International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 3, Jun 2016, 39-44
TJPRC Pvt. Ltd.

PRODUCTION OF POLYCLONAL ANTISERA AGAINST CASSAVA

MOSAIC VIRUS IN CASSAVA
NAIR A. B
Research Scholar, Department, of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, Kerala, India
ABSTRACT
Serological techniques are commonly used for the detection and characterization of plant viruses. These
methods employ the use of antisera produced by highly purified preparations. The present study was carried out for the
production of polyclonal antibody against Srilankan cassava mosaic virus (SLCMV). Partial purification of the virus was
successfully done from the infected young cassava leaves collected from the plants maintained in insect proof glass house.
The antiserum against Srilankan cassava mosaic virus was produced by immunizing New Zealand white female rabbit by
giving five intramuscular injections at weekly intervals with partially purified virus preparation mixed with an equal
volume of Fruends incomplete adjuvant. Finally, the blood of the animal was collected 21 days after the last injection
and processed by following the procedure described in materials and methods. The antiserum thus produced was used for

titre of antibody. Different dilutions of antiserum viz. 1:128, 1:256, 1:512, 1:1024, 1:2048 and 1:4096 tested gave a titre of
1: 512..
KEYWORDS: SLCMV, Antibodies, Titre, Rabbit

Received: Mar 23, 2016; Accepted: Apr 07, 2016; Published: Apr 12, 2016; Paper Id.: IJASRJUN20166

Original Article

further studies after cross absorption. DAC- ELISA was carried out to confirm working of antiserum and to determine the

INTRODUCTION
Cassava (Manihot esculenta Crantz), the staple food for about one fifth of the worlds population is found
susceptible to several pests and diseases of which Cassava Mosaic Disease (CMD) is found to be the most serious
one causing severe yield reduction. Cassava mosaic disease (CMD) is reported to be wide spread in Kerala which is
a limiting factor for cassava production and is caused by a virus included in the genus Begomovirus (Family:
Geminiviridae) that are transmitted by the whitefly, Bemisia tabaci and disseminated through the stem cuttings used
routinely for propagation. CMD causes severe mosaic, leaf distortion and stunted growth of plants, resulting in
considerable reduction of yield. In India, the disease can cause a yield loss of 17-88% depending on the cultivars
grown (Malathi et al., 1985). This disease is reported to be widespread in South India mainly in Kerala, Tamil Nadu
and Andhra Pradesh (Narasimhan and Arjunan, 1976).
Cassava mosaic begomoviruses (CMBeVs) are characterized by a circular single stranded DNA (ssDNA)
genome that consist of two components, termed DNA-A and DNA-B encapsidated in a twinned (geminate) particle
of approximately 20 x 30nm (Zhang et al., 2001). In India, the CMD is caused by two viruses namely Indian
cassava mosaic virus (ICMV) and SriLankan cassava mosaic virus (SLCMV) (Malathi et al., 1983).
A short duration high yielding cassava variety, Vellayani Hraswa has been evolved through selection at the
Instructional Farm, College of Agriculture, Vellayani, but this variety is found highly susceptible to CMD. Since

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Nair A. B

Cassava is vegetatively propagated, the cassava mosaic disease is carried from one crop cycle to the next, through the
cuttings (setts) used as planting material and in the field mostly through whitefly transmission. Yield losses are greatest
when plants are derived from infected cuttings and infection of upto 10% was reported with whiteflies (Colvin et al.,
2004). Detailed information on the disease along with methods of early detection and elimination of the virus through
meristem culture can improve its performance in the field. A simple and sensitive method for detection and quantification
of virus level in the plant is ELISA. The present study involved the production of polyclonal antisera against Cassava
mosaic virus for the serological diagnosis of the virus from infected samples since this method is found more reliable
compared to the conventional methods of detection mainly based on symptomatology.
Fargette et al. (1987) purified ACMV isolate obtained from diseased cassava plants propagated in N benthamiana
as per the procedure of Walter (1980). Mathew and Muniyappa (1992) purified ICMV from cassava and from systemically
infected Nicotiana benthamiana leaves. Perera and Dasssanayake (2002) purified CMBeV using young cassava leaves with
symptoms, extracted in 0.1 M Tris HCl, pH 8.4 + 0.2 % monothioglycerol and clarified with half volume of chloroform. It
was precipitated with 4 % PEG and purified by two cycles of differential centrifugation. CMBeV specific polyclonal
antiserum was successfully produced by Fargette et al. (1987). A rabbit was given six subcutaneous injections of 1-2 mg of
virus fixed in 1% glutaraldehyde and mixed with an equal volume of Freunds incomplete adjuvant. Two MAbs developed
to ACMV by Givord et al. (1994) were found useful in screening cassava plants in field tests for the presence of ACMV, in
detecting whitefly transmitted geminiviruses from various plants and various geographical origins, in assessing virus
concentrations in plants and in searching for natural reservoirs of the viruses.
Perera and Dassanayake (2002) reported the production of polyclonal antiserum by immunizing Newzealand
white rabbit with partially purified CMBeV. Abouzid et al. (2002) produced polyclonal rabbit antisera to the coat protein of
Bean golden mosaic virus Brazil isolate (BGMV), cabbage leaf curl virus (CabLCV), tomato yellow leaf curl virus
(TYLCV), and tomato mottle virus (ToMoV). The results indicated that polyclonal antisera prepared to express
begomovirus coat proteins were useful for the detection of begomoviruses. Fargette et al (1987) got an antiserum against
CMBeV of titre 1:256 when tested by agar double diffusion. Perera and Dassanayake (2002) got an antiserum dilution of
1:4000 against CMBeV which had the capacity to detect diseased samples by indirect enzyme linked immunosorbent
assay.

MATERIALS AND METHODS

Purification of the Virus
Purification of the Srilankan cassava mosaic virus (SLCMV) infecting cassava variety, Vellayani Hraswa was
carried out using the method described by Perera and Dassanayake (2002) with slight modification. The stock culture of
virus was maintained in insect proof glass house in pots as infected plants. Systemically infected leaves showing clear
mosaic symptoms were used for purification of the virus. Fifty gram of infected leaves were put in a poly bag and kept at 20C for four days. The frozen leaves were ground in a kitchen blender under chilled condition using 0.1 M Tris HCl buffer
(pH 8.4) containing 0.2% sodium sulphite. The homogenate was then squeezed through double layered muslin cloth and
the sap extract was added with half volume of chloroform and stirred for ten minutes in an ice bath. The clarified sap was
then centrifuged at 8000 rpm for 20 minutes in a refrigerated centrifuge (Hettich EBA 12R). The supernatant was collected
and 0.2 M sodium chloride and four per cent polyethylene glycol (PEG, MW 6000) were added. The extract was stirred for
90 minutes in an ice bath. Sap was then centrifuged at 14000 rpm at 4C for 90 minutes. Pellet was collected and
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Production of Polyclonal Antisera Against Cassava Mosaic Virus in Cassava

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resuspended in 0.01 M TE buffer, pH 8.0 containing 0.005 M EDTA. Then the resuspended pellet was homogenized for
several minutes and centrifuged at 2000 rpm at 4C for five minutes. The supernatant was collected, stored at 4C and was
used as antigen source for immunization of rabbits.
Production of Antiserum against Virus
Antiserum was produced against the virus in New Zealand white female rabbit by giving five intra-muscular
injections with the partially purified virus at weekly intervals. Before injection, the virus preparation was emulsified with
one ml of Freunds incomplete adjuvant (1:1 v/v). Ten days after the last injection the rabbit was bled through marginal ear
vein. Blood was collected in a sterile test tube and was kept in a slightly slanting position for one hour at room
temperature. After that the tube was kept overnight without disturbing at 4C. The clear serum was pipetted out and
centrifuged at 5000 rpm for 30 minutes at 4C. The supernatant was pipetted out using a micropipette and dispensed into
1.5 ml eppendorf tubes. A pinch of sodium azide was added to the clarified serum to prevent microbial contamination. The
vials were stored under refrigerated condition.
Determination of Antibody Titre
As partially purified sap was utilized for the production of antibody, cross absorption was done to remove any
antibodies of plant protein present in the serum. Apparently healthy sap was extracted by grinding in carbonate buffer (1:5
w/v) using a mortar and pestle. The antiserum was diluted in the extracted healthy sap (1:128) and was incubated at 37C
for one hour. After incubation, it was centrifuged at 5000 rpm for 15 minutes. Supernatant was collected and this absorbed
antiserum was used for preparing further dilutions.
Titre of the antiserum was determined by DAC-ELISA. The procedure described by Huguenot et al. (1992) was
followed for the detection.
One gram of infected young leaf was homogenized in 5 ml of coating buffer (carbonate buffer) containing two per
cent (w/v) PVP under chilled condition. Healthy plant extract was prepared by using leaves of healthy plants.
The homogenate was centrifuged at 5000 rpm for 10 minutes at 4C. Samples were dispensed at the rate of 100 l
into Nunc immunological plates. The treatments were replicated thrice. After incubation for two hours at 37C the wells
were washed with phosphate buffer saline- tween (PBS-T) three times each for duration of three minutes using a plate
washer (PW-40, BIORAD). Blocking was done with 100 l of five per cent spray dried milk (SDM) for 30 minutes at
37C. After incubation blocking agent was removed, plates were washed with PBS-T as before. The antibody raised in
rabbit was diluted in healthy plant sap for cross absorption and 100 l of each dilution was added to the well. Dilutions of
1:128, 1:256, 1:512, 1:1024, 1:2048, 1:4096 and 1:8192 were used for the determination of the titre of the antiserum
developed. Three replications were maintained for each treatment and incubated overnight at 4C. The plates were washed
with PBS-T and then treated with 100 l of goat-antirabbit immunoglobulin (SIGMA - Aldrich) diluted in PBS-T Polyvinyl
pyrrolidone ovalbumin (PBS-TPO) (1:10,000 v/v) and incubated for two hours at 37C. Wells were washed with PBS-T as
before. The substrate p-nitro phenyl phosphate (p-NPP) in diethanolamine buffer (1 mg per ml) was added to each well
(100 l per well) and incubated for one hour at 37C. Reaction was stopped by adding 50 l of four per cent NaOH. The
absorbance was read at 405 nm in an ELISA reader (Microplate Reader 680, BIORAD).

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Nair A. B

RESULTS AND CONCLUSIONS

Partial purification of the virus was successfully performed as per the procedure described by Perera and
Dasssanayake (2002) from the infected young cassava leaves collected from the plants maintained in insect proof glass
house.
The antiserum against Cassava mosaic begomovirus was produced by immunizing New Zealand white female
rabbit by giving five intramuscular injections at weekly intervals with partially purified virus preparation mixed with an
equal volume of Fruends incomplete adjuvant. Finally, the blood of the animal was collected 21 days after the last
injection and processed by following the procedure described in materials and methods. The antiserum thus produced was
used for further studies after cross absorption.
The cross absorbed antiserum was used for preparing further dilutions. DAC- ELISA was carried out to confirm
working of antiserum and to determine the titre of antibody. Different dilutions of antiserum viz. 1:128, 1:256, 1:512,
1:1024, 1:2048 and 1:4096 tested gave a titre of 1: 512 (Table 1).
Table 1: Determination of Titre of the Antibody
Developed Against the Virus
Treatment
1:128
1:256
1:512
1:1024
1:2048
1:4096

Absorbance @ 405 Nm
Healthy
Infected
0.451
0.455
0.294
0.178
0.140
0.21
0.127
0.099
0.112
0.127
0.086
0.083

Purification of virus infecting cassava was attempted by several workers. Fargette et al. (1987) purified ACMV
isolate obtained from diseased cassava plants propagated in N benthamiana as per the procedure of Walter (1980). Partially
purified virus was used for the production of antiserum.
The titre of the antiserum developed was determined and was found to be 1:512 in the present study. The low titre
value of the antiserum in the present study might be due to lower concentration of viral particles in the purified mixture
during immunization or it might probably be due to moderate immunogenecity of the virus. Fargette et al. (1987) got an
antiserum against CMBeV of titre 1:256 when tested by agar double diffusion. Polyclonal antibodies produced using
purified virus can be used in the detection of begomovirus detection.
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NAAS Rating: 3.53

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