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Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

Dietzgen, R.G. and Twin, J. and Talty, J. and Selladurai, S. and Carroll, M.L. and Coutts, B.A. and Berryman, D.I. and Jones, R.A.C. (2005) Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples. Annals of Applied Biology, 146 (4). pp. 517-530.

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Article Link(s): http://dx.doi.org/10.1111/j.1744-7348.2005.040155....

Publisher URL: http://www.aab.org.uk/

Abstract

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Item Type:Article
Business groups:Agri-Science, Crop and Food Science
Additional Information:© Association of Applied Biologists.
Keywords:TSWV; real time PCR; molecular diagnosis; serological tests; sensitivity; application; horticultural crops; N gene variability; phylogenetic analysis.
Subjects:Plant culture > Horticulture. Horticultural crops
Science > Biology > Genetics
Plant pests and diseases
Deposited On:24 Dec 2007
Last Modified:26 Oct 2011 06:08

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