Biolistic Inoculation of Selected Host Plants with different Begomoviruses and their associated Betasatellites: using partially digested rolling circle amplified Viral/Satellite DNA

Mehwish Rauf, Muhammad Zia-Ur-Rehman, Muhammad Saleem Haider


Background: The foundation of modern diagnostics of plant pathogens are based on the confirmation of Koch's postulates. However, following the rules of Koch's postulates is not always straightforward for the viruses because of their non-culturability and difficulties associated with the inoculation approaches.

Methods: Biolistic inoculation of plants with partially digested rolling circle amplified (RCA) viral components was demonstrated in this study. The verified DNA bands were excised from the gel and self-circularized using the rapid DNA ligation kit. The self-ligated viral DNA components were enriched by RCA and partially digested with the respective fast digest restriction enzymes. The RCA prep (partially digested 3 µg of virus and 3 µg of beta-satellite) was biolistically inoculated into healthy plants of Solanum lycopersicum, Nicotiana tabacum and Capsicum annum at the two-true leaf stage.

Results: By using this method, extracted begomoviruses from cotton [ (Cotton leaf curl Kokhran virus-Burewala strain (CLCuKοV-Bu), Tomato leaf curl New Delhi virus (ToLCNDV) and Chili leaf curl virus (ChiLCV) and their associated Cotton leaf curl Multan betasatellite (CLCuMB)] were successfully inoculated and propagated from the inoculation site. Furthermore, it has been noted that these viruses exhibit diverse behaviors in various host plants. The capacity of these viruses to infect systemically in C. annum without causing the typical disease symptoms is of interest.

Conclusion: Biolistic inoculation using partially digested RCA of viral or satellite DNA enables viruses to infect plants with either monopartite or bipartite genomes. This method is easy and quick, and allows begomovirus cultures to be created without the use of Agrobacterium tumefaciens or whiteflies.

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