Detection of Cucumber mosaic cucumovirus in infected cowpea plants (Vigna unguiculata L.) from northern Egypt

Cucumber mosaic cucumovirus (CMV) was isolated from naturally infected Cowpea plants (Vigna unguiculata L.) showing different symptoms of mosaic; mottle, dwarfing, and vein clearing, grown at certain locations of Alexandria governorate, Egypt, during the growing seasons from 2011- 2012. CMV isolate was transmitted by Aphid nerii, A. gossypii, and Myzus persicae in non-persistent manner. The virus was partially purified using polyethylene glycol (PEG) 6000, and differential centrifugation. The ratio of A 260/280 was 1.622 and A 280/260 was 0.617. Whereas, the ratio of A max/ min was 1.915. Concentration of the virus in the preparation was estimated using an extinction coefficient of E 2600.1% = 5. Yield of purified virus was about 6.88 mg/ 100g fresh weight of leaves of Nicotiana glauca. Antiserum titer was determined by Indirect enzyme linked immunosorbent assay (ELISA). Positive ELISA values were obtained up to dilutions of 1: 25600. The virus was detected by indirect ELISA in infected sap at 8, 16 and 24 days after inoculation; and by Tissue blot immunoassay (TBIA) on nitrocellulose membrane after the same period. The unused face of the processed nitrocellulose membrane already printed with plant tissues was tested. Results revealed that both faces of nitrocellulose membrane and Canson paper could be used as solid carriers in TBIA, for detection of CMV in infected leaves. According to Reverse transcription polymerase chain reaction (RT-PCR); the size of amplification of the obtained product was approximately 870 bp for CMV isolate; and was assigned accession number of LN606587. The Phylogenetic tree was generated using partial sequence of CMV isolate, with those of other CMV isolates obtained from GenBank. The aims of the current work were; to produce specific polyclonal antiserum against the purified CMV isolated from cowpea plants, and to register this isolate in GenBank.