الملخص الإنجليزي
Abstract Pepper (Capsicum annum) is cultivated in Al-Batinah, Al-Sharqiya and Dhofar regions in the Sultanate of Oman during the winter season to meet the high demand for fresh produce in the domestic market. To identify the causal agent of a widespread disease associated with infestations of the whitefly Bemisia tabaci (Genn.), leaves were collected from hot pepper and sweet pepper plants showing symptoms characteristic of the begomovirus disease in southern and northern regions of Oman during 2010 and 2011. Typical symptoms of chili leaf curl disease were observed on infected pepper plants that includes shortening of stem internodes, interveinal yellowing, upward curling of the leaf blade, downward rolling of the leaf blade, reduction of the leaflet area, fruit discoloration and fruit size reduction. The disease incidence varied from farm to farm ranging 0-100% irrespective of geographic locations but depending on farmer's management practice. In general pepper seedlings protected from whiteflies in nursery and for few weeks post transplanting showed little or no begomoviral symptoms. On the contrary seedlings infested with whiteflies in nursery and field exhibited severe symptoms with disease incidence of 80-100%. Total nucleic acids were isolated from the symptomatic pepper leaves and used as the template for polymerase chain reaction (PCR) to confirm the presence of begomovirus. Seventeen out of 34 symptomatic samples were tested positive with begomoviral specific PCR primers. Subsequently, nucleic acid from PCR positive symptomatic samples was used for 029 DNA polymerase amplification of begomoviral circular DNA. Putative full unit length begomoviral DNA multimers were digested with Xbal and cloned into the plasmid vector pUC19. The complete nucleotide (nt) sequence was determined as 2758 base pairs (bp), indicative of a monopartite begomoviral genome. A comparison of the genome sequences of twelve field isolates indicated that they shared 97 – 100% nt identity. The virus from Oman was found to be most closely related to Chili leaf curl virus-Multan (ChLCV-Multan) sharing >91% nt identity, a monopartite begomoviral isolate described previously from Pakistan. A satellite DNA (DNA B) was amplified by PCR using begomovirus beta satellite specific degenerate primers. Full-length satellite DNAs were cloned and their DNA sequence were determined. Beta satellite were found to have circular ssDNA genome with single ORF (C1) know to function as pathogenicity determinant. Analysis of the complete nt sequence of 1327 bp indicated that the DNA
nce
B shared 95% nucleotide similarity with its closest relatives, Tomato yellow leaf curl betasatellite-Al-Batinah isolated from tomato in Oman. The genome organization of all isolates of Chili leaf curl virus from Oman was similar to those of other monopartite begomoviruses. All ChLCV isolates showed circular, ssDNA genome with six ORFs (V1, V2, C1, C2, C3, C4) of which two were arranged in virion sense and four in complimentary sense. The amino acid sequence analysis revealed that Vi and V2 ORFs have highest similarity with PepLCV-Lahore and ChLCV-Lahore, respectively and C1-C4 ORFs showed highest similarity with ChLCV-Multan indicating the presence of recombination. Using the RDP3 recombination detection program, a recombination event involving Pepper leaf curl virus-Lahore as a minor parent, and Chili leaf curl virus-Multan and Papaya leaf curl virus-India as major parents were detected in ChLCV-OM sequence. Infectious clone consist of the partial tandem repeat of the viral genome were constructed in the binary vector PCAMBIA 1301 and agroinoculated to Nicotiana benthamiana. The viral clone produced severe upward leaf curl of the small, newly emerged leaves and vein thickening compared to controls.
