Molecular characterization of seed borne virus causing mosaic disease in cowpea
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Date
2010-05-20
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Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
Abstract
Cowpea (Vigna unguiculata L. Walp.) is an important leguminous food grain crop attacked
by wide array of diseases of biological origin especially viruses which cause devastating
effects and are a really constraint to increased yield of cowpea. Management of the viral
diseases is based primarily on the development of cowpea resistant varieties. The present
work is aimed at development of molecular diagnostic methods of some economically
important seedbome viruses-of cowpea to provide a basis for the control of these viruses.
Seed samples of cowpea were collected and tested for the presence of the blackeye
cowpea mosaic strain of bean common mosaic virus, cowpea aphid-borne mosaic virus,
cowpea mosaic virus, cucumber mosaic virus and bean yellow mosaic virus using growingon
test, enzyme linked immunosorbent assay, tissue print immune assay, differential host test,
and reverse transcription polymerase chain reaction. Sodium dodecyl sulphatepolyacrylamide
gel electrophoresis showed that partially purified viruses contained coat
protein polypeptide with an approximate molecular weight ITT 34 kDa. DAS ELISA kit
specific for BlCMV showed positive results for BlCMV antigen. Total RNA extracted from
infected tissues, using total RNA extraction kit was used for reverse transcription and
subsequent amplification of viral sequences. Alignments of potyvirus and comovirus coat
protein gene sequences allowed the selection of degenerate PCR primers in conserved coat
protein region. BlCMV coat protein gene was successfully amplified by reverse transcriptionpolymerase
chain reaction (RT-PCR) using degenerate primers. The amplified genome
fragment 290 base pairs in size, amplified from the coat protein gene of virus, was analyzed
by gel electrophoresis and visualized by Ethidium bromide staining.