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

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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.

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Abbreviations

RCA:

Rolling circle amplification

SDT:

Species demarcation tool

ToLCPMV:

Tomato leaf curl Palampur virus

MEGA:

Molecular evolutionary genetics analysis

NW:

New world

CMV:

Cucumber mosaic virus

SqMV:

Squash mosaic virus

WMV-1:

Watermelon mosaic I

WMV-2:

Watermelon mosaic 2

ZYMV:

Zucchini yellow mosaic

DNA:

Deoxyribose nucleic acid

CTAB:

Cetyltrimethylammonium bromide

ICTV:

International committee on taxonomy of viruses

GUS:

β-Glucuronidase

DIG:

Digoxygenin

CP:

Coat protein

BLAST:

Basic local alignment search tool

PCR:

Polymerase chain reaction

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Acknowledgements

We acknowledge all the members of virology lab at Institute of Agriculture Sciences, Punjab University Lahore, Pakistan for their help in performing experiments.

Funding

This research was funded by Institute of Agriculture Sciences, Punjab University Lahore, Pakistan. Sponsors have no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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Authors and Affiliations

  1. Institute of Agricultural Sciences (IAGS), University of the Punjab, Lahore, Pakistan

    Muhammad Shafiq, Mukhtar Ahmad, Ayesha Nisar, Muhammad Tariq Manzoor, Arslan Abid, Sehrish Mushtaq, Muhammad Ilyas & Saleem Haider

  2. Virology Lab, Center of Agricultural Biochemistry and Biotechnology, University of Agriculture, PO Box 38040, Jail Road, Faisalabad, Pakistan

    Adeel Riaz, Waseem Sarwar, Muhammad Shah Nawaz-ul-Rehman & Muhammad Mubin

  3. Laser Matter Interaction and Nano-sciences Lab, Department of Physics, University of Agriculture, Faisalabad, Pakistan

    Ayesha Younus

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  1. Muhammad Shafiq
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  2. Mukhtar Ahmad
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Contributions

MS, SM, AR, WS, MI were involved in performing experiments. MI, MSH, MSNR and MM were involved in data analysis, experimental design. MSH, MSNR, AY and MM wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Muhammad Mubin.

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The authors declare that they have no competing interests.

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Shafiq, M., Ahmad, M., Nisar, A. et al. Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan. 3 Biotech 9, 204 (2019). https://doi.org/10.1007/s13205-019-1727-3

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