First Report of Chickpea chlorotic dwarf virus Infecting Tomato Crops in Pakistan

During 2012, severe leaf curl symptoms reminiscent of geminivirus infection were observed in commercial tomato (Lycopersicon esculentum Mill.) plantings (70% incidence) in Vehari, Pakistan. Leaves (3 to 4) were collected from 6 symptomatic plants in 3 fields and subjected to total DNA isolation using the CTAB method (Doyle, 1991). Rolling circle amplification (RCA) (TempliPhi kit; GE Healthcare, USA) was carried out, and the RCA product was digested with a range of restriction enzymes to identify one or more enzymes that would yield a putative, full-length geminivirus genome or genomic components of ∼2.6 to 3 kbp in size. EcoRI digested ∼2.6-kbp fragments were obtained from 2 of 6 samples, and used for cloning into the plasmid vector, pGEM-3Zf+ (Promega, Madison, WI). Capillary DNA sequencing of both cloned inserts by primer walking yielded 2,586 nucleotide fragments that were identical, and so only one sequence (KP881605) was used for further analyses. The tomato virus isolate sequence and top 30 GenBank hits were subjected to SDT 1.0 analysis (Muhire et al. 2013) to estimate pairwise nucleotide identities. The cloned geminiviral genome from tomato shared its greatest nucleotide identity, at 99%, with Chickpea chlorotic virus (CpCDV) (KM377673) strain C, a member of the genus Mastrevirus, previously identified in Pakistan infecting lentil plants (Kraberger et al. 2014). Based on the criteria for Mastrevirus species and strain demarcation (Muhire et al. 2013), the cloned virus genome represents the first isolate of the C strain of CpCDV from tomato. The presence of the Mastrevirus CpCDV in symptomatic tomato plants was confirmed by Southern blot hybridization using a digoxygenin (DIG)-labeled probe based on a partial fragment of the replication-associated protein (nucleotides 1533-2411) of the CpCDV isolate identified in tomato plants from Vehari. CpCDV has been reported infecting pepper crops in India and Oman (Akhtar et al. 2014), corroborating other solanaceous species as hosts. The apparently broader-than-expected host range of CpCDV, which now includes legume and solanaceous species, has economic implications to Asia and the Arabian Peninsula. The discovery suggests that the host range of CpCDV is broader than expected.