Development and validation of a specific real-time PCR protocol for the detection of tomato leaf curl New Delhi virus

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting cucurbitaceous and solanaceous crops in the Mediterranean basin since the 2012. It has been categorized as quarantine pest according to the new EU Plant Health Regulation 2016/2031. In order to obtain a reliable and specific diagnostic method to detect the virus, a new primers and probe set, targeting the BC1 region of the component B of the ToLCNDV genome, was designed and the real-time amplification protocol validated according to the guidelines reported in the EPPO standard PM 7/98 (4). The protocol was tested for analytical sensitivity, analytical specificity, selectivity, repeatability and reproducibility obtaining consistent results for all the validation parameters. The protocol is also suitable for detecting ToLCNDV from its vector, the insect Bemisia tabaci (a whitefly able to transmit begomoviruses) using DNA extracted from a single viruliferous adult. Detection of ToLCNDV in insect vector could be an efficient way to early identify the virus in imported material or in new agricultural areas, before its massively spreading. To our knowledge, this is the first validated detection method for ToLCNDV according to the validation parameters required by the EPPO standard PM7/98 (4). This real-time PCR can be successfully applied to the specific detection of ToLCNDV satisfying the phytosanitary requirements of the new legislation on plant health.

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

Fig. 1
Fig. 2

References

  1. EFSA Panel on Plant Health (PLH). (2014). Scientific Opinion on the pest categorisation of Tomato yellow leaf curl virus and related viruses causing tomato yellow leaf curl disease in Europe. EFSA Journal, 12(10), 3850, 27 pp. https://doi.org/10.2903/j.efsa.2014.3850.

    Article  Google Scholar 

  2. EPPO. (2019). PM 7/98 (4) – specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. Bulletin OEPP/EPPO Bulletin, 49(3), 530–563.

    Article  Google Scholar 

  3. EPPO Reporting Service. (2019a). First report of Tomato leaf curl New Delhi virus in Estonia. EPPO Reporting Service no. 07–2019. Num. article: 2019/146.

  4. EPPO Reporting Service. (2019b). First report of Tomato leaf curl New Delhi virus in Portugal. EPPO Reporting Service no. 08–2019. Num. article: 2019/164.

  5. Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using phi 29 DNA polymerase and multiply primed rolling circle amplification. Genome Research, 11, 1095–1099.

    CAS  Article  Google Scholar 

  6. Fortes, I. M., Sánchez-Campos, S., Fiallo-Olivé, E., Díaz-Pendón, J., Navas-Castillo, J., & Moriones, E. (2016). A novel strain of tomato leaf curl New Delhi virus has spread to the Mediterranean basin. Viruses, 8, 307. https://doi.org/10.3390/v8110307.

    CAS  Article  PubMed Central  Google Scholar 

  7. Kheireddine, A., Sifres, A., Sáez, C., Picó, B., & López, C. (2019). First report of tomato leaf curl New Delhi virus infecting cucurbit plants in Algeria. Plant Disease, 103(12), 3291.

    Article  Google Scholar 

  8. Mnari-Hattab, M., Zammouri, S., Belkadhi, M. S., Bellon Doña, D., ben Nahia, E., & Hajlaoui, M. R. (2015). First report of tomato leaf curl New Delhi virus infecting cucurbits in Tunisia. New Dis Reports, 31, 21. https://doi.org/10.5197/j.2044-0588.2015.031.021.

    Article  Google Scholar 

  9. Orfanidou, C. G., Malandraki, I., Beris, D., Kektsidou, O., Vassilakos, N., Varveri, C., Katis, N. I., & Maliogka, V. I. (2019). First report of tomato leaf curl New Delhi virus in zucchini crops in Greece. J Plant Pathol (early view)., 101, 799. https://doi.org/10.1007/s42161-019-00265-y.

    Article  Google Scholar 

  10. Padidam, M., Beachy, R. N., & Fauquet, C. M. (1995). Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. J Gen Virol, 76, 25–35. https://doi.org/10.1099/0022-1317-76-1-25.

    CAS  Article  PubMed  Google Scholar 

  11. Panno, S., Iacono, G., Davino, M., Marchione, S., Zappardo, V., Bella, P., Tomassoli, L., Accotto, G. P., & Davino, S. (2016). First report of tomato leaf curl New Delhi virus affecting zucchini squash in an important horticultural area of southern Italy. New Dis Rep, 33, 6. https://doi.org/10.5197/j.2044-0588.2016.033.006.

    Article  Google Scholar 

  12. Radouane, N., Tahiri, A., El Ghadraoui, L., Al Figuigui, J., & Lahlali, R. (2018). First report of tomato leaf curl New Delhi virus in Morocco. New Dis Rep, 37, 2. https://doi.org/10.5197/j.2044-0588.2018.037.002.

    Article  Google Scholar 

  13. Ruiz, L., Simón, A., Velasco, L., & Janssen, D. (2017). Biological characterization of tomato leaf curl New Delhi virus from Spain. Plant Pathol, 66, 376–382. https://doi.org/10.1111/ppa.12587.

    CAS  Article  Google Scholar 

Download references

Acknowledgments

The authors thank Immacolata Dragone and Michele Mancini for their technical assistance. This work has been founded by the European Commission in the frame of the Project EU Reference Laboratory for viruses, viroids and phytoplasmas (EURL-VIR), GRANT SI2.809532 and the Latium Region in the frame of the National Project CUCURBIOMID, ID85-2017-15024 (Call ‘Progetti di Gruppi di Ricerca’, L.R. 13/2008 – art. 4).

Availability of data and material

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Funding

This research was supported by the following projects: EU Reference Laboratory for viruses, viroids and phytoplasmas (EURL-VIR), GRANT SI2.809532 and CUCURBIOMID (ID85–2017-15,024).

Author information

Affiliations

Authors

Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Luigi Marta, Manglli Ariana, Bertin Sabrina and Donati Livia. The first draft of the manuscript was written by Luigi Marta and Faggioli Francesco and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to L. Ferretti.

Ethics declarations

Conflicts of interest/competing interests

The authors declare that they have not conflicts of interest.

Ethical statement

This work did not involve any animal and/or human participants.

Consent to participate

Not applicable.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Luigi, M., Manglli, A., Bertin, S. et al. Development and validation of a specific real-time PCR protocol for the detection of tomato leaf curl New Delhi virus. Eur J Plant Pathol (2020). https://doi.org/10.1007/s10658-020-02038-1

Download citation

Keywords

  • Quarantine pest
  • Begomovirus
  • Detection
  • Real time PCR
  • Validated protocol