In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection.
Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation.
The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites.
The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.
Fig. 4 Southern blot analysis of DNA extracted from agroinoculated N. tabacum plants. Blots were probed for the presence of ChiLCV (a) and ToLCB (b). The samples run on the gels were extracted from Nicotiana glutinosa, N. benthamiana, and N. tabacum plants (as indicated) inoculated with only ChiLCV (lane 1 panel A) or co-inoculated with ChiLCV and ToLCB (lane 2 in panels A and B). In addition DNA extracted from one of the field-infected tobacco plants (F) and from a healthy, non-inoculated tobacco plant (H) was included as controls. In each case approx. 10 μg of DNA was loaded. For each blot a photograph of the ethidium bromide-stained genomic DNA band on the gel is shown to confirm equal loading. The positions of viral/betasatellite single-stranded (ss), supercoiled (sc) and open-circular DNA forms are indicated.