Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer’s field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch’s postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.