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Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR

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Abstract

Citrus concave-gum associated virus (CCGaV) and citrus virus A (CiVA) are two negative-stranded RNA viruses infecting citrus. Both viruses have been identified by high-throughput sequencing in Italy and their simultaneous presence in the same host documented. While CCGaV has been associated with citrus concave gum disease, the association of CiVA with symptoms is still unclear. Since the two virus genomes share about 75% sequence identity, their identification by RT-PCR is not obvious. Here, a multiplex RT-PCR to simultaneously detect and identify them is reported. Amplification of the citrus cytochrome oxidase (COX) gene transcript was included as internal control to minimize the risk of false negatives. Specificity and sensitivity of multiplex RT-PCR were comparable to that of conventional uniplex RT-PCR set up in previous studies for the independent detection of the two viruses. The method was validated by a field survey performed testing almost 300 trees grown in different areas of southern Italy. The assay revealed infection by CCGaV and CiVA in 17.7% and 18.4% of the tested trees, respectively, with mixed infection recorded in 5.0% of the samples. The novel mRT-PCR method saves time and costs and has the potential to be used for large-scale surveys and certification programs.

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Acknowledgements

We wish to express our sincere thanks to Professors Maria Laura Cestaro and Patrizia Porretta, the Directors of the Istituto Tecnico Agrario Statale ‘G. Fortunato’ and Istituto Tecnico Agrario Statale ‘E. De Cillis’, respectively, for providing access to the citrus collections, during the field survey. This work was partially supported by the projects ‘URCOFI’ (funded by Regione Campania, DGR 690/2016, 6th December 2016) and ‘INNOCI’ (funded by Regione Puglia, DGR n. 903/2012, 15th May 2012). This article reflects only the author’s view and the Agencies are not responsible for any use that may be made of the information it contains.

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Correspondence to Beatriz Navarro or Daniela Alioto.

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Minutolo, M., Di Serio, F., Cinque, M. et al. Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR. J Plant Pathol 102, 655–661 (2020). https://doi.org/10.1007/s42161-020-00542-1

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  • DOI: https://doi.org/10.1007/s42161-020-00542-1

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