A simple technique was developed to separate Cowpea chlorotic mottle virus (CCMV) from Cucumber mosaic virus (CMV) in natural mixed infections. Sap from cowpea leaves infected naturally with a mixture of CCMV and CMV was inoculated mechanically on the first tri-foliolate leaf of cowpea seedlings. Both inoculated and non-inoculated upper leaves were sampled 3 or 8 days post-inoculation and tested by reverse transcription polymerase chain reaction (RT-PCR) using primers specific to CCMV and CMV. RT-PCR analysis showed the presence of only CCMV in the inoculated leaf and both viruses in the non-inoculated systemically infected upper leaves. Total RNA from the inoculated leaves positive to CCMV only was further confirmed upon re-inoculation to cowpea seedlings. Typical CCMV symptoms were produced within 1 week and RT-PCR analysis showed only the presence of CCMV in both inoculated and non-inoculated systemically infected upper leaves. Systemically infected upper leaves of the same plants were used for CCMV purification. RT-PCR analysis of the purified virion and RNA extracted from the virion further confirmed the absence of CMV contamination. To our knowledge, this is the first report of a method separating CCMV directly from mixed infections with CMV in cowpea.
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