1Dept. of plant pathology, Faculty of Agriculture, Kafrelsheikh University, 33516, Kafrelsheikh, Egypt.
2Plant Virus Lab, Institute of Himalayan Bioresource Technology (CSIR), Palampur-176 061, India.
Naturally infected leaf samples of squash showing vein clearing and vein banding symptoms typical of virus infection were collected from Kafrelsheikh governorate, Egypt. The virus isolates were mechanically transferred back to squash plants grown in green-house conditions and the lab host produced the same symptoms. Inoculated samples as well as the field samples were tested for potyvirus infection using potyvirus group specific ELISA kit (Agdia, USA) as per manufacturer's instructions. All symptomatic samples were found positive. Total RNA was isolated from one of the positive samples using RNAqueous Kit (Ambion, USA) and used in RT-PCR using potyvirus universal primer pair which amplifies partial coat protein and 3’UTR region of the potyviral genome. Expected amplification of ~650bp was obtained and the amplicon was gel purified using QIAquick® gel extraction kit (Qiagen, USA) and cloned into pGEMT® Easy vector (Promega, USA). Both strands of the positive clone were sequenced using BigDye® Terminator Cycle sequencing kit (Applied Biosystems, USA) in ABI PRISM® 3100 Genetic Analyzer DNA sequencer (Applied Biosystems, USA). In BLAST search obtained sequence showed d”96% similarity with
