Detection, Amplification and Sequence Analysis of Begomovirus(es) from different Plant Species

Abstract

Begomovirus, the largest known genus of plant viruses, is a real challenge for plant virologists due to its great diversity, wider host range, and ability to undergo frequent recombination. Begomoviruses cause devastating yield losses in economically important crops. The present investigation aims to identify and amplify a begomovirus from commonly cultivated ornamental plants, specifically Duranta. repens, Hibiscus, and Jasmine. Leaf samples of one Duranta repens, showing leaf curl, two Hibiscus rosa-sinensis, exhibiting vein-thickening and a Jasmine, expressing leaf mottling and yellowing symptoms, indicative of begomoviruses and seemingly non-symptomatic samples, were collected in the vicinity of the National University of Sciences and Technology (NUST), Islamabad in 2022. Total nucleic acid was extracted via the CTAB method. Using diagnostic primer pair (CLCV1/CLCV2), expected size bands of approx. 1.1kb were obtained from symptomatic samples while non-symptomatic samples didn't yield any bands. An amplification product of approx. 2.8kb was obtained, using primer pair BurxF/BurxR, from Hibiscus (isolate HAHN), and was sequenced to its entirety. The total nucleotide sequence obtained had 2751 bases and shared maximum nucleotide identity with an isolate of Cotton leaf curl Multan virus (CLCuMuV), representing a variant of CLCuMuV. A sequence of 1892 bases was obtained from a full-length product, of Hibiscus (isolate HAH), which shared 97% sequence identity to CLCuMuV. Two full-length products (approx. 2.8kb each) were amplified from D. repens, using primers pairs BurxF/BurxR and SonF/SonR. The partial sequence, 1251 nucleotide bases, obtained from the product amplified with BurxF/BurxR primers shared 94% nt sequence identity to an isolate of Papaya leaf crumple virus (PLCV), which has not previously been reported from Pakistan. While partial sequence, 1608 bases, derived using SonF/SonR primers shared 95% nt sequence identity to an isolate of Chili leaf curl India virus (ChiLCINV). The partial sequence obtained from Jasmine, 1164 bases, amplified with primer pair SonF/SonR shared the highest nucleotide sequence identity of 98% to an isolate of ChiLCINV. Full-length betasatellite (approx. 1.3kb) were amplified from Hibiscus, and D. repens using primer pair Beta01/02 while no amplification was obtained from Jasmine. The complete sequence of betasatellite obtained from Hibiscus was determined to be 1346 nt and shared 96% nt sequence identity to Cotton leaf curl Multan betasatellite (CLCuMB) while the partial sequence, 358 bases, obtained from Duranta shared 91% nucleotide identity with CLCuMB. The study showed that Hibiscus, Duranta and Jasmine are reservoir host plants for economically important viruses, they may serve as sources of inoculum, and recombination sites xi for the emergence of new begomoviruses. The study revealed the first report of CLCuMuV infecting Hibiscus rosa-sinensis in Pakistan and mixed infection of PLCrV and ChiLCINV in Duranta repens