Characterization and Molecular Detection of Tomato spotted wilt Tospovirus Infecting Tomato in Egypt
Characterization and Molecular Detection of Tomato spotted wilt Tospovirus Infecting Tomato in Egypt
Hayam S. Abdelkader1; Gamalat M. Allam1; T. A. Moustafa2; and M. El-Hanunady2
ABSTRACT
Tomato spotted wilt virus was originally isolated from naturally infected tomato fruits. The virus infected a wide host range showing different symptoms varied from local necrosis, systemic necrosis, systemic mosaic yellowing, bronzing, or stunting. The virus transmitted by mechanical means as well as by Thrips tabaci. Its TIP was between 45-50oC, DEP was 10-5 and LIV was between 5-6 hrs. Local lesion technique for biological purification and electron microscopic analysis confirmed the production of mature infectious virus particles underlining the conclusion that a full infection cycle was completed in this system. Both the structural viral proteins nucleoprotein (N) and the envelope glycoproteins GI and G2 and the nonstructural viral proteins NSs and NSm were accumulated to amounts sufficient for detection and cytopathological analysis. Electron micrograph of the virus showed that it is quasi-spherical and its diameter ranges from 80 to 100 nm. The virus caused thickening of the cell wall and changes in the chloroplast structure. Virus identification was confirmed by Dot blot immunoassays. RT-PCR assays using primers complementary to the nucleocapsid protein gene (NPs) were used to detect two isolates of TSWV from Lycopersicum esculentum and Physalis peruviana plants. Total nucleic acids were reverse transcribed using Retrotool reverse transcriptase enzyme and the PCR reactions were performed for 30 min in a capillary thermal cycler. RFLP analysis of the PCR products was performed using Msel restriction enzyme. The results showed a dimorphic restriction digestion profile that "as suitable for identifying the two isolates. The method described here is rapid. reliable and highly recommended to be used in such virological and pathological studies.
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