Full Length Research Paper

Isolation and molecular characterization of Tomato spotted wilt virus (TSWV) isolates occurring in South Africa

B. J. Sivparsad and A. Gubba*

Discipline of Plant Pathology, School of Biochemistry, Genetics, Microbiology and Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, Private Bag X01, Scottsville, 3209, South Africa.

*Corresponding author. E-mail:  gubbaA@ukzn.ac.za . Fax: +27332605919. Phone: +27332605795)

Accepted 5 June, 2008

   Abstract

Tomato spotted wilt virus (TSWV), a Tospovirus, causes high economic losses in many crops worldwide.  A typical Tospovirus was isolated from naturally infected pepper (Capsicum sp.) in KwaZulu-Natal (KZN), South Africa (SA). The identity of the virus was confirmed as TSWV using an enzyme-linked immunosorbent assay (ELISA), electron microscopy, and protein analysis.  Genetic diversity of TSWV in SA was investigated. A total of six TSWV isolates originating from different regions in SA were used in this study. Total plant RNA, isolated from Nicotiana rustica infected with the SA TSWV isolates were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using primers specific to the nucleocapsid (N) gene of TSWV. The amplified 760 bp products was then cloned and sequenced.  Nucleotide sequence comparisons of the N gene revealed high similarity (> 90%) between the SA isolates.  Phylogenetic analysis based on the multiple alignments of N gene sequences of the SA TSWV and isolates occurring in different geographical locations in the world revealed the branching of TSWV isolates into two distinct clusters designated the American and European groups. The SA isolates showed high sequence similarity with TSWV isolates from Europe. The information generated in this study will be useful in formulating effective control measures using genetic engineering approaches for this economically important virus.   

Key words: Electron microscopy, molecular characterization, phylogenetic analysis, South Africa, Tomato spotted wilt virus, Tospovirus.