Serological Detection of Cardamom Mosaic Virus Infecting Small Cardamom, Elettaria cardamomum L.

Int. J. Life. Sci. Scienti. Res., 2(4): 333-338 (ISSN: 2455-1716) Impact Factor 2.4 JULY-2016
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Serological Detection of Cardamom Mosaic Virus
Infecting Small Cardamom, Elettaria
cardamomum L.
Snigdha Tiwari 1*
, Anitha Peter2
, Shamprasad Phadnis3
1
Ph. D Scholar, Department of Molecular Biology and Genetic Engineering, G. B. Pant University of Agriculture and
Technology, Pantnagar, Uttarkhand, India
2
Associate Professor, Department of Plant Biotechnology, UAS (B), GKVK, Bangalore, India
3
Ph.D. Scholar, Department of Plant Biotechnology, UAS (B), GKVK, Bangalore, India
*
Address for Correspondence: Snigdha Tiwari, Ph. D Scholar, Department of Molecular Biology and Genetic
Engineering, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarkhand, India
Received: 20 April 2016/Revised: 19 May 2016/Accepted: 16 June 2016
ABSTRACT- Small Cardamom (Elettariacardamomum L. Maton) is one of the major spice crops of India, which were
the world’s largest producer and exporter of cardamom till 1980. There has however been a reduction in production,
mainly because of Katte disease, caused by cardamom mosaic virus (CdMV) a potyvirus. Viral diseases can be managed
effectively by early diagnosis using serological methods. In the present investigation, CdMV isolates were sampled from
Mudigere, Karnataka, ultra purified, and electron micro graphed for confirmation. Polyclonal antibodies were raised
against the virus and a direct antigen coating plate Enzyme linked immunosorbent Assay (DAC-ELISA) and Dot-ELISA
(DIBA) standardized to detect the virus in diseased and tissue cultured plants. Early diagnosis in planting material will aid
in using disease free material for better yields and hence increased profit to the farmer.
Key-words- Cardamom mosaic virus (CdMV), Electron microscopy, Direct antigen coating Enzyme linked
immunosorbent Assay (DAC-ELISA), Dot-ELISA (DIBA)
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INTRODUCTION
Small cardamom (Elettariacardamomum L. Maton),
popularly known as ‘Queen of spices’, is one of the major
spice crops and enjoys a unique position in the international
spice market. It is one of the highly prized spices of the
world and is the third most expensive spice after saffron
and vanilla. It belongs to the family Zingiberaceae and is
indigenous to southern India [1]. India was the world’s
largest producer and exporter of cardamom till the 1980s
but, by 1990s Guatemala emerged as the leading producer
and exporter of cardamom [2]. The production area of
cardamom in India has drastically come down from 1.05
lakh ha (1987-88) to only 71, 110 ha (2011-12)
(www.mcxindia.org).
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Viral diseases of cardamom is one of the major reason for
low productivity of cardamom (145 kg/ha) [3-4]. ‘Katte’ or
Mosaic or Marble (Annamalai) disease being one of the
most destructive among them [5]. The disease is caused by
Cardamom mosaic virus (CdMV), belonging to the genus
Macluravirus of family Potyviridaewhich appears as
flexuous filamentous particles of about 650 nm in length
and 10-12 nm in diameter [4]. In diseased plants, the
morphological characteristics include interrupted pale
green stripes which run along the veins and parallel to each
other from midrib to margin of the leaf. In advanced stages
the pale green stripes are distributed evenly over the leaf
surface giving a distinct mosaic pattern. The disease
incidence was found to be 0.01 to 99.00 per cent in
cardamom growing tracts in South India [6]. The virus is
transmitted through the banana aphid, Pentalonia
nigronervosa and infected rhizomes [7]. The virus exists as
a symptomless carrier or with mild symptoms in the
planting material [8].
Management of ‘katte’ is difficult as there is no vector
control, alternate control or appropriate cultural practices.
Disease resistant cardamom varieties released against Katte
disease are met with little success, as they showed poor
Research Article (Open access)

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 4
agronomic character. Breeding for resistance and cross
protection are the traditional methods of plant virus control
[9] which shows only limited success in this crop as there
isn’t a single source of viable resistance in the species of
Elettaria. Other related genera which are infected by the
same virus are Ammomum, Alpinia, Curcuma, etc.
Incompatibility barriers have prevented the formation of
fruits in intergeneric hybridization [2]. Though tissue
culture techniques have been attempted for producing virus
free cardamom plantlets, it is unsuccessful [10].
In this scenario, one of the promising methods of managing
the disease is through early diagnosis using serological
techniques. This paper reports the purification and electron
microscopy of the virus and the standardization of a plate
Enzyme Linked Immunosorbent Assay (ELISA) and a
Dot-ELISA (DIBA) for the detection of the virus.
MATERIALS AND METHODS
Virus isolation, purification and Electron
microscopy
Cardamom plants infected with CdMV var. Mudigere-1 and
Mudigere-2 showing characteristic symptoms of cardamom
mosaic: alternate light yellow patches and dark green
patches with a characteristic mosaic pattern on upper
surface of leaf (Fig.1) were collected from Zonal
Agriculture Research Station, Mudigere, Karnataka, 2008.
The experiments were conducted in dept. of Biotechnology,
UAS, Bangalore, IVRI, Bangalore and NIMHANS,
Bangalore, 2008.
The virus was extracted by modification of procedure given
by [11]. Infected leaves were ground in liquid nitrogen and
homogenized with two and-a-half volumes (1g/2.5ml) of
0.1 M potassium phosphate buffer, pH 8.0 containing 0.1 %
2-mercaptaethanol and 0.225 per cent sodium diethyl
diethiocarbamate (DIECA). The extract was filtered
through double layered muslin cloth. Ten per cent cold
chloroform (v/v) was added to the filtrate and emulsified
for 15 min. The emulsion was broken by centrifugation at
10,000 rpm for 30 min. The upper aqueous phase
containing virus was collected and precipitated by adding
six per cent polyethylene glycol (PEG, molecular weight
6000) and 0.2 M sodium chloride. After stirring for 45 min.
in ice bath, the preparation was kept in refrigerator
overnight. The suspension was centrifuged at 10,000 rpm
for 30 min. and precipitate was resuspended in 0.05 M
borate phosphate buffer, pH 8.3, containing 0.2 M urea
(BPU) and was stirred for 90 min. The suspension was
vortexed and centrifuged at 10,000 rpm for 30 min. The
supernatant was collected with the help of Pasteur pipette
and centrifuged at 24,500 rpm (60,000 g) for 2 h. in SW 28
rotor. The supernatant was discarded and the liquid was
drained by keeping the tubes in an inverted position on a
filter paper. The pellet was resuspended in minimum
volume of BPU buffer and centrifuged at 10,000 rpm for 15
min. The supernatant was collected and made upto a
required volume by using BPU buffer and centrifuged at
30,000 rpm (1, 10, 000 g) for 2 h. The supernatant was
discarded and the liquid was drained off by keeping the
centrifuge tubes in an inverted position. The pellet was
resuspended in minimum volume of BPU buffer and is
layered on 20% sucrose cushion pad and centrifuged at
24,500 rpm for 2 h. Virus pellet was resuspended in 1 ml of
0.05 potassium phosphate buffer, pH 7.0 and centrifuged at
5,000 rpm for 10 min. The supernatant containing virus
was collected. The virus suspension was diluted with 0.05
M potassium phosphate buffer. The purified virus
suspension was used for electron microscopy.
A drop of purified virus preparation was placed on the
carbon-coated grids and allowed to settle for 2-3 min.
Excess sample was removed and a small droplet of dye
(uranyl acetate) was placed on it for 2-3 min. Excess stain
was drained after 15-30 min. and the grids were placed in
desiccator and examined under Biotechnai G-2,
Transmission electron microscope (TEM) at NIMHANS,
Bangalore.
Antiserum Production
New Zealand white rabbit was injected intramuscularly
with five 0.5 ml virus injections at weekly intervals. The
first was with equal volume of Freund’s complete adjuvant
and subsequent ones with Fruend’s incomplete adjuvant.
Test bleed was after the fourth injection, after a ten day gap
the final injection was given and final bleeding done ten
days later. The serum was separated after overnight
incubation at room temperature and antiserum collected
[12].
Enzyme linked immunosorbent assay (ELISA)
Indirect plate ELISA was standardized, using different
dilutions of infected plant sap (ips) or ultrapurified virus
(uv) as antigen, primary antibody was the raised antiserum
and secondary antibody was an alkaline phosphatase
conjugate. 100 µl of suitably diluted virus in coating buffer
was added to the wells of the polystyrene plate, covered
and incubated at 40
C overnight. The plate was then soaked
and washed thrice in PBS-T for 3 min. 200 μl of blocking
solution was added to the each well of the plate and
incubated followed by 100 µl each of suitably diluted
primary antibody and secondary antibody alkaline
phosphatase conjugate (Goat antirabbitIgG) in blocking
buffer. The incubation temperature and duration for each
step was 370
C for 1½ h. The incubation temperature for
each step was 370
C for 1½ h. Wash procedure was repeated
after each step. Finally 100 µl of enzyme substrate (para
nitro phenyl phosphate) was added and incubated at 370
C
for ½ h. Reaction was terminated with 50 µl of 5N KOH
after color development. Visual observation and absorbance
values using ELISA reader was taken at 405 nm.
The first protocol standardized for ELISA involved the
non-addition of PVP and ovalbumin in the blocking buffer
(PBS-T) containing 3% fat free milk (ELISA1) and the
second with PVP and ovalbumin in the blocking buffer
(ELISA2) [13]. The details of the dilutions of the antigen,
primary and secondary antibody for ELISA1 and ELISA2

are given in Table 1 and 2 respectively.
DIBA
Dot-Blot was done on nitrocellulose membrane which was
dipped in boiling water and equilibrated in PBS. Two
microlitre of 1:10, 1:100 and 1:500 dilution of ultrapurified
virus was loaded on the membrane using the impression of
wells from a manifold with a control, which was the buffer.
The dots were deep dried using a vacuum pump for 30 min.
The membrane was slowly removed and washed thrice with
PBS-T (0.05% TWEEN-20 in PBS) and air-dried. It was
then blocked with the blocking solution as above and
dotted with different dilutions of primary antibody and
followed by a wash step and then coating with the
secondary antibody (Table 3). Incubation for each step was
at 37˚C for 1½ h. Wash step was repeated after each step
(three washes, 3 min. per wash). The membrane was then
treated with the substrate bromocresoindoyl pyrophosphate
(BCIP) for 10 min in dark to develop color. The primary
antibody dilutions used were 1:500 and 1:1000 while that
of secondary antibody was 1:1000.
Disease diagnosis
Disease diagnosis was done in infected, healthy and
procured tissue cultured plants.
RESULTS AND DISCUSSION
Virus Purification and Electron microscopic study
The virus collected from diseased plants with typical
mosaic symptoms (Fig. 1) was subjected to purification
protocol described by [11] for peanut mottle virus with
modifications, namely using potassium phosphate buffer
(pH 8.0) containing 0.225% DIECA, 0.1%,
2-mercaptaethanol and PEG precipitation followed by
differential ultracentrifugation and 20 per cent sucrose
cushion pad. This method was found to be successful to
isolate the virus. The concentration of the virus however
was low in the present purification, which may be attributed
to the presence of fibrous matrix of the host tissue which
interferes with the various stages of purification of the virus
which is one of the major drawbacks encountered in many
potyvirus purification procedures [14], who has also
reported aggregation of virus particles at early stages of
purification of CdMV. Similar problems in purification of
potyviruses has been reported in Lettuce Mosaic Virus [15],
Soybean Mosaic Virus [16], Potato Virus Y[17], Tobacco
Etch Virus [18], Pea Seed-borne Mosaic Virus [18], Maize
Dwarf Virus [20], and Poplar Mosaic Virus [21]. To avoid
this, detergents such as Triton X-100 was used which
disrupt the particles of elongated viruses that tend to
aggregate and bind to plant organelles. Aggregation on
storage is also a deterrent in the purification of many
filamentous viruses like CdMV [5,9-10,14], Tobacco
Mosaic Virus [22] and Potato Virus Y [23]. The use of urea
in extraction and resuspension buffer can also reduce
aggregation considerably [9-10,18]. 20% sucrose gradient
was used in the present investigation, which is a
modification over the original protocol of [11] and [10] to
preserve the integrity of the virus particles as reported by
earlier workers [24-25].
Electron microscopy studies confirmed the presence of
flexuous rod shaped viral like particles at different
magnifications. The size of the virus particles measured was
700nm in length and 12-13 nm in width (Plate 1). The
electron microscopy of the purified preparation of CdMV
by using two per cent uranyl acetate revealed aggregated
numerous flexuous rod shaped particles intertwined
together which made it difficult to measure. However
average length of 700 nm was obtained among the
measured particles. This partial size closely resembled the
observation of 700-720nm [10], 720 nm [14], 710 nm [9].
Antibody production and serological tests
Polyclonal antibodies (PAbs) were successfully raised in
rabbit against CdMV by giving 2 intramuscular and
subcutaneous injections with the purified virus. Successful
production of PAbs against CdMV in rabbits and mice
respectively although with a different injection schedule
have also been reported [9-10].

Standardization of antibody titer raised against CdMV virion for ELISA
Standardization of antibody titer raised against CdMV virus for ELISA was done to know the suitable concentration of
virus and primary antibody for testing the virus. Titre fixation using different dilutions of antiserum with the different
concentration of antigen was done in ELISA plates in two runs namely ELISA 1 and ELISA 2 as mentioned earlier.
ELISA 1
The antiserum dilutions used were 1:500, 1:1000, and 1:2000. The concentrations of antigen were undiluted, 1:10, 1:50,
1:100, 1:200, 1:500 and 1:1000. Secondary Antibody dilutions were 1:1000 and 1:2000. In all combinations colour
development was observed. Optimum OD reading of 0.886 was observed in the combination of 1000 ng of antigen and
1:500 dilutions of primary antisera with 1:2000 dilutions of secondary antibodies (Table 1).
Table 1: ELISA 1 readings (OD) at 405 nm for the standardization of antibody titre raised against CdMV
Antigen Dilution
Primary
Antibody
Dilution
1000 ng 500ng 200ng 100ng 50 ng 10 ng +ve -ve
Secondary Antibody Dilution of 1:1000
1:500
0.895 0.789 0.632 0.589 0.421 0.190 1.460 0.076
1:1000
0.824 0.579 0.522 0.395 0.276 0.176 1.207 0.067
1:2000
0.657 0.498 0.426 0.350 0.210 0.123 1.060 0.087
1:500
0.886 0.635 0.512 0.492 0.376 0.312 1.346 0.082
1:1000
0.762 0.642 0.502 0.448 0.346 0.293 1.207 0.067
1:2000
0.683 0.539 0.483 0.384 0.306 0.216 1.060 0.087
ELISA 2 with PVP and Ovalbumin
The antiserum dilutions used were 1:500, 1:1000, and 1:2000. The concentrations of antigen were undiluted, 1:10, 1:50,
1:100, 1:200, 1:500 and 1:1000, with secondary antibody dilutions of 1:1000 and 1:2000. In all combinations colour
development was observed. Optimum OD reading of 0.960 was observed in the combination of 1000 ng of antigen and
1:500 diluted primary antisera with 1:2000 dilutions of secondary antibodies. This combination of 1:100 of antigen and
1:500 dilution of primary antiserum and 1:2000 of secondary antibody was used in further studies (Table 2).
Table 2: ELISA 2 readings (OD) at 405 nm for the standardization of antibody titre raised against CdMV
Antigen Dilution
Primary Anti-
body Dilution
1000 ng 500ng 200ng 100ng 50 ng 10 ng +ve -ve
1:500 1.004 0.894 0.835 0.711 0.624 0.514 1.756 0.078
1:1000 0.947 0.635 0.487 0.437 0.349 0.351 1.558 0.052
1:2000 0.828 0.459 0.363 0.339 0.310 0.296 1.410 0.032
1:500 0.960 0.600 0.432 0.419 0.384 0.331 1.329 0.089
1:1000 0.629 0.477 0.411 0.289 0.276 0.270 1.218 0.056
1:2000 0.624 0.391 0.299 0.250 0.235 0.183 1.254 0.067
Standardization of an indirect-ELISA for detecting CdMV at 5µg/ml concentration of primary antibody and 1:1000 and
1:2000 dilution of secondary antibody, and a direct-ELISA at same concentration of primary antibody and 1:400 and
1:800 dilution of secondary antibody [10]. It has been reported that CdMV could be detected at 1:1000 dilution of antigen
(leaf extract) and 1:6000 dilution of primary antibody but optimum was 1:100 dilution of antigen and 1:2000 of primary
antibody [9]. The results of the present investigation are in accordance with these reports. The ELISA results also revealed
that the use of PVP and OA in blocking buffer enhanced the efficiency of ELISA. This is in accordance with the

standardized protocols for serological detection of plant viruses [13] and also the protocol used for a related virus, viz the
pepper vein banding mosaic virus [26].
Standardization of antibody titre raised against CdMV virus for Dot ELISA
Standardization of antibody titre raised against CdMV virus for ELISA was done to know the suitable concentration of
virus and primary antibody raised in rabbit. Titre fixation using different dilutions of antiserum with the different
concentration of antigen was done in nitrocellulose membrane. The antiserum dilutions used were 1:500, 1:1000 and
1:2000 with secondary antibody concentration of 1:2000 and antigen concentration of 2µg/ml. In all combinations colour
development was observed in all dilutions except in negative control (containing only coating buffer) (Plate 2). Optimum
colour development observed was in 1:1000 diluted antisera. This combination of 1:1000 dilution of antiserum was used
in further studies (Table 3).
The colour development in all the dots helped in fixation of
antibody titre against CdMV. The titre was also confirmed
using Dot-ELISA.
Disease diagnosis
The virus was detected in infected plants and not in healthy
and procured tissue cultured plants by using standardized
ELISA (Plate 3, Table 4 and Figure 2). The virus was
detected in infected plants collected from the field and not
in healthy plants and the procured tissue cultured plants
using ELISA. This can be compared with the findings of
direct [10] and indirect ELISA and DOT-ELISA [9]
standardized for detection of CdMV in infected plants.
Detection of virus in plants is very difficult and hence
attempts have been made to understand the virus and its
serological detection by using ELISA for early infection.
Early diagnosis in mother plants through conventional
multiplication (rhizomes) and micropropagation is required
to curtail crop loss and avoid inoculum build and spread.
Table 3: Visual scoresof Dot-Blot for Standardization
of antibody titre raised against CdMV
Antiserum
dilutions
Control 1:500 1:1000 1:2000
Scores - ++++ ++++ +++
++++= Strongly positive, += weakly positive, -=Negative
Secondary antibody: 1:1000 dilution

Table 4: ELISA reading (OD) at 405 for detection of CdMV in tissue culture plants
Secondary antibody of 1:1000 dilution
CONCLUSION
The present study was successful in isolating and purifying
the CdMV from the infected leaves of cardamom collected
from Mudigere, electron microscopic studies of the purified
preparations of Katte virus revealed the flexuous rod
shaped particles and in standardization of DAC-ELISA and
DIBA against CdMV and its detection in tissue cultured
plants.
ACKNOWLEDGMENT
We are grateful to the Zonal Agriculture Research Station,
Mudigere, Karnataka, India. This work was supported by
DBT from the Department of Biotechnology, University of
Agricultural Sciences, Bangalore, India.
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Source of Financial Support: DBT-GOI
Conflict of interest: Nil
Sample
Antibody -ve +ve Infected leaves Tissue culture 1 Tissue culture 2 Healthy
1:500 0.0795 1.789 1.480 0.0889 0.0867 0.0876
1:1000 0.0724 1.579 1.257 0.0860 0.0880 0.0879
1:2000 0.0657 1.498 1.043 0.0840 0.0838 0.0836

Serological Detection of Cardamom Mosaic Virus Infecting Small Cardamom, Elettaria cardamomum L.

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