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Development of a generic method for inspection of tospoviruses

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Abstract

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.

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Acknowledgments

We are grateful to Dr. Chung-Jan Chang, emeritus professor of the University of Georgia, and Dr. Kuang-Ren Chung for their critical review of the manuscript. Our gratitude also goes to Michael Burton, Asia University. This study was partially supported by grants from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, the Ministry of Science and Technology (MOST 105-2313-B-005-021-MY3) and the Ministry of Education, Taiwan, R.O.C. under the ATU plan. We thank the virus providers: Dr. R. Provvidenti for TSWV; Dr. Dennis Gonsalves for GRSV; Dr. James W. Moyer for INSV; Dr. Richard Kormelink for ANSV, IYSV and TYRV; Dr. Mitsuru Okuda for CSNV; Dr. Prem A. Rajagopalan for GBNV and WBNV; Dr. Ioannis E. Tzanetakis for SVNaV; Dr. Jiahong Dong for HCRV, TNSaV and TZSV; Dr. Massimo Turina for PolRSV; the DSMZ Plant Virus Collection, Germany for TCSV; and the permission of the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan for the virus importation.

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Authors and Affiliations

  1. Department of Plant Pathology, National Chung Hsing University, Taichung, 40227, Taiwan

    Kuo-Shiou Huang & Fuh-Jyh Jan

  2. Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taipei City, 10070, Taiwan

    Kuo-Shiou Huang

  3. Department of Biotechnology, Asia University, Wufeng, Taichung, 41354, Taiwan

    Siang-Ling Li, Jing-Hua Sun, Yun-Chi Wang & Tsung-Chi Chen

  4. Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 40402, Taiwan

    Tsung-Chi Chen

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  1. Kuo-Shiou Huang
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Correspondence to Fuh-Jyh Jan or Tsung-Chi Chen.

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This study was funded by the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan (106AS-9.5.1-BQ-B7), and the Ministry of Science and Technology (MOST 105-2313-B-005-021-MY3). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Huang, KS., Li, SL., Sun, JH. et al. Development of a generic method for inspection of tospoviruses. Eur J Plant Pathol 150, 457–469 (2018). https://doi.org/10.1007/s10658-017-1295-5

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