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

Abstract

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.

First report of begomoviruses infecting Cucumis sativus L. in North America and identification of a proposed new begomovirus species

“…This new species is the first BGV originally isolated from C. sativus. The seven previous BGVs species were isolated from other crops such as tomato (Mizutani et al, 2011;Shafiq et al, 2019), mungbean (Shahid et al, 2018), melon (Romay, Lecoq & Desbiez, 2015) and squash (Table S4).…”

“…At the time of writing this article, only seven species of BGVs infecting cucumber had been reported, of which six species were identified in South Asia. Interestingly, none of the seven BGVs species that affect cucumber were originally isolated from this crop; they were originally isolated from other crops such as, tomato, mungbean, squash and melon (Ramirez et al, 2004;Mizutani et al, 2011;Shahid et al, 2018;Shafiq et al, 2019).…”

High-Throughput Sequencing Identified Distinct Bipartite and Monopartite Begomovirus Variants Associated with DNA-Satellites from Tomato and Muskmelon Plants in Saudi Arabia

“…The ToLCPalV isolates also showed very high nt sequence identity with ToLCPalV isolates reported from Iran [ 17 ]. ToLCPalV is a bipartite begomovirus widely spread in India and Pakistan, where it has been reported to infect many host plants, including tomato and cucurbits [ 37 , 38 , 39 , 40 , 41 ], and recently a weed [ 42 ]. Later, it was spread into Iran in 2009 [ 43 ], and until now, it has been reported from common bean, cucumber, melon, pumpkin, and watermelon crops in Iran [ 17 , 18 , 43 , 44 ].…”