Molecular characterization of katte mosaic virus of cardamom ( Elettaria cardmomum Manton )
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Date
2016
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara
Abstract
Cardamom ( Elettaria cardamomum Maton) considered as “Queen of Spices” belongs to the family Zingiberaceae. Due to its aroma, cardamom is one among the most expensive spices in the world. Cardamom is infected by several fungal, bacterial and viral diseases. Katte or Cardamom mosaic disease is the most destructive viral disease affecting cardamom plantations worldwide. The disease is caused by Katte mosaic virus or Cardamom mosaic virus (CdMV) and spreads through infected suckers or through aphid Pentalonia nigronervosa Coq. The loss in yield due to katte disease ranges from 38 to 69 per cent and when infection occurs at early stage, the loss is complete. Management of katte disease totally depends on the use of disease free planting materials. As the infected plant often remains symptomless, identification and diagnosis of the virus becomes difficult at early stages. The present study was undertaken to develop serological and PCR based methods for identification and characterization of CdMV of cardamom. The infected and healthy samples were collected from four locations of Idukki and two locations of Wayanad districts of Kerala. The viral protein was isolated and purified from infected samples and SDS-PAGE analysis of purified protein from infected plants revealed the presence of 37 kDa band of viral coat protein. The purified protein from the infected plants was further used as antigen for the production of polyclonal antibody against CdMV in 6-9 month old rabbit. The rabbit was immunized with 5 mg of purified protein. The blood sample from the immunized rabbit was collected and the antibody was purified. The ODD (Ouchterlony Double Diffusion) assay was performed to standardize the titre of the antibody and results had shown that antigen-antibody complex was formed in 1:10, 1:100 and 1:150 dilutions of primary antibody. The indirect ELISA was carried out with 1:10, 1:100 and 1:150 dilutions of primary antibody and 1:200 dilution of secondary antibody. It was found that virus was easily detected in the crude extract of infected leaves with 1:100 dilution of primary antibody. Indirect ELISA was also performed for investigating the serological relationship of Katte mosaic virus with other viruses. The result of indirect ELISA revealed that the crude sap of infected cardamom leaves cross reacted with the antibody specific for Banana Bract Mosaic Virus (BBrMV) which also belongs to same potyvirus group. For the detection of CdMV through RT-PCR, the total RNA was isolated from the infected and healthy plants using Trizol and converted to cDNA. A total of 11 primers were designed for the amplification of coat protein gene of the virus using Primer 3 software. The primers designed were used for synthesis of second strand of cDNA and presence of virus was detected. Out of 11 primers, 8 primers were able to amplify the coat protein gene of the virus in infected plants. The band size of 250, 650, 750 and 950 base pairs were observed in infected plant but not in healthy plant samples. The viral amplicons of 250, 950, 750, 650 base pairs were generated with primers CP-2, CP-9, CP-10 and CP-11 respectively. These amplicons were further eluted, reamplified and sequenced. The nucleotide sequence annotated using bioinformatics tools BLASTn and BLASTx. The result of BLASTn showed 90 to 100 per cent nucleotide sequence similarity with CdMV whereas; the result of BLASTx revealed that the sequence had 55-100 per cent similarity with the coat protein of CdMV. The phylogenetic analysis was performed using 950 bp products generated with CP-9 primer. The phylogenetic tree was developed using MEGA.7 software by utilizing neibhourhood joining method. The result of phylogenetic analysis revealed that the isolates of Ambalavayal, Meppadi and Myladumpara are closely related and also related to isolates of Irettyar and Paravalam whereas, Pampadumpara isolate showed more variation to the above isolates. The methods developed in the present study are useful in detecting the Katte mosaic virus or Cardamom mosaic virus in the infected leaf samples of cardamom. The method will be useful in virus indexing, quarantine management in germplasm exchange, germplasm management, supply of disease free planting materials to the farmers and also for selecting the resistant line or cultivar for large scale production or incorporation in breeding programme for crop improvement.
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173732