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MOLECULAR DIAGNOSIS OF THE VIRUSES ASSOCIATED WITH CHIRKE AND FOORKEY DISEASES OF LARGE CARDAMOM

KrishiKosh

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Title MOLECULAR DIAGNOSIS OF THE VIRUSES ASSOCIATED WITH CHIRKE AND FOORKEY DISEASES OF LARGE CARDAMOM
Ph.D.
 
Creator S. VIJAY ANAND RAJ
 
Contributor Bikash Mandal
 
Subject proteins, cardamoms, diseases, biological phenomena, genomes, planting, pcr, viruses, dna, genes
 
Description Chirke and foorkey diseases are important constraints of large cardamom
cultivation in eastern sub-Himalayan mountains. However the genome of the
chirke virus has not been fully characterized and molecular diagnostics were not
available. In this study 3’ terminal genome sequence (5961 nucleotides) of the
virus associated with chirke disease containing the partial CI, 9k, NIa-VPg, NIa-
Pro, NIb, CP and the 3' terminal untranslated region (UTR) was determined. The
partial polyprotein nucleotides (4401) and amino acids (1346) containing the 9k,
NIa-VPg, NIa-Pro, NIb and CP showed maximum sequence identity of 41.4 %
and 69.2 % with Cardamom mosaic virus (CdMV) causing katte disease in small
cardamom, respectively. The deduced amino acids sequences of the 9K protein
showed a maximum sequence identity of 83.7 % with CdMV. The viral genome
contains the RNA helicase motif, conserved tyrosine residue, Cysteine protease,
GDD motif as found in the macluraviruses and potyviruses. Phylogenetic analysis
based on the five polyproteins showed that virus was closer to CdMV, Alpinia
mosaic virus, and Chinese yam necrotic mosaic virus subclade. The deduced
amino acid sequence of putative coat protein (CP) gene showed maximum
similarity of 65.8% with the CdMV and other macluraviruses. The results suggest
that the virus associated with the chirke disease of large cardamom is a new
species under the genus Macluravirus in the family Potyviridae for which the
name large cardamom chirke virus (LCCV) is proposed.
Purification of LCCV from large cardamom tissues is difficult and
therefore immunodiagnostic reagents were not available. In the present study, we
have successfully expressed CP gene of LCCV in Escherichia coli. The
purification of expressed protein by Ni-NTA affinity chromatography was
inefficient due to precipitation of protein during renaturation. We have optimized
a simple, inexpensive and efficient method for purification of the expressed CP
through gel extraction with 5% SDS followed by renaturation in Milli-Q water,
which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher
titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced,
which strongly recognized LCCV in crude leaf extract and showed minimal
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background reaction with the healthy leaf extract in enzyme linked
immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The
sensitivities of the ELISA and DIBA were 5 ng and 0.1 ng of expressed protein,
respectively. Both the ELISA and DIBA were validated with 100% accuracy in
detecting LCCV in field samples. The results of ELISA and DIBA were further
confirmed by IC-RT-PCR at a dilution of 1:500 of polyclonal antibody. The PAb
differentiated Cardamom mosaic virus (CdMV), another close relative of LCCV.
Our study is first to report highly efficient immunodiagnosis with PAb to E. coli
expressed recombinant CP of a virus under the genus Macluravirus. The antigen
expression construct and PAb developed in the present study will be useful in
production of virus free planting materials of large cardamom.
Cardamom bushy dwarf virus (CBDV) belongs to the genus Babuvirus,
family Nanoviridae causes foorkey disease in large cardamom. In the present
study, the coat protein gene of CBDV was cloned in prokaryotic expression vector
pET28a and expressed in the rosetta E.coli (DE3) strain. The expressed CBDV CP
protein was purified using gel extraction using 5% SDS, which yielded 4.2 mg/ml.
The PAb specifically detected the CBDV CP in crude and purified preparations in
the western blot. The sample/healthy ratio indicates that the produced PAb reacts
with the inner tender meristem tissue at a dilution of 1:800. The antiserum
produced against the expressed CBDV specifically detected the CBDV in the
inner meristem tissues of the CBDV infected plantlets and a marginal positive
reaction with the base of the stem, whereas the PAb didn’t detect the CBDV in the
leaf, leaf sheath. PCR based detection of CBDV using conserved stem and loop
primers was optimized using fast Taq DNA polymerase and a fluorescent dye was
successfully used for rapid detection of CBDV. Both the ELISA and PCR based
detection of CBDV were validated with 100% accuracy in field samples. Our
study is first to report sensitive immunodiagnosis with PAb to E. coli expressed
recombinant CP of a virus under the genus CBDV.
This study for the first time characterized the virus associated with chirke
disease of large cardamom and ~80% of the genome sequence was generated.
Molecular diagnostic reagents developed in this study will be useful for the
production of virus free planting materials.
 
Date 2016-03-11T14:56:13Z
2016-03-11T14:56:13Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/65090
 
Language en_US
 
Format application/pdf
 
Publisher IARI, Division of Plant Pathology, New Delhi