European Journal of Plant Pathology

, Volume 132, Issue 4, pp 469–475 | Cite as

Simultaneous detection of eight viruses and two viroids affecting stone fruit trees by using a unique polyprobe

  • Ana Peiró
  • Vicente Pallás
  • Jesús Ángel Sánchez-Navarro


The use of riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the simultaneous detection of up to six different pathogens using a non-radioactive molecular hybridization procedure. In the present report, we describe the development of a unique polyprobe (poly10) with the capacity to detect viruses and viroids commonly found infecting fruit trees. The poly10 covers eight viruses: Apple mosaic virus (ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV), Plum bark necrosis and stem pitting-associated virus (PBNSPaV) and two viroids: Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Poly10 is comparable to the individual riboprobes in terms of end-point dilution limit and specificity, allowing the detection of up to 2.5 picograms of viral or viroidal RNA. However, the polyprobe requires a hybridization temperature of 60°C instead of the standard 68°C. The validation of the new simultaneous detection strategy was confirmed by the analysis of 60 field samples, which came from seven different hosts. The use of the polyprobe as an alternative to other routinely used detection methods is discussed.


Multiplex Non-radioactive molecular hybridization Stone fruit tree viruses and viroids Dig-RNA probe 



A. P. M. is the recipient of a JAE-Pre contract from the Consejo Superior de Investigaciones Científicas (CSIC). We thank L. Corachán for her excellent technical assistance. This work was supported by grant BIO2008-03528 from the Spanish granting agency DGICYT and by grant ACOMP/2010/214 from the Generalitat Valenciana.

Supplementary material

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  1. Astruc, N., Marcos, J. F., Macquaire, G., Candresse, T., & Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal of Plant Pathology, 102, 837–846.CrossRefGoogle Scholar
  2. Clark, M. F., & Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology, 34, 475–483.PubMedCrossRefGoogle Scholar
  3. Cohen, O., Batuman, O., Stanbekova, G., Sano, T., Mawassi, M., & Bar-Joseph, M. (2006). Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees. Virus Genes, 33, 287–292.PubMedGoogle Scholar
  4. Herranz, M. C., Sánchez-Navarro, J. A., Aparicio, F., & Pallás, V. (2005). Simultaneous detection of six stone fruit viruses by non-isotopic molecular hybridization using a unique riboprobe or ‘polyprobe’. Journal of Virological Methods, 124, 49–55.PubMedCrossRefGoogle Scholar
  5. James, D., Varga, A., Pallás, V., & Candresse, T. (2006). Strategies for simultaneous detection of multiple plant viruses. Canadian Journal of Plant Pathology-Revue Canadienne de Phytopathologie, 28, 16–29.CrossRefGoogle Scholar
  6. Lin, L., Li, R., Mock, R., & Kinard, G. (2011). Development of a polyprobe to detect six viroids of pome and stone fruit trees. Journal of Virological Methods, 171, 91–97.PubMedCrossRefGoogle Scholar
  7. López, M. M., Bertolini, E., Olmos, A., Caruso, P., Gorris, M. T., Llop, P., et al. (2003). Innovative tools for detection of plant pathogenic viruses and bacteria. International Microbiology, 6, 233–243.PubMedCrossRefGoogle Scholar
  8. Más, P., & Pallás, V. (1995). Non-isotopic tissue-printing hybridization: A new technique to study long-distance plant virus movement. Journal of Virological Methods, 52, 317–326.PubMedCrossRefGoogle Scholar
  9. Más, P., Sánchez-Navarro, J. A., Sánchez-Pina, M. A., & Pallás, V. (1993). Chemiluminescent and colorigenic detection of cherry leaf roll virus with digoxigenin-labelled RNA probes. Journal of Virological Methods, 45, 93–102.PubMedCrossRefGoogle Scholar
  10. Matic, S., Sánchez-Navarro, J. A., Mandic, B., Myrta, A., & Pallás, V. (2008). Tracking three ilarviruses in stone fruit trees throughout the year by ELISA and tissue-printing hybridization. Journal of Plant Pathology, 90, 137–141.Google Scholar
  11. Pallás, V., Mas, P., & Sánchez-Navarro, J. A. (1998). Detection of plant RNA viruses by nonisotopic dot-blot hybridization. Methods in Molecular Biology, 81, 461–468.PubMedGoogle Scholar
  12. Saade, M., Aparicio, F., Sánchez-Navarro, J. A., Herranz, M. C., Myrta, A., Di-Terlizzi, B., et al. (2000). Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridisation and multiplex reverse-transcription polymerase chain reaction. Phytopathology, 90, 1330–1336.PubMedCrossRefGoogle Scholar
  13. Saldarelli, P., Barbarossa, L., Grieco, F., & Gallitelli, D. (1996). Digoxigenin-labelled riboprobes applied to phytosanitary certification of tomato in Italy. Plant Disease, 80, 1343–1346.CrossRefGoogle Scholar
  14. Sánchez-Navarro, J. A., Canizares, M. C., Cano, E. A., & Pallás, V. (1999). Simultaneous detection of five carnation viruses by non-isotopic molecular hybridization. Journal of Virological Methods, 82, 167–175.PubMedCrossRefGoogle Scholar
  15. Sánchez-Navarro, J. A., Canizares, M. C., Cano, E. A., & Pallás, V. (2007). Plant tissue distribution and chemical inactivation of six carnation viruses. Crop Protection, 26, 1049–1054.CrossRefGoogle Scholar

Copyright information

© KNPV 2011

Authors and Affiliations

  • Ana Peiró
    • 1
  • Vicente Pallás
    • 1
  • Jesús Ángel Sánchez-Navarro
    • 1
  1. 1.Instituto de Biología Molecular y Celular de PlantasUniversidad Politecnica de Valencia-CSICValenciaSpain

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