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. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/5475 |
| Date | January 2006 |
| Creators | Sivparsad, Benice. |
| Contributors | Gubba, Augustine. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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