armigera, DNA extraction from fourth to fifth instar
larvae from different field crop and amplification of DNA in PCR for SSR primer with
following objectives.

1. To study the biology and biomtrics of Helicoverpa armigera on different field

crops.

2. Molecular characterization of Helicoverpa armigera based on feeding behavior

using SSR primer.

4
REVIEW OF
LITERATURE
 CHAPTER - II
REVIEW OF LITERATURE

Cotton bollworm, Helicoverpa armigera(Hubner) is a pest of cotton and other

crops in India and elsewhere in general and in particular in Maharashtra cotton growing
area resulting in high crop loss regularly. Versatility in rapidly evolving resistance to
almost all classes of insecticide molecules and its ability to survive on several hosts, there
must be a strong molecular basis regulating the behavior of H. armigera in making it a
serious pest on several crops (Subramanian, S. and Mohankumar, S. 2006).

2.1. Helicoverpa armigera: a pest of ‘national importance’ in India

H. armigera has been observed feeding on 181 cultivated and uncultivated plant
species belonging to 45 families in India (Manjunath et al., 1989). H. armigera larvae are
extremely damaging pest because they prefer to feed and develop on the reproductive
structures (fruit, flower) of crops which are rich in nitrogen, protein. (Fitt, 1989).

In India, where H. armigera destroys over half the yield of pulse crops, pigeon
pea and chickpea, losses were estimated at over $US 300 million per annum (Reed and
Pawar, 1982).The losses due to H. armigera are estimated to be 20-60% in cotton, 14-
85% in pigeonpea, 18-25% in sorghum, 30-60% in sunflower, 15- 46% in tomato
(Ranasingh and Mahalik, 2008).

This pest having agriculture importance due to its wide geographical presence and
heavy economic losses imparted to a variety of crops such as cereals, pulses, cotton,
vegetables, chickpea, pigeon pea, sorghum, sunflower, soya bean and groundnuts fruit
crops (Cunningham et al., 1999).

This pest having versatility due to mobility, polyphagy, rapid and high
reproductive rate, and diapause. (Ravi et al., 2005) recorded that the relative abundance
of H. armigera in redgram and chickpea was much higher than in cotton and other host
crops in a South Indian cotton ecosystem.

5
 As discussed earlier, the preference of H. armigera is the harvestable flowering
parts of high-value crops results in both high economic cost, and a high socioeconomic
impact in subsistence agriculture. Severe crop losses have been reported due to variability
of H. armigera attack but cost of controlling this pest sometimes more of crop production
(Thomas, et al., 1997). Farmers spend up to 50% of their annual income to buy chemicals
for H. armigera. The rise in the prices of insecticides and the replacement of the low cost
pyrethroids with more expensive alternatives to counter pyrethroid resistance also rised
the cost to millions of farmers on annual basis (Kranthi, et al., 2001).

2.2 Polyphagous nature of Helicoverpa armigera

Patankar et al., (2001) observed Helicoverpa armigera is a devastating pest of

cotton and other important crop plants all over the world. A detailed biochemical
investigation of H. armigera gut proteinases is essential for planning effective proteinase
inhibitor (PI)-based strategies for management of the insect infestation. They report the
complexity of gut proteinase of H. armigera fed on four different host plants, viz.
chickpea, pigeon pea, cotton and okra, and during larval development. H. armigera fed
on chickpea observed more than 2.5- to 3-fold proteinase activity than those fed on the
other host plants.

Polyphagyous nature of H. armigera is due to complex and diversity of digestive

and detoxifying enzymes. In insects, proteases are a major group of hydrolytic enzymes
divided as endo- and exo-proteases which involved in digestive processes, proenzyme
activation, metamorphosis, release of physiologically active peptides and complement
activation (Terra and Ferreira, 1996; Huang et al., 2010).

During larval development, the gut proteinase activity increases in which the
highest activity seen in the fifth instar larvae, followed by decline in the sixth instar. Over
88% of the gut proteinase activity of the fifth instar larvae is of serine proteinase type and
the second instar larvae show presence of other proteinase classes like metalloproteases,
aspartic and cysteine proteinases along with serine proteinase activity (Patankar et al.,
2001). Trypsin and chymotrypsin like proteases from H. armigera gut have been purified
and characterized (Johnston et al, 1991; Telang et al, 2005). The activity of gut protease

6
genes and the composition different according to diet and developmental stage of the
larvae (Patankar et al, 2001).

2.3 Biology of H.armigera(Hubner)

2.3.1 Eggs

The egg duration of H.armigera varied from 2.76 ± 0.43 to 5.17 ± 0.38 days on
gram (Shrivastava ans Saxena, 1958a, HSU et al., 1960, Reed, 1965a and Singth,1970 ).
The incubation period of H. armigera was observed 2.6 to 3.6 days on tomato (Singh and
Singh, 1975), 4 days on pigeon pea, groundnut and cotton and 5 day on tomato, gram,
cabbage and potato (Anonymous,1990), 4 to 6 on gram (Shahid et al.,1990) and 3.0 to
3.7 days on cotton (Venkataiah et al., 1994).The average incubation period of H.
armigera from 3.7 to 4.2,3.7 to 4.40,3.5 to 4.30, 3.5 to 4.30, 3.60 to 4.60, 3.70 to 4.50,
3.90 to 4.64 and 4.2 to 4.50 days on pigeon pea leaves, pigeon pea pods, black gram
leaves, black gram pods, gram leaves, gram pods, pea leaves and pea pods respectively
reported by Ameta and Bhardwaj (1996).

Akashe et al., (1997) reported the incubation period of H. armigera from 2 to 4

days on sunflower, 3 days each on Lucerne and sunflower (Patel and Koshiya,1998a and
1998b), Jallow and Matsumura,(2001) reported the incubation period of H. armigera
from 3.0, 2.7,2.5 and 2.6 days at 25.0°C, 27.7°C, 30.2°C, 32.5°C and 8.6, 5.4,3.4 and 2.7
days on gram at 15°C,20°C, 25°C, 30°C temperatures, respectively (Tiwari and
Rahalkar,2005).

The incubation period of H. armigera from 2 to 4 days on chickpea reported by

Pandey amd Kumar (2007), 3.36 ± 0.08 days on chickpea (All et al., 2009), 5 to 7, 6 to 7
and 4 to 6 days in the first ,second and third generation on tomato (Sharma et al.,
2011).The egg period of H.armigera ranges from 2 to 4 days with an average of 2.86 ±
0.45 days on groundnut reported by Gadhiya et al., (2014). Yadav et al., (2015) reported
the incubation period of H. armigera was 3.94, 3.70, 4.50, 3.67, 3.92 days on pigeon pea,
chickpea, sorghum, tomato, cotton.

7
2.3.2 Larvae

The larval duration of H. armigera was reported 20 to 28 days on gram in Uttar

Pradesh (Shrivastava and Saxsena,1958), 33.50 days on sunflower (Coaker,1959), 13 to
16 days on chickpea (Menolanche et al., 1959), 20 to 22 days in cotton (HSU et al.,
1960), 21.2 days on cotton during april under temperature of 21 to 27°C (Reed,1965),20
to 22°C days on cotton (Reed,1965b) and 17.6 days on maize, 18.2 days on cotton flower
buds and 18.4 days on sunflower(Pretorius, 1976).The shortest larval period (22.2 days)
of H.armigera on gram and longest period (25.3 days) on cotton observed by Gaikwad et
al., (1977). Singh and Sidhu (1980) observed the total larval duration pg H. armigera
varied from 8 to 28 days on young leaves of cotton with comparatively short duration
April to July and prolonged duration during winter. Dubey et al., (1981) observed the
larval duration of H. armigera 18.4, 19.6, 19.8 days on chickpea, pigeon pea and sweet
pea respectively.

The 6 larval instars of H. armigera on arhar, cotton, tomato, linseed and pea with
larval duration varying from 10.2 to 17.96 days reported by Goyal and Rathore (1988).
Tripathi and Singh (1989) observed highest growth index of H. armigera on chickpea
(5.03) and lowest on lentil (1.18).H. armigera completed 5 larval instars when reared on
young leaves of 7 Hirsitum and 1 arboreum cotton cultivars. Singh et al., (1992) reported
the larval duration of H. armigera is different in all eight varieties of cotton respectively.
The mean larval period of H. armigera to extend of 17.8, 15.3, and 14.5 days on gram
leaves, gram pods and Melilotus alba, 19.0, 19.4, 19.6, 19.7 days o chickpea, sweet pea,
pigeon pea and lentil and 9.0 to 20.5 days chickpea,black gram, soyabean, and chilli was
reported by Choudhary et al.,(1993) and Venkataiah et al., (1994).

Bilapate (1987) obsevered that the larval growth of H. armigera was found to be
fastest (13.43 days) on maize and slowest on cotton (14.99 days) with highest growth
index (7.01) on chickpea. The faster larval growth of H. armigera on arhar pods (13.2)
followed by gram pod (14.0 days), gram leaves (14.6 days) and tomato leaves (14.9
days).

8
 Patel and Koshiya (1998a) observed 18 days of larval period of H. armigera on
sunflower. The shortest larval period of H. armigera to extend of 13.44 days was
recorded on pigeon pea followed by artificial diet (15.97), maize (16.45), sorghum
(19.22), cowpea (25.65) and marigold (30.54) days reported by Bantewad and Sarode
(2000). The total larval development of H. armigera on tomato was found be 16.2, 13.3,
11.4, and 11.2 days when reared at 25°C, 27°C, 30°C and 32°C reported by Jallow and
Matsmura (2001).

Kakimoto et al., (2003) observed that the larval duration of H. armigera reared at
25°C on okra, cotton, soyabean, tomato and green pepper for 13.2, 13.4, 13.8, 14.8, 20.7
days respectively. Tiwari and Rahalkar, (2005) reported that the larval period of H.
armigera on gram was observes to be maximum (35.60 days) at 15°C temperature
followed by 30.22 days at 20°C temperature, 19.30 days at 25°C temperature, 14.89 days
at 30°C temperature respectively.

The larval period of H. armigera varied from 15 to 26 days with an average of

22.48 ± 4.45 days when larvae feed on okra observes by Parnar (2006). Pandey and
Kumar (2007) observed that the average larval period of H. armigera was 24.50 ± 1.40
days when reared on chickpea. Reported the larval duration of H. armigera was shortest
(14.6 days) on the chickpea flour diet and longest (42.4 days) on the water flour diet. It
was observed to be 15.3, 15.3, 15.7, 16.6, 16.8 and 21.5 days on mungbean, soyabean,
chickpea leaves and pods (control) wheat, maize and cotton seed.

Ali et al., (2009) reported that the larval duration of H. armigera was completed
through 6 instars and the average duration of first, second, third, fourth, fifth and six
instars larvae were 2.26, 2.47, 2.87, 2.98, 3.49 and 3.47, respectively when reared on
chickpea at 25°C temperature and 64% relative humidity. The H. armigera reared on BG-
I took 21.26 ± 2.09 days to complete its larval growth followed by 21.16 ± 2.15 days on
BG-II while it was found to be 20.21 ± 1.37 days on non-bt cotton repored by Ranjith et
al., (2010).

The longest larval duration of H. armigera was 26.20 ± 1.63 days on soyabean
cultivar L-17 observed by Fathipour and Naseri (2011). H. armigera passed through 5

9
larval instars when reared on tomato with average duration of first, second, third, fourth
and fifth larvae to extent of 8.39, 9.0, 4.32, 4.0, and 4.70, 9.24, 9.22, 9.67, 6.22 and 6.70
and 5.6, 3.20, 3.20, 3.80 and 4.40 days, in the first, second and third generation,
respectively. Its total larval period was 30.39, 38.70 and 21.34 days, in the first, second
and third generation, respectively observed by Sharma et al., (2011).

The average duration of first, second, third, fourth and fifth larvae of H. armigera
ranged from 5.60 to 9.44, 3.23 to 9.67, 3.12 to 6.88, 3.80 to 6.57 and 4.39 to 6.45
days respectively on tomato reported by Kumar et al., (2013).The larval development of
H. armigera was completed in 18.98, 18.24, 19.43, 16.32, 18.32, 19.45, 16.4, 16.67 and
20.12 days on carnation, pigeon pea, chickpea, sorghum, tomato, cotton, cowpea, bathua
and capsule of castor observed by Yadav et al., (2015).

2.3.3 Pupae

The pupal period of H. armigera was found to be 37.5 days on tomato in U.S.E.
observed by Wilcox et al., (1957). Reed (1965b) observed that the pupal duration of H.
armigera was 15.6 and 13.8 days for male and female on cotton, while it was 16.3 and
14.7 days on pigeon pea, respectively and 5 to 8 days with average of 6.2 ± 0.32 days on
tomato. Pretorious (1976) reported that the pupal duration of H. armigera was 12.3, 11.4
and 11.8 days on sunflower, maize cobs and cotton flower. The pupal period of H.
armigera was 8.02 ± 0.98 to 8.64 ± 1.34 days on cotton reported by Singh and Sindhu
(1980).

Patel and Talati (1987), Tripathi and Singh (1989b), Anonymous (1990),
Shrivastava and Shrivastava (1990), Singh et al., (1992), Bajpal and Sehgal (1993),
Choudhary et al., (1993) and Venkataiah et al., (1994) observed that the pupal duration of
H. armigera was 9 to 17 days om sunflower, 7.8 to 21.2 days and 11.3 to24.45 days on
pigeon pea and sunflower, 9.55 days on cotton cultivars, 9.0 and 7.1 days on gram leaves
and pods, 14.0, 12.3, 10.3 and 14.3 days on chickpea, sweet pea, pigeon pea, and lentils
and 9.3 and 9.6 day on chickpea and blackgram, respectively. Ameta and Bhardwaj
(1996) observed that the average pupal period of H.armigra was 9.33 to 9.80, 8.23 to
8.88, 9.42 to 0.45, 9.50 to 9.71, 9.66 to 10.3, 9.87 to 10.45 days on pigeon pea leaves,

10
pigeon pea pods, gram leaves, gram pods and pea leaves, pea pods, respectively. Sharma
and Singh (2001) observed that the mean pupal duration of H. armigera varied from 9.34
± 0.15 to11.34 ± o.22 days on sunflower and 15.10 to 16.30 days and 13.20 to 14.30 days
on leaves and bolls of cotton cultivars.

Pandey and Kumar (2007) reported the average pupal duration of H. armigera
was 14.32 ± 1.4 days on chickpea. The pupal duration of H. armigera was higher (15.25
± 1.45 days) in BG-I compared to BG-II (14.17 ± 1.16 days) and non Bt (14.45 ± 1.34
days) observes by Ranjith et al., (2010). Yadav et al., (2015) evaluated that the pupal
period of H. armigera was completed in 14.6, 14.53, 14.99, 14.45, 14.81, 13.43, 15 81,
14.45, 14.56 and 14.68 days on carnation, pigeon pea, bathua, chickpea, sorghum,
tomato, capsule of castor, cotton, mouthbean and cowpea respectively.

2.3.4 Feeding habit is affected on growth and development of H.armigera

Dubey et al., (1981) reported the larval body weight and pupal weight of H.
armigera on chickpea, berseem, pigeon pea, and sweet pea were observed to be 375 ±
62.03 and 302 ± 35.08, 369.8 ± 56.44 and 300.5 ± 17.6, 355.5 ± 57.43 and 300.5 ± 3.32
and 265 ± 5.67 and 250 ± 36.44 mg, respectively. The head capsule width and body
length and width of H. armigera to turn 0.28, 1.35 and 0.30mm,0.51, 4.35 and 0.68mm,
1.65, 6.92 and 1.06mm, 2.58, 12.45 and 2.22mm and 2.98, 24.13 and 3.10 mm for I to V
instars on cotton observed by Bilapate et al., (1982). It was reported by Bilapate (1987)
that the general pupal length and width of H.armigera was found to be 17.45 and 5.23,
17.68 and 5.66 and 19.39 and 5.87, 18,.12 and 5.40, 18.44 and 5.24, 17.99 and 4.48mm
on pigeon pea, safflower, chickpea, cotton, sunflower and maize, respectively. Their
pupal weight on these respective host plant were 281.10, 272.23, 260.45, 257.24, 257.16
and 260.90 mg.

Shrivastava and Shrivastava (1990) observed that the larval and pupal weight of
H. armigera ranged from 333.0 to 356.2mg and 231.3 to 252.3 mg on different genotype
of gram. The larval and pupal weight of H. armigera was calculated when grown on
chickpea, pigeon pea, sweet pea and lentil was 298.3 and 218.3mg, 245,6 and 143mg,
215,3 and 147.3mg, 257.4 and 152.3 mg reported by Choudhary et al., (1993).

11
 Venkataiah et al., (1993) observed the larval length, weight and pupal weight of
H.armigera on chickpea, black gram, soybean and chilli was 3.05, 2.04 and 1.77 mm,
0.35, 0.25 and 0.20mm, 430, 230 and 220 mg and 300, 210 and 200 mg respectively.
Tripathi and Singh (1999) observed that the larval length, width and weight of
H.armigera was 31.04 ± 2.45mm, 5.67 ± 0.74mm and 410 77 ± 45.99mg on chickpea at
vegetative stage, it was 31.96 ± 1.94 mm, 6.24 ± 0.87mm and 421 78 ± 68.45 mg on
chickpea at flowering stage.

Balkrishnan et al., (2004) reported the larval weight of H. armigera ranged from
482. 30 to 570.90 mg on cotton cultivars. Parmer (2006) observed that the length and
breadth of first, second, third, fourth and fifth instar larvae of H. armigera on okra was
1.75 ± 0.12 and 0.30 ± 0.02 mm, 4.85 ± 0.43 and 0.49 ± 0.03 mm, 8.45 ± 0.47 and 1.01 ±
0.17mm , 17.42 ± 0.75 and 2.21 ± 0.20 mm and 28.76 ± 1.05 and 3.56 ± 0.14 mm,
respectively. The width of head capsule of first to fifth instar larvae was 0.28 ± 0.01, 0.51
± 004, 0.70 ± 0.04, 1.27 ± 0.04 and 2.60 ± 0.02mm, respectively.The length of male
pupae range from 18.40 to 20.50 mm with an average of 20.32 ± 1.31 mm.The length of
female pupae range from 19.30 to 24.00 mm with an average of 21.76 ± 1.41 mm. the
breadth of male pupae range from 5.00 to 6.50 mm with an\ average of 5.68 ± 0.57 mm.

Dhurgude et al., (2009) concluded the biometrics of H. armigera reared on pods

of three gram cultivars viz., Virat, G-12 and BDN-9-3. They calculated of larval body
length and weight of H. armigera on Virat varied from 1.36 to 24.44mm and o.79 to
198.88mg, value on G-12 and BDN-9-3 ranged 1.29 to 17.88mm, 0.65 to 198.55mg and
1.23 to 16.45mm, 0.66 to 202.45mg respectively. Ranjith et al., (2010) concluded that the
pupal weight of H. armigera was less on BG-I and BG-II (216.13 ± 23.31 and 212.34 ±
22.14 mg) as compared to non- Bt (248.16 ± 22.15 mg).

Deepa and Srivastava (2010) observed that the average length and breadth og
first, second, third, fourth, fifth and sixth instar of larvae of H. armigera was 1.23 ± 0.03
and 0.37 ± 0.02mm, 2.88 ± 0.21 and 0.68 ± 0.01mm, 6.90 ± 0.17 and 1.63 ± 0.03mm,
11.73 ± 0.48 and 2.25 ± 0.04 mm, 19.98 ± 0.15 and 3.18 ± 0.04mm and 30.45 ± 0.36 and
3.33 ± 0.02mm, respectively on pigeon pea. The average length and breadth of pupa was
17.12 ± 0.23 and 5.62 ± 0.09mm.Patel et al., (2012) reported that the average body length

12
and breadth of I, II, III,IV, V and VI instar larvae of H.armigera on tomato was found to
be 1.45 ± 0.03 and 0.51 ± 0.02mm, 3.52 ± 1.08 and 0.82 ± 0.01mm, 9.45 ± 0.66 and 2.81
± 0.02mm, 23.09 ± 1.34 and 3.43 ± 0.02mm, 34.76 ± 1.25 and 5.11 ± 0.04mm and 43.89
± 1.24 and 6.59 ± 0.67mm. Average pre-pupal length and width was 25.09 ± 1.34 and
4.88 ± 0.03mm.

Sarate et al., (2012) described developmental and digestive flexibilities in the

midgut of a polyphagous pest, the cotton bollworm, Helicoverpa armigera. Larvae fed on
diets which is rich in proteins or carbohydrates (pigeonpea, chickpea, maize, and
sorghum) showed higher larval mass and developed more quickly than larvae fed on diets
with low protein and carbohydrate content (rose, marigold, okra, and tomato). Low
calorific value diets like rose and marigold observed in higher mortality (25-35%) of H.
armigera. Highly varying development efficiency and larval/pupal survival rates in
Helicoverpa armigera.

Kuntar et al.,(2013) the average length and breadth if I, II, III, IV and V instar
larvae of H.armigera on tomato was to be 1.41 ± 0.06 and 0.45 ± 0.02mm, 3.77 ± 0.11
and 0.75 ± 0.01mm, 7.80 ± 0.15 and 2.28 ± 0.04mm, 12.75 ± 0.43 and 2.87 ± 0.04mm
and 20.98 ± 0.62 and 4.03 ± 0.05mm.Gadhiya et al., (2014) reported that the length
breadth of egg of H.armigera on groundnut was 0.47 ± 0.02 and 0.47 ± 0.02mm. 1.80 ±
0.11, 4.67 ± 0.38, 8.46 ± 0.47, 17.60 ± 0.84 and 28.76 ± 1.05 mm. The breadth were o.31
± 0.02, 0.62 ± 0.04, 1.03 ± 0.17, 2.31 ± 0.15 and 3.76 ± 0.33 mm, respectively.

Gill et al., (2015) observed the maximum larval weight of H. armigera on cotton
(532.36 ± 4.34 mg), it was minimum on sunflower (402.07 ± 4.45mg). They are longer
larval length on cotton (25.87 ± 0.31mm), while it was 24.56 ± 0.24mm on pigeon pea.
The male and female pupae were heavier on cotton (254.77 ± 1.42 and 283.76 ± 2.64
mg). Male and female pupal length was longest on cotton (17.05 ± 0.04 and 17.32 ± 0.25
mm).

Kumar et al., (2017) studied the feeding response is effect on growth and
development of Helicoverpa armigera (Hub.). In experiment, five different diets was
used such as (Chickpea soaked grains, Tomato fruits, Pigeon pea soaked grains, Pea

13
soaked grains and Artificial diet). Eight different parameters such as larval period, pre-
pupal period, pupal period, percent pupation, pupal weight and percent adult emergence
were compared growth and development. Reported that artificial diet is more effective in
all parameters.

Hu et al., (2018) described feeding experiences on hot pepper and tobacco could
induce positive feeding preference, while those on cotton often induced negative effect,
reported that innate feeding preference is not related to the direction of host plant induced
preference.

2.4 Molecular diversity of Helicoverpa armigera(Hubner)

Baker, et al., (1965) and Templeton, et al., (1980) concluded that molecular
differences within and between geographic populations of an ecosystem. The population
changeability patterns as effected by various ecological factors in the past and the
historical pressures on the genome.

Zhou et al., (2000) observed a low level of genetic distances among Israeli and
Turkish H. armigera populations. H. armigera moths across six locations in eastern
Mediterranean analysis RAPD and a high level of gene flow, reported that 84 out of 88
amplified DNA fragments generated by three primers were found to be polymorphic at
99 per cent.

Explanation of molecular diversity in geographical populations is an important

factor to study the pest populations and their management. Within an ecosystem, the
expanse of molecular diversity between geographical populations depends on gene flow
between populations, host range and time since separation (Fakrudin, et al., 2004a;
Chandrashekhar, et al., 2004; and Fakrudin, et al., 2004b).

Fakrudin, et al., (2004a) determined genetic variability within and between

geographic populations of cotton bollworm, H. armigera inducing in south Indian cotton
ecosystems using RAPD PCR. Results that the mean similarity coefficient across
populations ranged from 0.75 to 0.82 Samples from Madhira had the highest (0.82) and
those from Nagpur, Nanded and Nalgonda had the lowest similarity (0.75). In

14
populations also, it ranged from 0.73 to 0.86 indicating amount of genetic heterogeneity
significantly. Clustering analysis showed two major groups, each six populations of
southern and northern parts of South Indian cotton ecosystem.

S. Subramanian and S. Mohankumar (2006) reported Genetic variability of the

bollworm, Helicoverpa armigera, occurring on different host plants. Out of 10 SSR
markers in which nine of the SSR primers indicated high variability across the different
host plants and generated polymorphism ranging from 75 to 100 per cent in population.
H. armigera collected and reared on cotton and other host plants by using the un-
weighted pair-group method analysis.

Behere et al., (2007) reported genetic diversity of H. armigera and its natural
relationship with H. zea. The analysis of four major Helicoverpa pest species showed
that H. punctigera is basal to H. assulta and H. armigera basal to H. zea. Samples from
North and South America indicate that H. zea is also a single species across its
distribution. Their data showed short genetic distances between H. armigera and H. zea
which appears to established via a founder event from H. armigera stock at around 1.5
million years ago. They also revealed that mitochondrial DNA sequence data indicate the
single species status of H. armigera across Africa, Asia and Australia. The evidence for
inter-continental gene flow observed in their study was compatible with published
evidence of the ability o f this species to migrate over long distances.

Vijaykumar et al. (2008) studied genetic diversity among cotton bollworm,

Helicoverpa armigera populations from different geographic regions of South India using
mitochondrial DNA-specific markers. 167 PCR amplicons generated by thirteen selected
mtDNA universal primers, In which 162 were showed polymorphic across all 12
populations. An amplicons per primer was noted. Separated the 12 populations into
different groups based on band-sharing data after principal component analysis. All
populations could be separated from one another using specific primers; specific bands
could be used to differentiate individual populations. On a larger scale, genetic
differences among populations appear low. Result that the level of genetic variation
observed between the H. armigera populations with mtDNA PCR analysis reveals that it

15
is an effective marker technology for delineating genetic relationships amongst
populations and estimating genetic diversity.

Jain,et al., (2010) observed insects represent a major life form on earth. So far,
nearly 0.9 million insect species are discovered, 75% of all the recorded animal species.
Some of the insect species are easy to identify, while for others, it is difficult for identify
because its having small size and morphological similarity. Moreover, it is difficult to
identify morphological variation due to environmental factors. To control these problems,
the advanced molecular techniques used, such as Polymerase Chain Reaction (PCR),
Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA
(RAPD) and Arbitrary Fragment Length Polymorphism (AFLP). RAPD markers used in
gene mapping to characterize cultivars and species genetically, infer phylogeny and
biogeography of insect population and RAPD helped understand modes of evolution and
evolutionary trajectories. Thus, RAPD markers have the most common sticks for
measuring genetic differences between individuals, within and between related species or
population. The unprecedented evolutions in modern molecular biology, particularly in
those of DNA marker technology, have created technical know-how that finds advance
application in molecular ecology research in insects.

Khiaban et al., (2010) concluded that genetic diversity based on Nie s gene index
ranged from 0.188 to 0.250 by using 10 different SSR primers to differentiate H.
armigera larvae population collected during summer 2006-2007 from five different
places of Iran. Molecular variance analysis revealed significant variance within and
between population. The between and within geographical variance observed 13.88 and
86.12 percent of total molecular variance, respectively. Molecular data in geographical
population calculated by cluster analysis and divided pod borer moth populations into
two groups. In this grouping, group one indicate Golestan population. The maximum and
minimum genetic distances were revealed between Gorgan- Mughan (0.21853) and
Kermanshah- Shahindej (0.05789). Significant correction was not found between genetic
and geographic matrices observed by Mantel test.

Deepa,.M. and Srivastava, C. P. (2011) studied genetic diversity of Helicoverpa

armigera populations using RAPD markers which collected from different agro-climatic

16
zones of India. They isolated DNA from larvae and conducted polymerase chain reaction
using 25 RAPD primers. An average of 14.84 amplicon levels per primer, in which 14.76
were showing polymorphic that indicating high variability among H. armigera
populations. Genetic similarities among geographical populations from the data were
ranging from 0.15 to 0.40. Using the pair group method analysis, H. armigera collected
and reared in Varanasi stood out as unique in one cluster while the insects collected and
reared in other states group.

Yenagi, et al., (2012) observed genetic diversity among five populations of cotton
Helicoverpa armigera which collected from different geographic regions of North
Karnataka, India by using RAPD markers. Nineteen RAPD markers which generated a
total of 58 PCR amplicons, in which 26 were polymorphic across all five populations. An
average of 6.44 amplicons per primer noted. Result that all populations could be
differentiated from one another using specific primers; specific band (s) could used to
differentiate individual populations. The five populations differentiated into groups based
on band sharing data by principal component analysis. Populations showed degrees of
genetic similarity within a range of 0.11 to 0.84.

Rahman, et al., (2014) studied the genetic variability of Helicoverpa armigera

(Hübner) at different agroecological zones of Bangladesh and comparison with Indian
population which was conducted in India during September 2008 to February 2009. Out
of 12 H. armigera populations in which 10 populations collected from different
agroecological zones of Bangladesh and two populations from India were used for their
genetic variability. Eight out of the ten primers generated scorable PCR products and
PCR products were subjected for analysis. Eight RAPD primers amplified 138 amplicons
which produced polymorphic. The similarity coefficient based on 138 RAPD amplicons
ranged from 0.000 to 0.777. An UPGMA dendrogram based on Jaccard’s similarity
coefficient was generated for the 12 samples of H. armigera. The dendrogram revealed
that H. armigera population from Bangladesh had 25 to 45 percent similarity, and within
this range similarity remained found in Indian population.

17
2.5 Molecular marker

JI et al., studied polymorphic microsatellite loci for the cotton bollworm

Helicoverpa armigera (Lepidoptera: Noctuidae) and some remarks on their isolation.
Identified the five polymorphic tri- and tetranucleotide microsatellite loci suitable for
Helicoverpa armigera. Efficiency of microsatellite cloning in H. armigera is 2.5% which
is eightfold lower than that for the gadoid fishes (20%).

DI et al., reported isolation and characterization of microsatellite loci from the

pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae). Thirteen
polymorphic microsatellite markers were developed from an enriched partial genomic
library for the pink bollworm, Pectinophora gossypiella(Saunders). Heterozygosity at
these loci ranged from 0.042 to 0.83. In 50 individuals, although the presence of null
alleles in some loci is suspected, these polymorphic markers are provide useful tools for
genetic studies of this species.

JI et al., developed the five novel polymorphic microsatellite markers in the

cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) from an enriched
genomic library. They cross-amplified with other published loci was tested in a closely
related species, the tobacco budworm, H. assulta. Resulted was the expected
heterozygosity at these loci ranges from 0.62 to 0.91 in the cotton bollworm.

18
MATERIALS AND
METHOD
 CHAPTER - III

MATERIALS AND METHODS

The present investigation titled “Study of genetic variation on basis of feeding
behavior of bollworm (Helicoverpa armigera) attribute molecular marker.” was
carried out at Department of Plant Biotechnology, Vilasrao Deshmukh College of
Agricultural Biotechnology, Latur (M.S.) during the year 2019-20. The material and
laboratory procedure followed during this course of investigation are described in this
chapter.

3.1 MATERIALS

3.1.1. Insects

The first and fifth instar larvae of Helicoverpa armigera collected from different
field crops at Oilseed Research Station, Latur (M.S.) were used as experimental material.

3.1.2 Ethics statement

Helicoverpa armigera has not been notified under any act or laws and rules
thereof of the Government of India or State Government of Maharashtra as an
endangered or threatened species restricting or regulating its collection and observation.
No permits were required, for collecting the larvae from the field since H. armigera is not
an endangered species affecting the biodiversity status.

3.1.3 Leaves of different host plants

Leaves of different host plants were collected from different field crops at Oilseed
Research Station, Latur (M.S.) were used as experimental material.

3.2 METHODOLOGY

3.2.1 Biology of Helicoverpa armigera (Hubner) on different host plants.

The studies on biology of H. armigera (Hubner) was carried out on five

different host plants viz., pigeon pea, sunflower, cotton, chickpea and brinjal in a

19
 completely randomized design replicated three times respectively at the College
of Agricultural Biotechology, Latur during 2019- 2020.

The initial culture of H. armigera (Hubner) was developed by collecting

large number of larvae from the field of pigeon pea, sunflower, cotton and
chickpea, brinjal. The collected larvae were reared individually in the plastic
containers by feeding them plant parts of host plants every day. After emergence
from the pupae, more number of female were observed than males. The freshly
emerged adults were released in the oviposition cage of 50 × 30 sq.cm size
covered with black muslin cloth. Ninety freshly laid eggs of H. armigera were
obtained from the oviposition cage in order to study the biology on each of five
different host plants. The newly hatched larvae were reared individually in a clean
plastic container on plant parts of five different host plants. The developing green
pods of chickpea, part of capitulum of sunflower, boll of cotton, fruit of brinjal
and green pods of pigeon pea were used as food substrate of H. armigera.

The observations on larval duration, per cent larvae pupated, prepupal and
pupal durations, and percent adult emergence were recorded on respective plant
parts of five different host plants. The growth index was calculated by using
howes (1953) formula.

Growth index = Percent larvae pupated

Mean larval duration

3.2.2 Procedure adopted for feeding assays of Helicoverpa armigera on different host
plant:-

1) The present investigation was carried out in laboratory at temperature 27-290C,

the relative humidity was 55-75% and available photoperiod was 14-10 (L:D)h.
2) Five types of host plant leaves selected for this experiment are (1) Chickpea (2)
Pigeon pea (3) Sunflower (4) Cotton (5) Brinjal.

20
 3) Helicoverpa armigera on Pigeon pea and Cotton was used for comparative
studies of larval feeding behavior.
a. Ith Trial:- Ten H. armigera larvae from cotton crop were transferred to
another vials containing leaves of other four different host plant i.e, chickpea,
pigeon pea, sunflower, brinjal). Leaves were changed daily.
b. IIth Trial:- Ten H. armigera larvae from pigeon pea crop were transferred to
another vials containing leaves of other four different host plant i.e cotton,
chickpea, sunflower, brinjal). Leaves were changed daily.
4) Following observations were recorded during the feeding assays:- mass gained
by the larvae during first to third, fourth to fifth and sixth instar larvae.

3.2.3 Isolation of midgut from Helicoverpa armigera

Prior to dissection, larvae of H. armigera were immobilized by chloroform

(100%) and sterilized in 0.1% sodium hypochlorite and 70% aqueous ethanol for five
seconds to remove the adhering contaminants (Gebbardi, et al., 2001).

3.2.3.1 Dissection of midgut

Surface sterilized larvae of H. armigera was dissected on solid paraffin wax petri
plate by sterile scalpel blade in laminar air flow. The complete midgut of larvae was
transfer to the sterile 1.5 ml centrifuge tube.

3.2.4 DNA isolation form Helicovarpa armigera larvae

High quality DNA was isolated from Helicovarpa armigera larvae, as per
protocol given by Doyle and Doyle (1987) with some modifications.
3.2.4.1 CTAB Extraction buffer: To prepare CTAB buffer stock of following solutions
were prepared and then subsequent extraction buffer was prepared.
1. CTAB (10%): Ten gram CTAB was dissolved in sterile double distilled water and
volume made to 100 ml.

2. NaCl (5M): For preparation of 5 M stock solution, 23.4 g. Sodium chloride was
dissolved in sterile double distilled water and final volume made up to 100 ml.

3. Tris-HCl (1M) (pH 8.0): For preparation of 1M stock, 15.76 g. Tris-HCl was

21
dissolved in sterile doubled distilled water and pH was adjusted at 8.0 by adding 0.1N HCl
and final volume made to 100 ml.

4. EDTA (0.5M) (pH 8.0): EDTA (0.5M) was prepared by adding 14.61 g. of EDTA in
double distilled water and pH was adjusted to 8.0 by adding pellets of NaOH and final
volume made up to 100 ml.
After preparation of stock, all the stock solutions except CTAB were autoclaved.
Extraction buffer was prepared by taking following composition from stock solutions.

Table 3.1 Composition of 2% extraction buffer

Sr. No. Components Stock Quantity Final Concentration

1 CTAB 2% 20ml 2%

2 NaCl 1.4 mM 28ml 1.4mM

3 EDTA (pH 8.0) 0.5 M 4ml 20mM
4 Tris-HCl (pH 8.0) 1.0 M 10ml 100mM
5 Polyvinylpyrrolidone 2%

5 β-Mercaptoethanol - 200μl 0.2%

6 Double distilled water - 38 ml -

Total volume 100 ml

3.2.4.2 Reagents for agarose gel electrophoresis.

1. 50X TAE buffer

Tris base : 242 gm
Glacial acetic acid : 57.1 ml
EDTA : 37.2 g
Distilled water : 1000ml

2. Ethidium bromide (10 mg/ml)

10 mg of Ethidium bromide was dissolved in 10 ml of distilled water.
Tube wrapped in aluminum foil and stored at 4℃.

22
3.2.4.3 Protocol for DNA isolation form Helicovarpa armigera larvae
1. Larva of H. armigera was grinded in liquid nitrogen using plastic homogenizer in
1.5 ml eppendorf™ tube.
2. After grinding the larvae, 400 µl of prewarmed 2% CTAB extraction buffer was
added.
3. The mixture was incubated at 60–65°C in a dry bath for 1 h with gentle shaking at
10 min interval.
4. Equal volumes of chloroform: isoamyl alcohol (24: 1, v/v) was added and
centrifuged at 13,000–14,000 rpm for 15 min at room temperature.
5. The aqueous phase was transferred to a new eppendorf tube and then 0.6 volume
of isopropanol was added and incubate at –20°C for overnight.
6. The mixture was then centrifuged at 10,000 rpm for 10 min to pellet the DNA at
room temperature.
7. The pellet was washed twice with 70 % ethanol and centrifuged at 10,000 rpm for
10 min at 4°C to pellet the DNA.
8. The pellet was air-dried and dissolved in 50 µl of deionized sterile distilled water.
9. The isolated DNA was treated with 25 µl of RNAase and mixed by gentle tapping
to remove RNA.
10. The whole content was incubated at 37°C for 1 h.

3.2.5 Determination of quantity and quality of isolated DNA

Determination of quantity and quality of isolated DNA was done by

spectrophotometer .The instrument was set to a blank with 50 μl of distilled water. After
that 49 μl distilled water and 1 μl of sample were added in Eppendorf® cuvette and the
quantity and quality in Nano gram at A260/A280 nm was determined. The ratio higher
than 2.0 indicated the impurity of protein and less than 1.8 indicated RNA impurity in
sample. The amount of DNA was calculated by using the formula:

A260 X 50
DNA (g/l) = X dilution factor
1000

23
3.2.6 Agarose gel electrophoresis

Agarose gel electrophoresis unit was cleaned before use. Agarose gel (0.8%) was
prepared by dissolving 0.8g agarose powder in 100 ml 1X TAE buffer and heated in a
microwave oven. Then 10 mg/ml ethidium bromide was added to it after cooling down to
500C. The gel was poured in gel casting tray in which comb was inserted and kept for 45
min for solidification. After solidification the comb was removed. 5μl DNA was mixed
with 1μl to 6X gel loading dye and loaded on the gel. The electrophoresis was carried out
at 100 volts for 1.5 hr using 1 X TAE buffer. The extracted genomic DNA of the all H.
armigera larvae was used as template DNA for PCR.

3.2.7 Dilution of DNA sample

Part of DNA samples was diluted with appropriate quantity of sterilized distilled
water to yield a working concentration of 25ng/μl and stored at 40C until PCR
amplification.

3.2.8 Oligonucleotide primers

The oligonucleotides were reconstituted to 100pmol/μl stocks in sterile TE buffer.

The primers were used at working concentration of 100 pmol/μl in sterile nuclease free
water. The sequences of the primers were listed in (Table 3.2).

Table 3.2 List of SSR primers for molecular characterization

Sr. Total nu. of

No. Primer Sequence (5’ to 3’)
base
HaSSR1 F TAGGTGATTGTGGCTCAGTTTT 22
1
R CAAACCCATCAGCAAATGCAAC 22
HaSSR2 R AACACCCATTGAAGTCCCATGAA 23
2
R TTCCTATGTTCACTGCTAGTT 21
HaSSR3 F ATCCTTATGCTTTTAGCCGTTTA 23
3
R CAGTGGACTGCTATAGGCTGA 21
HaSSR4 F TGTTACTTGGGTTTCCTGAATA 22
4
R ACCACCGACACGTGCCGACTTC 22
5 HaSSR5 F GATAAGTTATTTCGGTTTAGTATT 24

24
 R AAGTACCTAATCCGTTTTTATTC 23
HaSSR6 F CATAGGAAGTGGTGAAGGGT 20
6
R CACATTCGTCTTTCATCGAC 20
HaSSR7 F ACGTCGATGAAAGACGAATGTGA 23
7
R AAGCTGGTCTGTGCTGCCAT 20
HaSSR8 F GCCGTAATGCCCTCAATTCTT 21
8
R TTCCCTCGGAGAGCCGT 17
HaSSR9 F TAGTCTGGGAATTTTGTCTGGTGT 24
9
R CGTGCCATTGAAATAGTAAGCCAT 24
10 HaSSR10 F TAAGTATGCCCTCGACTGTCGT 22
R CACTTTCCAATTAGCCTCGATGCT 24

3.2.9 Optimization of PCR reaction and components concentrations

A. Components of PCR

The genomic DNA, 10X PCR buffer, dNTPs, primers and Taq DNA polymerase
were optimized for DNA amplification. The master mixture for DNA amplification was
prepared through mixing of 20.9μl of Sterile H2O, 2μl of 10X PCR buffer (250mM Tris-
HCl, 10mM KCl, 1.5mM MgCl2), 0.4μl dNTPs, 0.2μl of each (F and R) primer, and 0.3μl
of Taq polymerase (Table 3.3). For one sample reaction, 1.0μl of diluted DNA of each H.
armigera larvae was mixed with 24.0μl of master mixture in PCR tube. DNA
amplification reaction was performed in a thermal cycler.

B. PCR Reaction

Following is the list of PCR reagents with their stock concentration used in this
study. The PCR reaction of 25µl volume was set for each genotype by using an
individual SSR primer.

C. PCR cyclic parameters for SSR markers

Optimum annealing temperature was determined by employing gradient PCR.

Amplification reaction was performed in 0.5ml tubes. Individual reaction (50μl)
contained 100ng of the extracted DNA, 1X PCR assay buffer (250mM Tris-HCl, 10mM

25
KCl, 1.5mM MgCl2), 100mM dNTP’s, 100ng/μl each of forward and reverse primers, 1
unit of Taq DNA polymerase. PCR was performed with forward and reverse primers with
an initial denaturation for 5 min at 940C, followed by 30 cycles of 940C denaturation for
45s, 520C for annealing for 45s and extension at 720C for 1 min.

Table 3.3 Reagents and stock solutions used for SSR primer

Sr. Stock Quantity

Reagents
No. solution ( µl)
1. Assay buffer 10X 2
2. dNTPs 25mM 0.4
3. Taq DNA polymerase 3.3U/µl 0.3
4. Forward Primer 100pM/µl 0.2
5. Reverse Primer 100pM/µl 0.2
6. Sterile water - 20.9
7. Template DNA(40 ng) 30ng/µl 1
Total 25

Finally the reactions were healed at 720C for 10 min. Specific and optimum
amplification of the gene was seen at 520C of annealing temperature. Subsequently the
DNA was amplified at 52 0C and the amplified PCR product (1.5 Kb) was purified from
low melting agarose gel, stained with EtBr (0.5μg/μl) using as per (Table 3.4).

1. Temperature profile
Table 3.4 PCR Programme used for SSR analysis

Sr. Temperature Time

Steps
No. (0 C ) requirement
1. Initial denaturation 94 4 min
2. Denaturation 94 45 sec
3. Annealing temp. 52 40 cycles 45 min
4. Extension 72 1 min
6. Final extension 72 10 min
7. Hold 4 ∞

26
2. Agarose Gel Electrophoresis

Agarose gel (1.5%) was prepared by dissolving 1.5 g of agarose in 100 ml 1X

TAE buffer and EtBr (10 mg/ml) was added. Further 5 µl of DNA was mixed with 1µl of
6X gel loading dye and loaded on 1.5% agarose gel. The genomic DNA was resolved on
1.5% agarose gel through electrophoresis.

3.2.10 Resolution of amplified product

The amplified products were resolved on 2.5% agarose gel for SSR markers and
1.5% for RAPD marker at 5V/cm for 1 to 1.5hr. After electrophoresis, the gel was taken
out for observation of banding pattern and photographed on a Gel Documentation System
(Alpha-Innotech, USA).

3.2.11 Data scoring and analysis

Data analysis was performed using NTSYS-pc (Numerical Taxonomy System,

Version 2.02i). The SIMQUAL program was used to calculate the Jaccard’s coefficient.
Dendrogram was constructed using Unweighted Paired Group Method for Arithmetic
Mean (UPGMA) based on Jaccard’s similarity coefficient. The polymorphic percentage
of the obtained bands and polymorphic information content of primers were calculated by
using following formulae,

1. Polymorphism % = No. of polymorphic bands X 100

Total bands

2. PIC = 1 − ∑𝑥𝑖2

Where, xi is the relative frequency of ith allele of marker loci.

27
RESULTS AND DISCUSSION
 CHAPTER - IV
RESULTS AND DISCUSSION

The results of the laboratory and field investigations carried out at Vilasrao
Deshmukh College of Agricultural Biotechnology, Latur Maharashtra during 2019-20 are
presented in this chapter. The detailed results of experiments as well as pooled data have
been furnished for impact as per the experiments planned and put for validation under the
following hands.

4.1 Biology of H. armigera (Hubner) on different host plant.

4.2 Procedure adopted for feeding assays of Helicoverpa armigera on

different host plant
4.3 Molecular characterization of Helicoverpa armigera based on feeding
behavior using SSR marker

4.1 Biology of H. armigera (Hubner) on different host plant.

4.1.1 Egg
The incubation period of H. armigera was varied when reared on different host
plants. However, incubation period of H. armigera was maximum on Chickpea (4.00
days) followed by pigeon pea (3.90 days), cotton (3.80 days), brinjal (3.36 days) and
sunflower (3.00 days). The incubation period of H. armigera on first two and latter two
host plants were various with each other. Significantly highest egg hatching to the extent
of 90.00 percent was observed on pigeon pea and sunflower followed by cotton (87.00
percent), chickpea (85.66) and brinjal (80.00 percent).

According to following table, the incubation period of H. armigera was extended

when reared on Chickpea. Yadav et al., (2015) reported the incubation period of H.
armigera was 3.94, 3.70, 4.50, 3.67, 3.92 days on pigeon pea, chickpea, sorghum,
tomato, cotton. Akashe et al., (1997) Reported the incubation period of H. armigera from
2 to 4 days on sunflower, 3 days each on Lucerne and sunflower (Patel and
Koshiya,1998a and 1998b).The results in respect of incubation period of H. armigera
reared on different host plants are in conformity with the results reported by above
referred research workers.

28
 Table 4.1: The mean incubation period, percent egg hatch, larval duration,
percent pupation and growth index of H. armigera on different host plant.

Name of the Mean Percent Mean Percent Growth

host plant incubation egg larval larvae Index
period hatch duration pupated
(days) (days) (%)

Sunflower 3.00 88.00 19.48 57.46 2.96

Chickpea 4.00 85.66 15.99 65.35 4.08

Cotton 3.80 87.00 22.20 53.32 2.39

Pigeon pea 3.90 89.66 17.57 70.60 3.86

Brinjal 3.36 80.00 15.16 44.55 2.96

S.E ± 0.05 0.82 0.32 0.39 0.02

C.D at 5% 0.16 2.50 0.97 1.20 0.06

C.V (%) 2.57 1.66 3.04 1.17 1.15

4.1.2 Larva
According to Table 4.1, that significantly the shortest mean larval duration of H.
armigera to the extent of 15.16 days was observed on brinjal and it was at per with that
on 15.99 days on chickpea. It was followed by 17.57 pigeon pea, 19.48 days sunflower
and 22.20 days on cotton. This indicated that among the host plants studied, longest
larval period of H.armigera was recorded on cotton while shorted was on brinjal.

29
 The larval duration of H. armigera was reported 18 to 21 days on sunflower
(Coaker, 1959), 13 to 16 days on chickpea (Menolanche et al., 1959), 20 to 22 days in
cotton (HSU et al., 1960). The longest period (25.3 days) on cotton observed by Gaikwad
et al., (1977).The larval development of H. armigera was completed in 18.98, 18.24,
19.43, 16.32, 18.32, 19.45, 16.4, 16.67 and 20.12 days on carnation, pigeon pea,
chickpea, sorghum, tomato, cotton, cowpea, bathua and capsule of castor observed by
Yadav et al., (2015).

The significantly lowest pupation of H. armigera was recorded on brinjal (44.55

percent) followed by cotton (53.32 percent). The growth index values varied from 2.39 to
4.08. The significantly highest growth index was observed in the case of larvae fed on
chickpea (4.08) over pigeon pea (3.86).

Table 4.2 : The mean larval instars duration of H. armigera on different host
plants.

Name of the Duration (days) Total Mean

host plants
Larval instars duration duration
(days) (days)
I II III IV V VI

Sunflower 3.17 2.92 3.33 3.09 3.45 3.52 19.48 3.24

Chickpea 1.63 2.00 2.45 3.15 3.50 3.26 15.99 2.66

Cotton 3.22 3.60 3.50 4.05 4.10 3.73 22.20 3.70

Pigeon pea 2.38 2.78 3.14 2.98 3.23 3.06 17.57 2.92

Brinjal 1.53 2.11 2.45 3.07 3.18 2.82 15.16 2.52

30
 S.E ± 0.09 0.07 0.06 0.10 0.11 0.07 - -

C.D at 5% 0.28 0.23 0.18 0.30 0.36 0.21 - -

C.V (%) 6.80 4.96 3.58 5.31 5.88 3.78 - -

The data in Table 4.2 indicated the instars- wise larval duration of H. armigera
when reared on different host plants. The duration of I, II, III, IV, V, VI larval instars
ranged from 1.53 to 3.22, 2.00 to 3.60, 2.45 to 3.50, 2.98 to 4.05, 3.18 to 4.10 and 2.82 to
3.73 days, respectively on different host plants under investigation. The mean larval
instar duration was lowest on brinjal (2.52) and maximum on cotton, followed by
chickpea (2.66), pigeon pea (2.92), sunflower (3.24). Jha et. al., (2012) reported that the
average first, second, third , fourth, fifth and sixth larval instar duration was 2.08, 3.77,
2.53, 4.22,5.32 and 4.71 days, respectively on hybrid sweet corn.

4.1.3 Pupa
The pre pupal and pupal duration, percent adult emergence and total
developmental period of H. armigera on different host plants are presented in Table 4.3.

Table 4.3: The mean pre pupal and pupal duration, percent adult emergence
and total development period of H. armigera on different host plants.

Name of the Pre- Pupal Per cent Total

host plants pupal duration adult developm
duration (days) emergence ent period
(days) (days)

Sunflower 2.26 15.11 69.60 39.87

Chickpea 2.07 9.09 72.21 31.16

31
 Cotton 2.52 11.31 65.60 39.85

Pigeon pea 2.11 9.55 71.46 33.14

Brinjal 2.02 11.78 60.09 31.73

S.E ± 0.04 0.13 0.48 0.47

C.D at 5% 0.14 0.41 1.48 1.44

C.V (%) 3.76 2.09 1.24 2.34

The significantly shortest mean pre pupal duration of H. armigera to the extent of
2.02 days was recorded on brinjal while shortest mean pupal duration to the extent of
9.09 days was recorded on chickpea. While the pre pupal duration of H. armigera were
highest on cotton and pupal duration of H. armigera were highest on sunflower (15.11)
followed by brinjal (11.78), cotton (11.31), pigeon pea (9.55) and chickpea (9.09).

Patel and Talati (1987), Tripathi and Singh (1989b), Anonymous (1990),
Shrivastava and Shrivastava (1990), Singh et al., (1992), Bajpal and Sehgal (1993),
Choudhary et al., (1993) and Venkataiah et al., (1994) observed that the pupal duration of
H. armigera was 9 to 17 days on sunflower, 7.8 to 21.2 days and 11.3 to24.45 days on
pigeon pea and sunflower, 9.55 days on cotton cultivars, 9.0 and 7.1 days on gram leaves
and pods, 14.0, 12.3, 10.3 and 14.3 days on chickpea, sweet pea, pigeon pea, and lentils
and 9.3 and 9.6 day on chickpea and blackgram, respectively.The results on the pre pupal
and pupal period of H. armigera on different host plants in good line with above referred
research workers. The highest adult emergence was observed in chickpea (72.21 percent)
followed by pigeon pea, sunflower, cotton and brinjal (Table 3). This indicates that the
chickpea was suitable host for emergence of adults.

32
Plate 4.1 Larval instars of H.armigera(Hubner)
4.2 Procedure adopted for feeding assays of Helicoverpa armigera on different host
plant

4.2.1 Larval body weight

The data in respect of larval body weight of H.armigera on different host plant in
(Experiment I) are presented in Table 4.4

Table 4.4: Larval body weight gained by H.armigera on different host plants
(Experiment I)

Name of the Instars-wise larval body weight (mg)

host plants
I II III IV V VI

Cotton 0.83 2.60 30.20 84.35 218.32 358.70

(control)

Pigeon pea 0.80 2.97 39.40 87.33 225.45 380.20

Chickpea 0.85 3.35 36.45 90.60 220.60 360.40

Brinjal 0.81 2.80 28.50 80.30 208.70 325.20

Sunflower 0.86 3.12 34.60 86.76 215.80 354.70

S.E ± 0.01 0.02 0.1 0.1 0.1 0.09

C.D at 5% 0.05 0.07 0.3 0.3 0.3 0.2

C.V (%) 3.21 1.37 0.5 0.2 0.08 0.04

33
 The data in Table 4, indicated intars-wise larval weight of H. armigera when
reared on different host plants. The weight of I, II, III, IV, V, VI larval instars ranged
from 0.80 to 0.86, 2.60 to 3.35, 28.50 to 39.40, 80.30 to 90.60, 208.70 to 225.45 and
325.20 to 380.20 mg, respectively on different host plants under investigation. The larval
body weight was highest on pigeon pea and lowest on brinjal as compared to cotton
(control).

4.2.2 The data in respect of larval body weight of H.armigera on different host plant
(Experiment II)

The data in Table 5, indicated instar-wise larvae weight of H.armigera from first
to six larvae instars when reared on different host plants. The weight of I, II, III, IV, V,
VI larval instars ranged from 0.81 to 0.90, 2.30 to 3.54, 28 to 32.40, 83.40 to 87.31,
187.48 to 218.12 and 298.22 to 310.42 mg, respectively on different host plants under
investigation. The larval body weight was highest on chickpea and lowest on brinjal as
compared to pigeon pea (control).

Table 4.5: Larval body weight gained by H.armigera on different host plant
(Experiment II)

Name of the Instars- wise larval body weight (mg)

host plants
I II III IV V VI

Pigeon 0.88 2.80 30.82 85.45 218.12 307.42

pea(control)

Chickpea 0.90 3.54 32.40 87.31 216.75 310.42

Cotton 0.82 2.65 29.76 85.10 208.25 306.18

Sunflower 0.85 3.22 31.50 86.60 215.41 305.13

34
 Brinjal 0.81 2.30 28 83.40 187.48 298.22

S.E ± 0.01 0.02 0.01 0.02 0.04 0.04

C.D at 5% 0.04 0.06 0.05 0.08 0.14 0.13

C.V (%) 3.12 1.21 0.10 0.05 0.03 0.02

4.3 Molecular characterization by using SSR primers of H.armigera.

A set of 10 SSR primers were used to carry out PCR amplification of 5 genomic
DNAs. Following SSR primers were found to be polymorphic, monomorphic and
selected for final PCR amplification. They are listed in Table 4.8.
The dendrogram Figure 4.1 showing phylogenetic relationship based on UPGMA
cluster analysis reveals that the genotype under present study could be divided into two
subcluster viz., cluster A and cluster B. cluster A consist of Cotton. Cluster B is divided
into B1 and B2. B1 is further grouped into B1a and B1b. Sub- subcluster B1a consist of
Sunflower and Pigeonpea. Sub-subcluster B1b consist of Chickpea. Subcluster B2 is
consist of Brinjal.
Table 4.6 List of H.armigera’s host plant for cluster analysis with DNA
based SSR markers

Name of Cluster Name of Sample

Cluster A Cotton

Sunflower
B1 (a)
B1 Piegonpea

Cluster B B1 (b) Chickpea

B2 Brinjal

35
Plate 4.2 A SSR profile of H.armigera on different host
plant with primer HaSSR1 , HaSSR 2, HaSSR3 compared
with 1 Kb DNA ladder. ( L- Ladder, 1- Sunflower, 2- Cotton,
3- Pigeon pea, 4- Chickpea, 5- Brinjal)
Plate 4.3 A SSR profile of H.armigera on different host plant
with primer HaSSR4 , HaSSR5, HaSSR6 compared with 1
Kb DNA ladder. ( L- Ladder, 1- Sunflower, 2- Cotton, 3-
Pigeon pea, 4- Chickpea, 5- Brinjal)
Plate 4.4 A SSR profile of H.armigera on different host plant
with primer HaSSR7 , HaSSR8, HaSSR9 compared with 1
Kb DNA ladder. ( L- Ladder, 1- Sunflower, 2- Cotton, 3-
Pigeon pea, 4- Chickpea, 5- Brinjal)
 Plate 4.5 A SSR profile of H.armigera on different host
plant with primer HaSSR10 compared with 1 Kb DNA
ladder. ( L- Ladder, 1- Sunflower, 2- Cotton, 3- Pigeon pea, 4-
Chickpea, 5- Brinjal)

Table No. 4.7 Similarity matrix of H.armigera on different host plants based on
NTSYS-pc UPGMA clustering method.

Sunflower Cotton Pigeon pea Chickpea Brinjal

Sunflower 1.0000000

Cotton 0.3684211 1.0000000

Pigeon pea 0.9090909 0.3157895 1.0000000

Chickpea 0.7333333 0.5789474 0.6666667 1.0000000

Brinjal 0.4736842 0.5238095 0.5000000 0.6842105 1.0000000

Figure 4.1 Dendrogram of H.armigera on different host plants by SSR Markers

Sunflower

Piegonpea

Chickpea

Brinjal

Cotton

0.45 0.56 0.68 0.79 0.91

Coefficient
 In the present study using SSR primers a total number of 25 amplicons were
generated by 10 SSR primers Table 4.8. 21 amplicons were found to be polymorphic
with an average polymorphism of 66.7 percent. On an average each primer produced 3.1
amplicons. The size of amplification product ranged from 300bp-100bp.
1. The SSR Primer HaSSR2 produced all monomorphic bands on 200bp. Hence
showed 0% polymorphism.

2. The SSR primers HaSSR3, HaSSR4, HaSSR5, HaSSR7, HaSSR8 and HaSSR10
produced 2 amplicons each. All or one the amplicons generated were polymorphic.
The fragment size ranged from 300bp-100bp.

Table 4.8 Polymorphic SSR primers used for analysis of H.armigera on different
host plants.

Amplification

Polymorphism
Monomorphic
Polymorphic
Amplicons

Amplicons

Amplicons
Annealing

generated

PIC Value
Name of

Temp ºc

Product
primer

Percent
Size of
No of

HaSSR1 52 3 3 - 300bp-100bp 100 0.52

HaSSR2 52 1 0 1 200bp 0 0.74
HaSSR3 52.5 2 1 - 350bp-100bp 50 0.64
HaSSR4 52 2 1 - 250bp-150bp 50 0.57
HaSSR5 53 2 2 - 250bp-150bp 100 0.32
HaSSR6 52 3 3 - 300bp-200bp 100 0.46
HaSSR7 54 2 2 - 300bp- 200bp 100 0.32
HaSSR8 52 2 1 1 200bp- 250bp 50 0.81
HaSSR9 54 3 2 - 350bp-150bp 67 0.47
HaSSR10 58 2 1 - 300bp-200bp 50 0.22

36
3. The SSR Primer HaSSR1, HaSSR6 and HaSSR9 Produced maximum number of
amplicons (3). All were polymorphic in nature. The fragment size is ranged from
300bp-150bp.

4. Similarity coefficient values obtain from SSR molecular marker data analysis.

The dice similarity coefficient of H.armigera on different host plant represented in

Fig. 4.1 ranged from 0.45 to 0.91 indicating that high diversity was present among those
H.armigera on different host plant. Maximum similarity coefficient of 0.91 was observed
between Sunflower and Pigeon pea. And minimum similarity coefficient was observed
between Cotton and Brinjal.
The PIC value generated by SSR primer ranged from 0.32 to 0.81. Highest PIC
value recorded in primer HaSSR8 (0.81). Whereas minimum PIC value recorded in Primer
HaSSR5 (0.32).
Subramanian, S and Mohankumar, S. (2006) reported among the ten SSR primer,
nine SSR primers indicated high variability across the different host with polymorphism
ranging from 75 to 100 per cent. Result was the primer HaSSR2 produced a single
monomorphic band for all DNA samples of different host plants. The coefficient values
ranged from 0.348–0.741. The H. armigera populations occurring on tomato and bhendi
were found to be closely related with a coefficient of 0.741, while the population
occurring on cotton and blackgram was found to differ widely with a coefficient value of
0.348. The population on cotton was found to be distantly related to the others with lower
dice coefficients.

37
SUMMARY AND
CONCLUSION
 CHAPTER V

SUMMARY AND CONCLISION

The laboratory experiments were conducted to study the biology, biometrics and
genetic diversity tables of H. armigera(Hubner) on different host plants viz., pigeon pea,
sunflower, chickpea, cotton and brinjal at the department of agricultural biotechology,
college of agricultural biotechology, latur during 2019-2020. The results obtained during
the investigation are summarized below.
Feeding behavior of H.armigera is help to prepare the best integrated pest
management strategy. For selection improvement in plant breeding, evalution of genetic
diversity is important. Genetic diversity is measured by genetic distance or genetic
similarity, both of which implies that there are either differences or similarities at genetic
level. Understanding the genetic variation among the H. armigera populations occurring
on different host plants has become important to understand the veriability in their
susceptibility to different insecticides, including Bacillus thuringiensis.

5.1 Biology of H. armigera (Hubner) on different host plant

Incubation period of H. armigera was highest on Chickpea (4 days) followed by

pigeon pea (3.90 days), cotton (3.80 days), brinjal (3.36 days) and sunflower (3 days).
The highest egg hatched 90.00 percent was observed on pigeon pea and sunflower
followed by cotton (87.00 percent), chickpea (85.66) and brinjal (80.00 percent).
Significantly the shortest mean larval duration of H. armigera to the extent of 15.16 days
was observed on brinjal followed by 15.99 days on Chickpea, 17.57 on pigeon pea, 19.48
days on sunflower and 22.20 days on cotton. The growth index values varied from 2.39 to
4.08. The significantly highest growth index was observed in the case of larvae fed on
chickpea (4.08) over pigeon pea (3.86).

The larval development of H. armigera was completed by passing through six

instars on different host plants viz., pigeon pea, sunflower , ckickpea, cotton and brinjal
under investigation The mean larval instar duration was lowest on brinjal (2.52) and
38
maximum on cotton, followed by chickpea (2.66), pigeon pea (2.92), sunflower
(3.24).The significantly pupal duration of H. armigera were highest on sunflower (15.11)
followed by brinjal (11.78), cotton (11.31), pigeon pea (9.55) and chickpea (9.09).

5.2 Procedure adopted for feeding assays of Helicoverpa armigera on different host
plant

In experiment I, indicated intars-wise larval weight of H. armigera when reared

on different host plants. The weight of I, II, III, IV, V, VI larval instars ranged from 0.80
to 0.86, 2.60 to 3.35, 28.50 to 39.40, 80.30 to 90.60, 208.70 to 225.45 and 325.20 to
380.20 mg, respectively on different host plants under investigation. The larval body
weight was highest on pigeon pea and lowest on brinjal as compared to cotton (control).

In experiment II, indicated instar-wise larvae weight of H.armigera from first to

six larvae instars when reared on different host plants. The weight of I, II, III, IV, V, VI
larval instars ranged from 0.81 to 0.90, 2.30 to 3.54, 28 to 32.40, 83.40 to 87.31, 187.48
to 218.12 and 298.22 to 310.42 mg, respectively on different host plants under
investigation. The larval body weight was highest on chickpea and lowest on brinjal as
compared to pigeon pea (control).

5.3 Molecular characterization

Five H.armigera on different host plant were screened for molecular

characterization using SSR markers. Set of 10 SSR primers used in this study, which
generated total 22 amplicons among which 21 amplicons were polymorphic, with an
average of 1.2 amplicons per primer. The PIC Value ranged from 0.32 to 0.81. With
highest PIC recorded in HaSSR8. The similarity coefficient between the genotypes
ranged from 0.45 to 0.91. Cluster analysis in which maximum similarity occur in
Sunflower and Pigeon pea .Whereas cotton and brinjal showed minimum similarity with
each other.

39
 Conclusions:

i. According to biology of H.armigera on different host plant, chickpea host plant was the
most suitable for H. amigera as compared to cotton, sunflower, brinjal and pigeon pea
in total development.
ii. Adopted feeding assays for Helicoverpa armigera on different host plant in which the
larval body weight was highest on pigeon pea and lowest on brinjal as compared to
cotton (control) and the larval body weight was highest on chickpea and lowest on
brinjal as compared to pigeon pea (control).
iii. The results of molecular studies had demonstrated that there are differences in genetic
diversity of genotypes. The SSR markers have proved to be suitable for characterizing
H.armigera.
iv. Molecular characterization of H.armigera on different host plant carried out using SSR
marker system. Shows clear differences among host plant of H.armigera. The analysis
showed that Cotton had independent cluster showing least similarity, whereas
Sunflower and Pigeon pea showed maximum similarity with each other.

40
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xii
ABSTRACT
 THESIS ABSTRACT

“ Study of genetic variation on

basis of feeding behavior of
A) Title of research topic :
bollworm (Helicoverpa armigera)
attribute molecular marker ”

B) Name of the student : Pakhare Pallavi Eknath

C) Degree to be awarded : M.Sc. (Agri. Biotechnology)

D) Major Subject : Agricultural Biotechnology

E) Number of pages in thesis : 76

F) Number of wordzs in : 316

Abstract

G) Signature of the Student :

(Pakhare .P.E.)

H) Signature, name and :

address of Major Advisor Dr. A.A. Bharose
Associate Professor
Vilasrao Deshmukh College of
Agril. Biotechnology, Latur,
(VNMKV, Parbhani)

I) Signature, name and address :

of forwarding authority Dr. H. B. Patil
Associate Dean and Principal
Vilasrao Deshmukh College of
Agril. Biotechnology, Latur,
(VNMKV, Parbhani)

I
 THESIS ABSTRACT
STUDY OF GENETIC VARIATION ON BASIS OF FEEDING BEHAVIOR
OF BOLLWORM (Helicoverpa armigera) ATTRIBUTE MOLECULAR
MARKER

Helicoverpa armigera is major important polyphagous insect pests in

agriculture which attacking at least 60 cultivated and 67 wild host plants from
different families and its has a worldwide distribution. The experiments were
conducted to study the biology, biometri and genetic diversity of H. armigera
(Hubner) on different host plants vis., sunflower, pigeon pea, chickpea, cotton,
brinjal.
The growth index and larval duration of H. armigera was found to be 2.96,
4.08, 2.39, 3.86 and 2.96 and 19.48, 15.99, 22.20, 17.57, 15.16 days on Sunflower,
chickpea, cotton, pigeon pea, brinjal, respectively. Feeding assays of Helicoverpa
armigera in experiment I was the larval body weight was highest on pigeon pea
and lowest on brinjal as compared to cotton (control) and in experiment II was the
larval body weight was highest on chickpea and lowest on brinjal as compared to
pigeon pea (control).
Set of 10 SSR primers used in this study, which generated total 22
amplicons among which 21 amplicons were polymorphic, with an average of 1.2
amplicons per primer. The PIC Value ranged from 0.32 to 0.81. With highest PIC
recorded in HaSSR8. The similarity coefficient between the genotypes ranged from
0.45 to 0.91. Cluster analysis in which maximum similarity occur in sunflower and
pigeon pea .Whereas cotton and brinjal showed minimum similarity with each
other. So, it is help to prepare the best integrated pest management strategy of H.
armigera and understand the veriability in their susceptibility to different
insecticides, including Bacillus thuringiensis.

Keywords: Helicoverpa armigera, biology, host plants, SSR primer

P. E. Pakhare Prof. A. A. Bharose

2018/BT/07/ML (Research Guide)

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