Isolation and molecular characterisation of tomato spotted wilt virus (TSWV) isolates occuring in South Africa.

Abstract

Tomato spotted wilt virus (TSWV), a Tospovirus, is one of the ten most economically destructive plant viruses worldwide, causing losses exceeding one billion U.S. dollars annually on several crops. In South Africa (SA), TSWV has become an important virus in many economically important crops. The main objective of this research project was to isolate, identify and characterise TSWV isolates occurring in SA. A review of current literature assembled background information on TSWV molecular biology, epidemiology, transmission, detection and control. A TSWV isolate infecting pepper (Capsicum sp.) occurring in KZN was isolated and partially characterised. The virus was positively identified as TSWV using the enzyme-linked immunosorbent assay (ELISA) and the presence of typical necrotic TSWV symptoms on Nicotinia rustica L. Symptomatic leaves were harvested and the virus was partially purified using standard procedures. Under the transmission electron microscope (TEM), typical quasi-spherical and dumbbell-shaped particles of 80-100nm in diameter were observed in negatively stained preparations of both crude and purified virus samples. In negatively stained ultra-thin virus infected leaf sections, an abundance of mature viral particles (100nm) housed in the cisternae of the endoplasmic reticulum (ER) were observed among typical viroplasm inclusions (30nm) and hollow tubules (200-300nm). A viral protein migrating as a 29kDa band, which corresponds to the TSWV nucleocapsid (N) protein, was observed after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Total plant RNA, isolated from N. rustica displaying typical symptoms was subjected to reverse-transcription polymerase chain reaction (RT-PCR) using .primers specific to the nucleocapsid (N) gene. An expected 760bp product was amplified. The results obtained in this study confirm the presence of TSWV in infected pepper plants from KZN. The genetic diversity of TSWV isolates occurring in SA was examined. The nucleocapsid (N) gene sequences of six SA TSWV isolates originating from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were determined and used in a phylogenetic tree comparison with TSWV isolates occurring in different geographical locations in the world. Nucleotide sequence comparisons of the N gene revealed high levels of similarity between the SA isolates and TSWV isolates from Asia and Europe. SA isolates showed a high degree of sequence similarity (99-100%) which was reflected in their distinct clustering pattern. The resistance of tomato (Lycopersicon escuJentum Mill.) plants with natural and transgenic resistance against mechanical inoculation with TSWV isolates occurring in SA was evaluated. The Stevens cultivar which has natural resistance conferred by the Sw-5 gene and the transgenic 13-1 line, which expresses the nucleocapsid (N) protein gene of the TSWV-BL isolate, was used as test cultivars. Plants were assessed for TSWV resistance using a disease severity rating scale and measurements of virion accumulation levels (A405nm). There were no significant differences among the reactions produced by the six TSWV isolates on the test plants. Although both plants were susceptible to the SA TSWV isolates by exhibiting similarly high viral accumulation levels, the transgenic tomato line showed milder disease severity compared to the natural resistant cultivar. Results suggest that transgenic resistance is a more effective approach in the control of TSWV in SA. The information generated in this study will be useful in formulating effective control measures using genetic engineering approaches for this economically important virus. Such approaches will be used as a tool to make strategic decisions in an integrated control programme for ISWV.