Skip to main content

Advertisement

SpringerLink
Log in

Developing a multiplex real-time RT-PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid

  • Original Paper
  • Published:
Australasian Plant Pathology Aims and scope Submit manuscript

Abstract

Tomato (Solanum lycopersicum L.) is one of the most important vegetables in the world and Asian countries are the center for hybrid tomato seed production. Seed-borne diseases, if not managed well through effective seed health assay, could seriously hamper the global seed trade. Pepper chat fruit viroid (PCFVd) and Columnea latent viroid (CLVd) are two emerging and economically important pospiviroid species infecting tomato and other solanaceous crops. With their greater sequence diversity from those of other common tomato-infecting pospiviroids, it is necessary to develop a new detection method that could effectively detect PCFVd and CLVd, useful for seed health assay. In the present study, we developed a multiplex real time (quantitative) reverse transcription-polymerase chain reaction (qRT-PCR) to detect PCFVd and CLVd in a single tube, thus to save time and money. Using known viroid-contaminated seeds, this multiplex qRT-PCR could detect CLVd and PCFVd from a single contaminated seed, suggesting its sensitivity for seed health assay. The effectiveness of this newly developed multiplex qRT-PCR for detection of PCFVd and CLVd was validated using field-produced tomato plant tissue or commercial tomato seeds.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Bakker D, Bruinsma M, Dekter RW, Toonen MAJ, Verhoeven JTJ, Koenraadt HMS (2015) Detection of PSTVd and TCDVd in seeds of tomato using real-time RT-PCR. EPPO Bull 45:14–21

    Article  Google Scholar 

  • Batuman O, Gilbertson RL (2013) First report of Columnea latent viroid (CLVd) in tomato in Mali. Plant Dis: Disease Notes 97:692. https://doi.org/10.1094/PDIS-10-12-0920-PDN

    Article  Google Scholar 

  • Bogaert NV, Olivier T, Bragard C, Maes M, Smagghe G, Jonghe KD (2015) Assessment of pospiviroid transmission by Myzus persicae, Macrolophus pygmaeus and Bombus terrestris. Eur J Plant Pathol 144:289–296. https://doi.org/10.1007/s10658-015-0766-9

    Article  CAS  Google Scholar 

  • Boonham N, Perez LG, Mendez MS, Peralta EL, Blockley A, Walsh K, Barker I, Mumford RA (2004) Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. J Virol Methods 116:139–146

    Article  CAS  PubMed  Google Scholar 

  • Bostan H, Nie X, Singh RP (2004) An RT-PCR primer pair for the detection of pospiviroids and its application in surveying ornamental plants for viroids. J Virol Methods 116:189–193

    Article  CAS  PubMed  Google Scholar 

  • Botermans M, van de Vossenberg BTLH, Verhoeven JTJ, Roenhorst JW, Hooftman M, Dekter R, Meekes ETT (2013) Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids. J Virol Methods 187:43–50

    Article  CAS  PubMed  Google Scholar 

  • Card SD, Pearson MN, Clover GRG (2007) Plant pathogens transmitted by pollen. Australas Plant Pathol 36:455–461

    Article  Google Scholar 

  • Chambers GA, Seyb AM, Mackie J, Constable FE, Rodoni BC, Letham D, Gibbs MJ (2013) First report of Pepper chat fruit viroid in traded tomato seed, an interception by Australian biosecurity. Plant Dis 97:1386

    Article  PubMed  Google Scholar 

  • Di Serio F, Flores R, Verhoeven JT, Li SF, Pallás V, Randles JW, Sano T, Vidalakis G, Owens RA (2014) Current status of viroid taxonomy. Arch Virol 159:3467–3478

    Article  PubMed  Google Scholar 

  • EFSA Panel on Plant Health (2011) Scientific opinion on the assessment of the risk of solanaceous pospiviroids for the EU territory and the identification and evaluation of risk management options. EFSA J 9:133

    Google Scholar 

  • Hadidi A, Flores R, Randles J, Semancik J (2003) Viroid: properties, detection, diseases and their control. CIRO Publishing, Melbourne 392 p

    Google Scholar 

  • Hammond R W (2017) Seed, pollen, and insect transmission of viroids. In Hadidi A, Flores R, Randles J W, Palukaitis P (eds) Viroids and Satellites. pp. 521–529

    Chapter  Google Scholar 

  • Hammond RW, Owens RA (2006) Viroids: new and continuing risks for horticultural and agricultural crops. APS Features. https://doi.org/10.1094/APSnetFeature-2006-1106

  • Hoshino S, Okuta T, Isaka M, Tutumi N, Miyai N, Ikeshiro T, Saito N, Ohara T, Takahashi T (2006) Detection of Potato spindle tuber viroid (PSTVd) in tomato (Lycopersicon esculentum) and potato (Solanum tuberosum) seeds. Res Bull Pl Prot Japan 42:75–79

    Google Scholar 

  • Kovalskaya N, Hammond RW (2014) Molecular biology of viroid-host interactions and disease control strategies. Plant Sci 228:48–60. https://doi.org/10.1016/j.plantsci.2014.05.006. Accessed 20 October 2014

    Article  CAS  PubMed  Google Scholar 

  • Kryczynski S, Paduch-Cichal E, Skrzeczkowski LJ (1988) Transmission of three viroids through seed and pollen of tomato plants. J Phytopathol 121:51–57

    Article  Google Scholar 

  • Luigi M, Costantini E, Luison D, Mangiaracina P, Tomassoli L, Faggioli F (2014) A diagnostic method for the simultaneous detection and identification of pospiviroids. J Plant Pathol 96:151–158

    Google Scholar 

  • Matsushita Y, Tsuda S (2016) Seed transmission of Potato spindle tuber viroid, Tomato chlorotic dwarf viroid, Tomato apical stunt viroid and Columnea latent viroid in horticultural plants. Eur J Plant Pathol 145:1007–1011. https://doi.org/10.1007/s10658-016-0868-z

    Article  CAS  Google Scholar 

  • Matsuura S, Matsushita Y, Usugi T, Tsuda S (2010) Disinfection of Tomato chlorotic dwarf viroid by chemical and biological agents. Crop Prot 29:1157–1161

    Article  CAS  Google Scholar 

  • Ministry of Agriculture and Cooperative (2007) Ministry of Agriculture and Cooperative Notification: Specification of plant pests as prohibited articles under the Plant Quarantine Act B.E. 2507 (1964) (No. 7) B.E. 2550 (2007)

  • Mink GI (1993) Pollen and seed-transmission viruses and viroids. Annu Rev Phytopathol 31:375–402

    Article  CAS  PubMed  Google Scholar 

  • Monger W, Tomlinson J, Boonham N, Marn MV, Plesko IM, Molinero-Demilly V, Tassus X, Meeles E, Toonen M, Papayiannis L, Perez-Egusquiza Z, Mehle N, Jansen CN, Nielsen SL (2010) Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids. J Virol Methods 169:207–210

    Article  CAS  PubMed  Google Scholar 

  • Naktuinbouw (2014) Reference protocol Naktuinbouw: real time RT-PCR (RT TaqMan PCR) for pospiviroids (CEVd, CLVd, MPVd, PCFVd, PSTVd, TASVd and TPMVd) on seeds of tomato (Solanumlycopersicum). The Dutch protocol SPN-VO439 p

  • Nixona T, Glovera R, Mathews-Berrya S, Dalya M, Hobdena E, Lambourneb C, Harju V, Skeltona A (2010) Columnea latent viroid (CLVd) in tomato: the first report in the United Kingdom. Plant Pathol 59:392. https://doi.org/10.1111/j.1365-3059.2009.02127.x

    Article  Google Scholar 

  • Owens RA, Diener TO (1981) Sensitive and rapid diagnosis of Potato spindle tuber viroid disease by nucleic-acid hybridization. Phytopathology 71:770

    Google Scholar 

  • Reanwarakorn K, Klinkong S, Porsoongnurn J (2011) First report of natural infection of Pepper chat fruit viroid in tomato plants in Thailand. New Dis Rep 24:6. https://doi.org/10.5197/j.2044-0588.2011.024.006

    Article  Google Scholar 

  • Sastry KS (2013) Seed-borne plant virus diseases. Springer, India 327 p

    Book  Google Scholar 

  • Sombat S, Ling KS (2014) Diagnostic methods for the detection of pospiviroid in solanaceous plants and seeds. Thai Agric Res J 32:164–177

    Google Scholar 

  • Spieker RL (1996) A viroid from Brunfelsia undulate closely related to the Columnea latent viroid. Arch Virol 14:1823–1832

    Article  Google Scholar 

  • Steyer S, Olivier T, Skelton A, Hobden E (2010) Columnea latent viroid (CLVd): first report in tomato in France. Plant Pathol 59:794

    Article  Google Scholar 

  • Tangkanchanapas P, Reanwarakorn K, Chanprame S, Hongprayoon R (2005) An RT-PCR primer pair for the detection of six pospiviroid in tomato plants. Thai Phytopathol 19:13–21

    Google Scholar 

  • Tsutsumi N, Yanagisawa H, Fujiwara Y, Ohara T (2010) Detection of Potato spindle tuber viroid by reverse transcription loop-mediated isothermal amplification. Res Bull Pl Prot Japan 46:61–67

    CAS  Google Scholar 

  • Verhoeven J Th J (2010) Identification and epidemiology of pospiviroid. Wageningen University, Ph.D thesis. 136 p

  • Verhoeven J, Th J, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW (2004) Natural infection of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. Eur J Plant Pathol 110:823–831

    Article  CAS  Google Scholar 

  • Verhoeven J, Th J, Jansen CCC, Roenhorst JW, Flores R, Peña MD (2009) Pepper chat fruit viroid: biological and molecular properties of a proposed new species of the genus Pospiviroid. Virus Res 144:209–214

    Article  CAS  PubMed  Google Scholar 

  • Verhoeven J, Th J, Roenhorst J, Owens R (2011) Mexican papita viroid and Tomato planta macho viroid belong to a single species in the genus Pospiviroid. Arch Virol 156:1433–1437

    Article  CAS  PubMed  Google Scholar 

  • Weller SA, Elphinstone JG, Smith NC, Boonham N, Stead DE (2000) Detection of Ralstonia solanacearum strains with a quantitative multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl Environ Microbiol 66:2853–2858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yanagisawa H, Shiki Y, Matsushita Y, Ooishi M, Takaue N, Tsuda S (2017) Development of a comprehensive detection and identification molecular based system for eight pospiviroids. Eur J Plant Pathol 149:11–23

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by Agricultural Research Development Agency (Public Organization) and Department of Agriculture, Thailand.

Author information

Authors and Affiliations

  1. Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom Province, Thailand

    Sukhontip Sombat & Kanungnit Reanwarakorn

  2. U.S. Department of Agriculture- Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, 29414, USA

    Kai-Shu Ling

Authors
  1. Sukhontip Sombat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kanungnit Reanwarakorn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kai-Shu Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Kanungnit Reanwarakorn or Kai-Shu Ling.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sombat, S., Reanwarakorn, K. & Ling, KS. Developing a multiplex real-time RT-PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid. Australasian Plant Pathol. 47, 615–621 (2018). https://doi.org/10.1007/s13313-018-0597-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13313-018-0597-1

Keywords

Search

Navigation