molecular characterization of tospoviruses infecting

Indian Journal of Biotechnology

Vol. 19, July 2020, pp 182-191

Molecular characterization of tospoviruses infecting Capsicum annuum L.

Rajamanickam S1*

, Haokip B D1, Caroline R

2 and Nakkeeran S

1

1Department of Plant Pathology, 2Department of Plant Molecular Biology and Bioinformatics, Tamil Nadu Agricultural University,

Coimbatore - 641 003, India

Received 15 April 2020; revised & accepted 26 May 2020

Chilli (Capsicum annuum L) a prime vegetable cum spice crop is infected by tospoviruses, which has become a serious

threat to it's cultivation worldwide. A survey was conducted for infection of tospoviruses on the basis of chlorotic and

necrotic symptoms in chilli growing areas of Tamil Nadu. The symptomatic leaves were positive for tospovirus-specific

antiserum tested through direct antigen coating - enzyme linked immunosorbent assay (DAC-ELISA), dot blot

immunobinding assay (DIBA) and tissue blot immunobinding assay (TIBA). The samples with typical symptoms were

successfully established for the chlorotic and necrotic lesions in assay host, cowpea and chilli upon artificial inoculation of

tospoviruses. Further, an expected complementary DNA (cDNA) fragment sequence of about 840 bp and 1.2 kb were

positively amplified using PCR correspond to coat protein (CP) gene of the respective viruses, which confirmed the

presence of groundnut bud necrosis virus (GBNV), capsicum chlorosis virus (CaCV) in chilli. However, there is a lack of

standard method to understand the genetic information of tospoviruses infecting chilli which requires the reliable, sensitive

and specific method. To address this issue, we have investigated an application based on CP gene of GBNV and CaCV to

perform molecular profiling. The nucleotide sequences of GBNV and CaCV showed maximum identity between the isolates

and amino acid sequences had more than 90% similarity. However, superimposed 3D prediction structures constructed using

MODELLER software revealed the single variation at 259th position comprising of glutamine and valine respectively. The

protein profiling of CP gene provides the basic knowledge on properties of CP proteins of two tospoviruses infecting chilli

and their structural relationships.

Keywords: Chilli, tospoviruses, serodiagnosis, CP gene sequences, 3D prediction structures

Introduction

Chilli (Capsicum annuum L.) belongs to the

Solanaceae family known as red pepper, is an

important condiment cum vegetable crop in India.

The chilli is known for its flavor, pungency and for its

medicinal value. India has produced around 8 lakh

tonnes of dry chilli from an area of 9.3 lakh hectares

from major chilli producing states of India including

Andhra Pradesh, Tamil Nadu, Karnataka, Kerala and

Maharashtra1-2

. As with other crops, chilli is affected

by various pathogen and pest that can cause

comprehensive loss in production. Among the various

pathogens infecting chilli, around 10 viruses cause a

serious threat affecting the yield3. The viral disease of

chilli causes 50-100 per cent yield reduction during

early stage of infection and often has social

consequences4-5

. The literature survey revealed that,

groundnut bud necrosis virus (GBNV) is an

economically important virus with a broad host range

and widespread distribution throughout the world. In

India, necrosis disease in vegetables is caused by a

virus collectively known as tospovirus group.

Tospoviruses are important among the plant viruses

worldwide causing significant economic losses in the

cultivation of chilli and other vegetable crops6. In the

early 2000, chilli leaf curl virus (ChiLCV), the type

species of geminivirus and potyviruses caused

significant losses on chilli in northern part of India7. In

the late 2000, a new tospovirus, GBNV, which caused

severe losses in the production of chilli and other

vegetable crops in India. The virus was first recorded

on groundnut during 1968 and it has become a major

constraint to the cultivation of several leguminous and

solanaceous hosts such as potato, tomato and chilli8-10

.

The another tospovirus, capsicum chlorosis virus

(CaCV) was reported for the first time in tomato in

northern India during 2007 and subsequently it has

been reported in peppers cultivated at southern India11

.

In addition, the distribution of CaCV infecting tomato in

southern and central India belongs to watermelon silver

mottle virus (WSMoV) serogroup. The tospoviruses are

known to be transmitted by different species of thrips in

a persistent and propagative manner. Tospoviruses form

——————

*Author for correspondence:

[email protected]

RAJAMANICKAM et al MOLECULAR ANALYSIS OF TOSPOVIRUSES INFECTING CHILLI

183

pleomorphic, spherical particles within plant cells and

are surrounded by a lipid envelope with two surface

glycoproteins projections, enclosing three nucleocapsids.

The virus consists of a tripartite genome with S, M and

L RNA segments.

The symptoms produced by CaCV mimic the same

as that of GBNV in chilli which includes, circular

chlorotic and necrotic spots on the leaves, stunting of

plants and misshapen leaves under field conditions.

So far, only morphological and symptom based

studies have been conducted on necrosis disease in

chilli and other vegetable crops. Furthermore, CaCV

can react to all the serogroup of GBNV, therefore it is

difficult to differentiate the specificity of tospovirus

infecting chilli through serological reactions11

. There

are few investigations focused on the molecular

characterization on different viruses infecting chilli

under field conditions. Strategies for the management

of viral diseases normally include control of vector

population using insecticides, use of virus-free

propagating material, appropriate cultural practices

and use of resistant cultivars. Recently, plant

virologists have focused their attention to develop

virus resistance transgenic plants or triggered the

host plant resistance against plant viruses through

different molecular approaches worldwide12-13

. The

enhancement of host plant resistance appears as one

of the best options because it offers an easy, cheap

and sustainable technology. Several researchers find

the active sites present in the viral genome which can

positively interact with the several proteins from

plants and microbial origin which leads to

enhancement of plant immunity14-15

. Under these

circumstances, it is highly imperative to develop

biological and molecular tools for the detection of

tospoviruses infecting chilli in Tamil Nadu. Hence,

the present study was aimed in serological, molecular

detection and computational analysis to find out the

protein profiling of tospoviruses infecting chilli.

Materials and Methods

Survey and Collection of Virus Infected Samples

Field survey was conducted during 2017-18 by

covering major chilli growing areas viz., Coimbatore,

Dharapuram, Pollachi and Udumalapet of Tamil Nadu

state to document the virus diseases. The per cent

disease incidence was recorded by counting the total

number of plants and virus infected plants in each and

every field of different areas. The plant samples

showing characteristic tospovirus-like symptoms were

collected separately for further analyses. Ten samples

from each field were collected in zip-loc bags and

labeled. Samples were transported to the laboratory

on ice and kept at 4°C. All samples were processed

within 24 h after collection.

Serological Assay of Tospovirus Isolates

The chilli plants showing the characteristic

symptoms of necrosis diseases were subjected to

direct antigen coating-enzyme linked immunosorbent

assay (DAC-ELISA) using the polyclonal antibodies

specific to tospoviruses16

. The polyclonal antibody

was diluted in antibody buffer and experiment was

carried out with standard protocol. The results were

quantitatively recorded in an ELISA reader at 405 nm

(Biotek EL X 800). The positive samples were further

tested through dot blot immunobinding assay (DIBA).

The infected tissue was extracted (1:10 w/v) in

antigen extraction buffer and filtered through double

layer of cheese cloth. The samples (5-10 µl) were

spotted on nitrocellulose membrane, air-dried and

tested17

. The results were assessed by comparing the

intensity of colour and photographed.

Inoculums Preparation and Mechanical Transmission

The positive samples of tospovirus in DAC-ELISA

were used for transmission studies in cowpea and

chilli hosts by mechanical sap inoculation. Cowpea

cv. C152 and chilli var. syngenta bullet were used as

propagating host for the virus inoculation. Cowpea

and chilli express typical chlorotic and necrotic lesion

within 3-4 days and 10 days after inoculation,

respectively. The plants were raised in the glass house

and maintained under insect-proof conditions. The

virus extract was prepared by macerating infected

chilli plant tissue with 0.1M sodium phosphate

buffer (pH 7.0) containing 0.1% β-mercaptoethanol,

using ice tray. Inoculation was carried out by gentle

rubbing with inoculum using broad end of the pestle

on the cotyledonary leaves on six day old cowpea

plants and in young leaves of one month old chilli

plants, which were previously dusted with 600 mesh

carborundum powder. After few min, the excess

inoculum was washed with a jet of sterile distilled

water using wash bottle. The inoculated plants were

incubated at room temperature for 3-7 days for

cowpea and for 10 days in case of chilli plants for

studying the expression of symptoms18

.

Total RNA Extraction, cDNA Synthesis and Amplification of

Coat Protein (CP) Genes

Total RNA of ~50 μg/μl concentration was

extracted from 100 mg leaves of infected chilli using

INDIAN J BIOTECHNOL, APRIL 2020

184

trizol plant extraction kit (Sigma Chemicals, USA)

according to the manufacturers protocol and

resuspended in 50 μl nuclease-free water. The total

RNA isolated from the virus infected field chilli

samples were subjected to PCR in

50 µl reaction volume containing cDNA and 2 units

of enzyme mix along with the primers specific

to GBNV (CPF-ATGTCTAACGTYAAGCAGCTC;

CPR-TTACAACTCTAGCGAAGGAC) and CaCV

(CPF-AACCAATAGTTTGCCTCCG; CPR - AGA

GCAATCGAGGCACTA) corresponding to coat

protein gene of GBNV and CaCV, respectively, to

amplify the complete coding region of CP genes19

.

The PCR settings comprised of 35 cycles of

amplification including denaturation at 94°C for

2 min, annealing at 52°C (GBNV) and 60°C (CaCV)

for 30 s, extension at 72°C for 1 min and a final

extension at 72°C for 10 min. PCR reaction was

carried out in Eppendorf master cycler gradient ES.

The amplified products were analysed on 1% agarose

gel, stained with ethidium bromide and photographed

under UV-gel doc system (Alpha Imager).

Prediction of CP Gene at Sequence Level

The amplicons of CP gene fragments were purified

using QIAGEN gel extraction kit (Qiagen Inc.,

Chatsworth, CA, USA), cloned into pGEM-T Easy

vector and transformed into Escherichia coli DH5α

following standard protocol20

. Plasmid DNA from

positive clones were sequenced, edited using the

BIOEDIT software. The nucleotide and amino acid

sequences were aligned with selected sequences from

National Centre for Biotechnology Information (NCBI)

database using MEGA 721-22

. Phylogenetic tree was

constructed using the neighbour-joining method with

bootstrap value of 1,000 replicates23

. Tomato zonate

spot virus was used as a reference out-group member

of the genus tospovirus for rooting the phylogenetic

tree. The protein sequences of GBNV and CaCV

were retrieved from NCBI with accession numbers

MK424873 and MK507959, respectively. The

sequence level comparison was carried by performing

pair-wise alignment and DOTPLOT was used for

performing this pair-wise alignment24

. The classical dot

plot algorithm uses a pair-wise comparison between

two sequences, and the results are presented as a dot-

matrix. A sliding window is often used to filter the

output for better visualization25

.

3D Structure Prediction

To compare the structure of GBNV and CaCV, a

protein model was generated using MODELLER

software26

. To model the protein structure, the target

sequence was aligned with the template structure

using 2D scripts in MODELLER and models for the

target were generated with discrete optimized protein

energy (DOPE) score. The modeled 3D structures

were visualized using PyMOL, an independent

software development project of DeLano Scientific, a

sole proprietorship based in San Carlos, California,

USA27

. The 3D structures were superimposed and

compared to find the structural difference.

RAMPAGE server was used for validating the

protein structure based on Ramachandran plot. The

Ramachandran plot displays the main chain

conformation angles (φ and Ψ) of the polypeptide

chain of a protein molecule28

.

Results

Serodiagnosis of Virus Isolates

During the survey, a highest per cent disease

incidence of tospovirus was recorded in Dharapuram

area of Tirupur district of Tamil Nadu state. The

severe disease symptoms of tospovirus infection were

observed under field condition in young plants, before

the flowering stage. During the field plants infected

by tospovirus showed the diagnostic symptoms of

chlorotic and necrotic ring spots on the leaves and

stems (Fig. 1). Further analysis, the infected samples

showed strong positive reaction with approximately

five-fold increase in absorbance values than the

apparently healthy samples analyzed through DAC-

ELISA. The positive samples were further tested

through dot blot immunobinding assay (DIBA) and

tissue blot immunobinding assay (TIBA). The

positive signals were detected by the development of

purple color on blotted area of nitrocellulose

membrane, while no such color development was

observed in the apparently healthy samples against

tospovirus antiserum. The result was assessed by

visual observation by comparing the intensity of

colour and photographed.

Biological Assay for Proliferation of Virus

Chilli plant samples showing the characteristic

symptoms of tospovirus were collected from field was

propagated on cowpea cv. C152 plants separately

through mechanical sap inoculation. The assay host

plant cowpea cv. C152 expressed distinct local

chlorotic lesions after 4 days post inoculation (dpi).

The inoculated cowpea cotyledonary leaves

developed necrotic lesions and then the systemic

veinal necrosis occurred. The veinal necrosis resulted


185

in severe stem necrosis and led to the death of

inoculated plants. Similarly, the assay host plant chilli

expressed distinct local lesions on 10 days after

inoculation. The inoculated leaves developed circular

necrotic lesions and necrotic patches. The systemic

infection was noticed after one month of expression

of local lesions on the inoculated leaves (Fig. 1c). The

virus produced brown necrotic rings spots and

necrosis symptoms after 10 days of inoculation and

100 per cent transmission of virus was recorded.

Identification of CP Genes

The cDNA derived from the extracted RNA

of chilli samples collected from field were used for

PCR amplification of CP genes of tospovirus.

The amplified PCR products from infected

samples with an amplicon size of approximately

840 bp and 1.2 kb corresponding to CP gene

of GBNV and CaCV respectively (Fig. 2). The

nucleotide sequence analysis using NCBI BLAST

confirmed the association of GBNV/CaCV. The CP

gene sequences of GBNV isolates were submitted in

NCBI GenBank database bearing the Accession

Nos. MK424873 and MK424874. Similarly, CP gene

sequences of CaCV isolates were submitted in

NCBI GenBank database bearing the Accession

Nos. MK507959 and MK507960.

Fig. 1 — Chilli plants expressing symptoms of necrosis disease caused by tospovirus (a and b); circular chlorotic patches on leaves of

inoculated cowpea upon artificial inoculation (c)

Fig. 2 — Amplification GBNV (a) and CaCV (b) from infected samples. Where, Lane 1 - 100 bp ladder; Lane 2 & 3 - Infected samples;

Lane 4 - Healthy control; Lane 5 - Positive control


186

Multiple Sequence Alignment and Analysis of CP Genes Analysis of CP gene sequences of GBNV and

CaCV isolates were compared to the reference sequence of with other tospovirus isolates at the nucleotide and amino acid sequence levels. The GBNV sequences of our isolates showed cent per cent sequence similarity between the isolates. Though, the nucleotide sequence recorded the maximum of 98.30% nucleotide homology with tomato GBNV isolate reported in India (AY463968). This was followed by sequence of our isolates had 97.8%, 97.7% and 92.70%, nucleotide homology with chilli GBNV isolate reported in India (AY882002, KX244330 and AY882001), respectively. Analysis of CP gene of CaCV of our isolates revealed that, the nucleotide sequences had 97.50% nucleotide homology between the isolates. The Coimbatore (MK507959) isolate exhibited the maximum of 96.40% nucleotide homology with CaCV (KY308186) reported in India. This sequence also had 96.0, 96.1 and 95.8% similarity from peanut and tomato CaCV (DQ022745, AY626762 and FJ947156) reported from Thailand, respectively. Similarly, Dharapuram isolate (MK507960) exhibited the maximum of 96.2% nucleotide homology with other groundnut-CaCV (KX078567) reported from China. Further, Dharapuram isolate showed 94.4, 94.3 and 94.2% nucleotide homology with other chilli-GBNV (DQ022745, AY626762 and FJ947156) reported from Thailand respectively (Table 1). Multiple nucleotide sequence alignment and phylogenetic analysis revealed very high homologies between the GBNV and CaCV isolates (Fig. 3). The tospoviruses formed two different clusters. Where, cluster I is comprised of GBNV isolates and cluster II is comprised of CaCV isolates. Further sequence analysis, higher homology between the nucleotide sequence of chilli, peanut and tomato isolates were observed. Multiple Sequence Alignment and Protein Level Comparison of CP Genes

The alignment and visualization of GBNV/CaCV sequences using MULTALIN programme revealed more than 90% amino acid sequence similarity between two tospoviruses (Fig. 4). Further, dotplot analysis revealed that CP gene of GBNV and CaCV had substantial region similarity, several dots align to form diagonal lines and local regions exhibited the long diagonal lines. The analysis also showed the other features such as repeats (which form parallel diagonal lines), insertions or deletions (which form


187

breaks or discontinuities in the diagonal lines). The

amino acid sequences exhibited more than 90%

sequence similarity formed a long diagonal line.

3D Structure Comparison of CP Genes

The template structure, 5IP2A chain was retrieved

using PDB BLAST based on the maximum query

coverage (87%) with more than 30% sequence

identity. Further, modelling process with the Python

script for model in MODELLER resulted in selection

of 3 best constructed models based on atomic charges,

Van der waals interaction and hydrophilicity. The

software generates the model with DOPE score and

the model with least DOPE scores of -25038.51 and -

24820.74 was selected as the best one for the targets

GBNV and CaCV, respectively. The generated 3D

structures visualized using PyMOL software revealed

the single amino acid variation at 259th

position and

was represented as ball and sticks in the model. The

modeled structure of GBNV was comprised with

glutamine and CaCV was comprised with valine at

259th position (Fig. 5).

Fig. 3 — Neighbour joining phylogenetic tree based on the nucleotide sequences of CP gene of GBNV, CaCV and tomato zonate spot

virus as a for out group


188

Discussion

Studies on emerging tospoviruses infecting chilli in

Tamil Nadu state are important since it causes crop

loss in terms of yield and quality, provides the basic

knowledge on disease diagnosis and genetic

information of the virus. The infected chilli samples

showed symptoms of tospoviruses including necrosis

and chlorosis on leaves and stems. Initially small

concentric rings appeared on the leaves, which later

coalesced to produce mosaic pattern in older leaves.

In some plants, circular necrotic rings were observed

on the young leaves, later which spread to the

terminal buds leading to complete drying of leaves

under field conditions. In advance stage, necrotic

streaks were observed on the tender part of the stem,

mimicking the GBNV as like that of infected tomato.

The chilli exhibiting typical symptoms of

tospovriuses were inoculated on cowpea cv. C152

plants, resulted in production of typical circular

chlorotic lesions on inoculated primary leaves on

4 dpi; advanced stage of infection led to death of

plants under glasshouse conditions. The development

of circular chlorotic and necrotic lesions on cowpea

are the characteristic symptoms of tospoviruses by

mechanical inoculation29

. In India, CaCV a genus in

tospovirus was first reported in tomato during the year

2007 and subsequently this virus also reported from

chilli since the virus has a wide host range. In our

study, chilli produced local and as well systemic

infection by developing concentric necrotic lesions on

the inoculated leaves, later coalesce to form large

patches, stunting of plants with small size leaves with

systemic infection irrespective of isolates upon

artificial inoculation with CaCV and GBNV. This

Fig. 4 — Protein sequence alignment of GBNV and CaCV using MultAlin

Fig. 5 — 3D structure representations of GBNV model (a) and CaCV model (b) and superimposed modelled structure (c)


189

result was supported by Kunkalikar et al. (2010) who

reported that tospovirus produced symptoms of

cholorotic and necrotic lesions in many plant species

which belong to Solanaceae, Amaranthaceae,

Leguminaceae and Fabaceae. They also stated the

symptoms produced by CaCV resemble those induced

by GBNV under field and greenhouse conditions,

which indicates the possible occurrence of CaCV for

long time in India. Recently, Haokip et al. (2016)

observed that CaCV from chilli produced circular

chlorotic and necrotic lesions on cowpea and

concentric necrotic rings on chilli.

Serological or immunological assays have been

adopted for the detection of plant viruses for a long

time. Infected chilli samples collected from field were

found to be positive for polyclonal antibody specific

to tospoviruses. However, chilli sample infected with

CaCV was found to react strongly than the one

infected with GBNV. This type of results were

supported by Kunkalikar et al. (2010) who reported

that CaCV infected tomato and chilli samples showed

strong reaction with CaCV-PAb, but were found to

react weakly with PBNV-PAb in ELISA. Similarly,

Haokip et al. (2016) raised the polyclonal antiserum

against the CaCV and reported the positive reaction of

infected samples of chilli in ELISA. In our study,

virus preparation has reacted with the antiserum

specific to tospoviruses and samples were also

produced positive reaction in DIBA and TIBA. This

suggested that it is difficult to differentiate the virus

infecting chilli under the genus tospovirus group

through symptomatology and serodiagnosis. This was

supported by the results of Jain et al. (2005) who

developed the antiserum specific to GBNV and

detected the virus from infected chilli under field

condition30

. Similarly, Gopal et al. (2011) surveyed

for GBNV infection in chilli under field condition at

Ranga Reddy district of Andhra Pradesh and they

confirmed the infection of GBNV using DAC-

ELISA31

. Sharma and Kulshrestha (2016) also

detected the GBNV infection in bell pepper from

Himachal Pradesh, through DAC-ELISA using

polyclonal antiserum specific to GBNV32

.

The present investigation was attempted for accurate

and highly specific molecular characterization of two

tospoviruses from chilli based on molecular profiling

of CP gene. During our survey in Tamil Nadu, chilli

plants were found to be diseased autonomously with

GBNV and CaCV, but there were no mixed infections.

In disparity, CaCV and TSWV infected the tomato

plant as mixed infection33

. The results of our study

represented the amplification of approximately

840 bp and 1.2 kb corresponding to CP gene of

GBNV and CaCV using specific primers. The GBNV

isolates shared 98.3% identities with tomato isolate

(AY463968) reported from India. The results were in

line with those obtained by Pavithra et al. (2016), who

diagnosed the presence of GBNV from chilli collected

from the farmer fields and characterized the CP gene

of GBNV using specific primers. Further, the

sequence analysis revealed more than 96% sequence

similarity with GBNV isolate reported from India.

The present study also demonstrated that, CaCV

isolates shared maximum of 97.50% and 96.4%

identities with Indian isolates. In earlier studies,

higher level of identities was obtained from CaCV

isolates with similarity of more than 98% and

99-100% at nucleotide and amino acid levels,

respectively

(Haokip et al. 2016). Similarly,

CaCV isolates from chilli and tomato are genetically

similar showing 96-100 % similarity with N gene

amino acid, indicated the wider host range with

possible spread of virus from one host to another

(Kunkalikar et al. 2010).

In our study, a combined phylogenetic tree

constructed by NJ algorithm using tospoviruses

revealed the formation of two different clusters. The

phylogenetic analysis enabled to describe the

relatedness of viruses infecting crops by generating a

distinct cluster and their gradual evolution34

. We have

investigated the molecular characterization and

profiling of CP gene of GBNV and CaCV isolates

involved in disease outbreaks in chilli. Since the

relative sequence analysis CP gene soft tospoviruses

infecting chilli and structural study of genes through

3D prediction structure provides precise identification

of variability at molecular level and provided a simple

method of classification of emerging viruses. This

type of study was conducted in mixed infection of

chilli vein mottle virus and chilli leaf curl virus (Sahu

et al. 2016) and cucumber mosaic virus (Biswas et al.

2013). In our study, GBNV and CaCV isolates were

exhibited the minimum level of similarities between

the viruses, which concluded that both viruses are

dissimilar at nucleotide levels. Whereas, MULTALIN

programme revealed that both the viruses had 90%

similarity at amino acid levels, which confirms the

presence of little variation between CaCV and GBNV

isolates. They were exhibited a single variation in

259th position at amino acid levels consisting of


190

glutamine and valine in GBNV and CaCV,

respectively. Similarly, Chavhan et al. (2018) also

documented the protein profiling of tobacco streak

virus, represented the active pocket site at 177th

amino

acid residue. The three dimensional model from ßC1

pathogenicity region of cotton leaf curl virus

suggested that plant flavonoid have strong binding

active site, as these molecules binds with active site at

viral protein which leads to plant defense against viral

infection (Sarwar et al. 2019). This study concludes

the occurrence of GBNV and CaCV in chilli at

serological and molecular levels. The sequence

structure analysis highlights the key residues involved

in the confirmation of the protein structure. It would

enhance the power of the analysis by drawing a

connection between primary sequence, 3D structure

and its function. Predicted 3D structures of viral

proteins allowed to find a consistent location

for conserved residues among all members of

tospovirus, which could be useful in developing

novel virus control strategies targeting the localized

residues in future.

Acknowledgements

Authors gratefully acknowledge the School of Post

Graduate Studies, Tamil Nadu Agricultural

University, Coimbatore, India for the financial

support of the project under Dr. A.P.J. Abdul Kalam

Fellowship. Also, acknowledge the DST-FIST, GOI,

New Delhi for providing infrastructure facilities.

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