First report of Chickpea chlorotic dwarf virus infecting cucumber in Pakistan

A number of plant viruses that infect cucurbit crops and cause significant yield losses have been reported. Field surveys were conducted during 2012 to 2013 to determine the incidence of cucurbit-infecting viruses in Punjab Province, Pakistan. Virus-like symptoms were observed in cucumber (Cucumis sativus L.) plants, including leaf curling, mosaic, and necrotic spots, in commercial fields in the Vehari district of Pakistan. Disease incidence ranged between 20 to 30%. Both whiteflies and leafhoppers were observed to colonize cucumber plants. Leaf samples were collected from seven symptomatic plants, and total DNA was isolated using CTAB method (Doyle 1991). The DNA was enriched for circular DNA using rolling circle amplification (RCA) (TempliPhi kit; GE Healthcare, U.S.A.), and RCA products were digested with several restriction enzymes. Samples digested with EcoRI yielded 2.6 kbp fragments, which were ligated into the pGEM-3Zf+ plasmid vector (Promega, Madison, WI) linearized with EcoRI and transformed in E. coli DH5 alpha cells. The DNA sequence was determined for three clones amplified by colony PCR, assembled, and annotated. The three sequences shared >99% nucleotide (nt) identity with one another, and so one genome sequence of 2,588 nucleotides in size was deposited in GenBank as accession number KT719388. The top 20 BLASTn hits were downloaded and SDTv1.2 (Muhire et al. 2013) was used to calculate pairwise nucleotide identities. The SDT analysis revealed that the isolated viral sequence highest nt identity, at 97.9%, with Chickpea chlorotic dwarf virus (CpCDV) strain C (AM900416) reported from Pakistan. The presence of CpCDV was confirmed by Southern blot hybridization using a probe based on a fragment of the replication-associated protein spanning the nt coordinates 1533 to 2411 of the CpCDV isolate (Zia-Ur-Rehman et al. 2015). CpCDV has also been reported infecting tomato plants in Pakistan (Zia-Ur-Rehman et al. 2015). To our knowledge, this is the first report of CpCDV infecting cucumber worldwide. This finding supports the hypothesis that the host range of CpCDV is increasing in the Punjab region in Pakistan.