First report of Chickpea chlorotic dwarf virus infecting okra in Pakistan

Chickpea chlorotic dwarf virus (CpCDV) is a member of the genus Mastrevirus, family Geminiviridae. Mastreviruses are leafhopper-transmitted, and have a single-stranded, circular DNA genome with four open reading frames, two each encoded in the virion-and complementary-sense orientation, separated by two intergenic regions. Okra (Abelmoschus esculentus) is an important cultivated vegetable in Pakistan and is reported to be infected by a number of begomoviruses. In June 2012, during a field survey in okra cultivations in the Vehari district of Punjab, Pakistan, approximately 15% of okra plants were exhibiting leaf curling, yellowing, and vein thickening symptoms, in two locations of the city. Total DNA was extracted from leaves collected from five symptomatic plants using the CTAB method (^†). The circular DNA fraction was enriched using rolling circle amplification (RCA) (TempliPhi kit; GE Healthcare, U.S.A.), digested with EcoRI to obtain a full-length viral-size genome(s) of ∼2,600 base pairs (bp), and cloned into the pGEM-3Zf+ plasmid vector (Promega, Madison, WI). The cloned inserts were subjected to Sanger DNA sequencing, yielding three 2,586-bp sequences that shared high nucleotide (nt) sequence identity with previously reported CpCDV isolates, based on BLASTn analysis against the NCBI-GenBank database. All complete CpCDV genome sequences were downloaded, and together with the chickpea-associated CpCDV-like sequences determined herein, were subjected to pairwise distance analysis using SDT 1.2 software (^†). The three sequences shared 100% nt identity with each other and 98.8% nt identity with CpCDV strain C (AM850136) isolated from chickpea (Cicer arietinum) from Layyah, Pakistan. As the three sequences representing both locations were identical, so the only one sequence was used in further analysis. Using the mastrevirus species and strain threshold of 78% and >94% nt identity, respectively (^†), the sequence described here (KT719391) represents the first CpCDV strain C isolate associated with okra in Pakistan. Based on phylogenetic analysis using maximum-likelihood (1,000 bootstrap iterations; >70% support), implemented in MEGA6, the sequence obtained here grouped with all other strain C isolates of CpDCV. The presence of CpCDV in three symptomatic okra plants was confirmed by Southern blot hybridization using a digoxygenin-labeled probe, as described (^†). Overall, 12 strains of CpCDV, A-L, have been reported from diverse dicot hosts (^†). CpCDV strain C has been most frequently isolated from chickpea, but most recently it has been isolated from tomato (^†) and cucumber (^†) in Pakistan. To our knowledge, this is the first report of CpCDV associated with symptomatic okra plants in Pakistan. This finding supports the hypothesis that CpCDV has a broader than expected host range, and consequently, may infect additional plant species.