[en] Citrus impietratura disease (CID) is a graft transmissible, virus-like disease observed in old-line citrus trees; its characteristic symptom is the appearance of gum in the albedo of the affected fruits. To identify the causal agent of the disease, high-throughput sequencing (HTS) was performed on symptomatic orange fruits. The analysis of the obtained data revealed in all samples mixed infections of viroids commonly found in citrus trees together with the recently described citrus virus A (CiVA). Examination of additional symptomatic fruits with conventional reverse transcription PCR led to the identification of a single CiVA infection in one tree, which was verified by HTS. Indexing of the single CiVA-infected tree on indicator plants resulted in the appearance of characteristic symptoms in the leaves that were correlated with virus accumulation. Moreover, a comparative analysis among symptomatic and asymptomatic fruits derived from the same trees was performed and included the single CiVA-infected orange tree. The analysis revealed a positive correlation between the appearance of symptoms and the accumulation of CiVA RNAs. To facilitate CiVA detection during certification programs of propagation material, a quantitative RT-PCR targeting the movement protein of the virus was developed and evaluated for reliable and sensitive detection of the virus. To the best of our knowledge this is the first study that associates CiVA with the appearance of CID symptoms.
ERDF - European Regional Development Fund [BE] COST - European Cooperation in Science and Technology [BE]
Funding text :
Funding: This work was supported by the Research Infrastructure “Upgrading the Plant Capital (PlantUp)” (MIS 5002803), which is implemented under the action “Reinforcement of the Research and Innovation Infrastructure,” funded by the operational program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). This work was also funded by the European Cooperation in Science and Technology (COST) action FA1407.
Albariño, C. G., Bird, B. H., and Nichol, S. T. 2007. A shared transcription termination signal on negative and ambisense RNA genome segments of Rift Valley fever, sandfly fever Sicilian, and Toscana viruses. J. Virol. 81: 5246-5256.
Asano, S., Hirayama, Y., and Matsushita, Y. 2017. Distribution of Tomato spotted wilt virus in dahlia plants. Lett. Appl. Microbiol. 64:297-303.
Bar-joseph, M., and Loebenstein, G. 1970. Leaf flecking on indicator seedlings, associated with citrus impietratura in Israel: a possible indexing method. Plant Dis. Rep. 54:643-645.
Bester, R., Karaan, M., Cook, G., and Maree, H. J. 2021. First report of citrus virus A in citrus in South Africa. J. Citrus Pathol. 8. https://escholarship.org/uc/item/5gr6p8zh.
Caruso, A., Davino, G. C., and Terranova, G. 1993. Observations on impietratura disease symptoms in four citrus species. International Organization of Citrus Virologists Conference Proceedings (1957–2010) 12:379-382.
Cook, G., van Vuuren, S. P., Breytenbach, J. H. J., and Manicom, B. Q. 2012. Citrus viroid IV detected in Citrus sinensis and C. reticulata in South Africa. Plant Dis. 96:772.
Diaz-Lara, A., Navarro, B., Di Serio, F., Stevens, K., Hwang, M. S., Kohl, J., et al. 2019. Two novel negative-sense RNA viruses infecting grapevine are members of a newly proposed genus within the family Phenuiviridae. Viruses 11:685.
Fox, A. 2020. Reconsidering causal association in plant virology. Plant Pathol. 69:956-961.
Galipienso, L., Carmen Vives, M., Navarro, L., Moreno, P., and Guerri, J. 2004. Detection of Citrus leaf blotch virus using digoxigenin-labeled cDNA probes and RT-PCR. Eur. J. Plant Pathol. 110:175-181.
Garrison, E., and Marth, G. 2012. Haplotype-based variant detection from short-read sequencing. arXiv 1207.3907.
Green, S. K., Kuo, Y. J., and Lee, D. R. 1988. Uneven distribution of two potyviruses (feathery mottle virus and sweet potato latent virus) in sweet potato plants and its implication on virus indexing of meristem derived plants. Trop. Pest Manage. 34:298-302.
Hou, W., Li, S., and Massart, S. 2020. Is there a “biological desert” with the discovery of new plant viruses? A retrospective analysis for new fruit tree viruses. Front. Microbiol. 11:592816. https://www.frontiersin.org/articles/10.3389/fmicb.2020.592816/full.
Ito, T., Ieki, H., and Ozaki, K. 2002. Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. J. Virol. Methods 106: 235-239.
Jridi, C., Martin, J.-F., Marie-Jeanne, V., Labonne, G., and Blanc, S. 2006. Distinct viral populations differentiate and evolve independently in a single perennial host plant. JVI 80:2349-2357.
Kominek, P., Glasa, M., and Kominkova, M. 2009. Analysis of multiple virus-infected grapevine plant reveals persistence but uneven virus distribution. Acta Virol. 53:281-285.
Kormelink, R., Garcia, M. L., Goodin, M., Sasaya, T., and Haenni, A.-L. 2011. Negative-strand RNA viruses: the plant-infecting counterparts. Virus Res. 162:184-202.
Mafra, V., Kubo, K. S., Alves-Ferreira, M., Ribeiro-Alves, M., Stuart, R. M., Boava, L. P., et al. 2012. Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7:e31263.
Malandraki, I., Beris, D., Isaioglou, I., Olmos, A., Varveri, C., and Vassilakos, N. 2017. Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR. PLoS One 12:e0180877.
Massart, S., Candresse, T., Gil, J., Lacomme, C., Predajna, L., Ravnikar, M., et al. 2017. A framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by NGS Technologies. Front. Microbiol. 8:45.
Minutolo, M., Di Serio, F., Cinque, M., Lombardi, P., Navarro, B., and Alioto, D. 2020. Simultaneous detection of citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA) by multiplex RT-PCR. J. Plant Pathol. 102:655-661.
Navarro, B., Minutolo, M., Stradis, A. D., Palmisano, F., Alioto, D., and Serio, F. D. 2018a. The first phlebo-like virus infecting plants: A case study on the adaptation of negative-stranded RNA viruses to new hosts. Mol. Plant Pathol. 19:1075-1089.
Navarro, B., Zicca, S., Minutolo, M., Saponari, M., Alioto, D., and Di Serio, F. 2018b. A negative-stranded RNA virus infecting citrus trees: the second member of a new genus within the order Bunyavirales. Front. Microbiol. 9:2340.
Noda, H., Yamagishi, N., Yaegashi, H., Xing, F., Xie, J., Li, S., et al. 2017. Apple necrotic mosaic virus, a novel ilarvirus from mosaic-diseased apple trees in Japan and China. J. Gen. Plant Pathol. 83:83-90.
Osman, F., Hodzic, E., Kwon, S.-J., Wang, J., and Vidalakis, G. 2015. Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus. J. Virol. Methods 220:64-75.
Rodio, M.-E., Delgado, S., Flores, R., and Serio, F. D. 2006. Variants of Peach latent mosaic viroid inducing peach calico: Uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant. J. Gen. Virol. 87:231-240.
Roistacher, C. N. 2004. Diagnosis and management of virus and virus like diseases of citrus. Pages 109-189 in Diseases of Fruits and Vegetables Volume I: Diagnosis and Management, S. A. M. H. Naqvi, ed. Springer, Dordrecht, the Netherlands.
Rott, M. E., Kesanakurti, P., Berwarth, C., Rast, H., Boyes, I., Phelan, J., and Jelkmann, W. 2018. Discovery of negative-sense RNA viruses in trees infected with Apple rubbery wood disease by next-generation sequencing. Plant Dis. 102:1254-1263.
Roy, A., Fayad, A., Barthe, G., and Brlansky, R. H. 2005. A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees. J. Virol. Methods 129:47-55.
Ruijter, J. M., Ruiz Villalba, A., Hellemans, J., Untergasser, A., and van den Hoff, M. J. B. 2015. Removal of between-run variation in a multi-plate qPCR experiment. Biomol Detect. Quantif. 5:10-14.
Steibel, J. P., Poletto, R., Coussens, P. M., and Rosa, G. J. M. 2009. A powerful and flexible linear mixed model framework for the analysis of relative quantification RT-PCR data. Genomics 94:146-152.
Svanella-Dumas, L., Marais, A., Depasse, F., Faure, C., Lefebre, M., Brans, Y., et al. 2019. First report of Citrus virus A (CiVA) infecting pear (Pyrus communis) in France. Plant Dis. 103:2703.
Thompson, J. R., Wetzel, S., Klerks, M. M., Vašková, D., Schoen, C. D., Špak, J., et al. 2003. Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control. J. Virol. Methods 111:85-93.
Timmer, L. W., Garnsey, S. M., and Graham, J. H., eds. 2000. Compendium of Citrus Diseases, 2nd ed. American Phytopathological Society, St. Paul, MN.
Villamor, D. E. V., Ho, T., Al Rwahnih, M., Martin, R. R., and Tzanetakis, I. E. 2019. High throughput sequencing for plant virus detection and discovery. Phytopathology 109:716-725.
Wang, X., Zhou, C., Tang, K., Zhou, Y., and Li, Z. 2009. A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China. Eur. J. Plant Pathol. 124:175-180.
Wright, A. A., Szostek, S. A., Beaver-Kanuya, E., and Harper, S. J. 2018. Diversity of three bunya-like viruses infecting apple. Arch. Virol. 163: 3339-3343.
