Development of a generic method for inspection of tospoviruses

Members of the genus Tospovirus which can infect plants in the family Bunyaviridae , possess quasi-spherical enveloped particles (80–120 nm in diameter) and a segmented tripartite single-stranded RNA genome, with large (L), medium (M) and small (S) RNA segments. Tospoviruses are vectored by thrips i...

Full description

Saved in:
Bibliographic Details
Published in:European journal of plant pathology 2018-02, Vol.150 (2), p.457-469
Main Authors: Huang, Kuo-Shiou, Li, Siang-Ling, Sun, Jing-Hua, Wang, Yun-Chi, Jan, Fuh-Jyh, Chen, Tsung-Chi
Format: Article
Language:English
Subjects:
Agriculture
Biomedical and Life Sciences
Ecology
Gel electrophoresis
Gene sequencing
Genomes
Inspection
Life Sciences
Melting curve
Plant Pathology
Plant Sciences
Polymerase chain reaction
Primers
Reverse transcription
Ribonucleic acid
RNA
Spot
Tomatoes
Vigna unguiculata
Viruses
Wilt
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Members of the genus Tospovirus which can infect plants in the family Bunyaviridae , possess quasi-spherical enveloped particles (80–120 nm in diameter) and a segmented tripartite single-stranded RNA genome, with large (L), medium (M) and small (S) RNA segments. Tospoviruses are vectored by thrips in a persistent manner and cause yield losses of numerous economic crops worldwide. Inspection is an important measure to prevent invasion of tospoviruses. However, the increase of new virus species makes inspection challenging. In this study, a degenerate primer pair dTospo-F2/dTospo-R2 was designed from the conserved regions of the tospoviral L RNA sequences and used for the tospovirus detection in SYBR Green I-based quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR). The designed primers could amplify specific products from all tested 20 tospovirus species. The specificity of amplifications was validated by melting curve assays, agarose gel electrophoresis and direct sequencing of amplicons. Furthermore, the degenerate primer pair was used in RT-qPCR to detect tospovirus infections from field cowpea and sweet pepper samples. Sequencing of the amplicons confirmed the identity of the viruses as Groundnut chlorotic fan-spot virus (GCFSV) from the cowpea and Tomato spotted wilt virus from the sweet pepper. Our results demonstrated that the degenerate primer pair dTospo-F2/dTospo-R2 is highly specific to tospoviruses, and when used in RT-qPCR, it greatly simplifies and enhances the efficacy of the inspection of tospoviruses. Additionally, this is the first report of GCFSV infected cowpea.
ISSN:0929-1873
1573-8469
DOI:10.1007/s10658-017-1295-5