European Journal of Plant Pathology

, Volume 133, Issue 3, pp 765–772 | Cite as

One-step multiplex RT-PCR for simultaneous detection of four pome tree viroids

  • Liming Lin
  • Ruhui Li
  • Ray Mock
  • Gary Kinard


Apple scar skin viroid (ASSVd), Apple dimple fruit viroid (ADFVd), Apple fruit crinkle viroid (AFCVd), and Pear blister canker viroid (PBCVd) infect pome fruit trees. These viroids are important quarantine pathogens for the international movement of pome germplasm. A single-step multiplex reverse transcription polymerase chain reaction assay (mRT-PCR) was developed for the simultaneous detection of these viroids. Four pairs of primers specific for each of the four viroids were used to amplify PCR products of different sizes that can be resolved by agarose gel electrophoresis. Amplification of a plant 18S rRNA was included in the assay as an internal control. Amplicons of 371 bp (AFCVd), 270 bp (ADFVd), 186 bp (ASSVd), 120 bp (PBCVd), and 844 bp (18S rRNA) were obtained in both uniplex and mRT-PCR assays. The identities of the amplification products were confirmed by sequencing. The specificities and limits of detection for all four viroids by uniplex and mRT-PCR assays were comparable. The assay was further validated using samples from pome trees inoculated with all four viroids, as well as field samples from commercial orchards in Colorado. All four viroids were detected from inoculated pear trees and up to three viroids were detected from inoculated apple trees. This is a simple, rapid and cost-effective technique to detect these four viroids in fruit trees. The procedure is especially applicable to certification and quarantine programs, where numerous samples must be tested for all four viroids.


Apscaviroid Apple dimple fruit viroid Apple fruit crinkle viroid Apple scar skin viroid Multiplex RT-PCR Pear blister canker viroid 



The authors would like to thank Mr. Sam Grinstead for maintenance of trees in the screenhouse and Dr. Ramesh Pokharel (Colorado State University) for pome samples from Colorado orchards.


  1. Boubourakas, I. N., Arambatzis, C., Kyriakopoulou, P. E., & Dovas, C. (2008). Amelioration of a reverse transcription polymerase chain reaction (RT-PCR) for the detection of ASSVd, PBCVd and PLMVd viroids, and their presence in cultivated and wild pome and stone fruits in Greece. Acta Horticulturae, 781, 519–527.Google Scholar
  2. Desvignes, J. C., Cornaggia, D., Grasseau, N., Ambrós, S., & Flores, R. (1999a). Pear blister canker viroid: host range and improved bioassay with two new pear indicators, Fieud 37 and Fieud 110. Plant Disease, 83, 419–422.CrossRefGoogle Scholar
  3. Desvignes, J. C., Grasseau, N., Boyé, R., Cornaggia, D., Aparicio, F., Di Serio, F., & Flores, R. (1999b). Biological properties of apple scar skin viroid: isolates, host range, different sensitivity of apple cultivars, elimination, and natural transmission. Plant Disease, 83, 768–772.CrossRefGoogle Scholar
  4. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., Desvignes, J. C., & Flores, R. (2001). Apple dimple fruit viroid: fulfillment of Koch’s postulates and symptom characteristics. Plant Disease, 85, 179–182.CrossRefGoogle Scholar
  5. Di Serio, F., Malfitano, M., Alioto, D., Ragozzino, A., & Flores, R. (2002). Apple dimple fruit viroid: sequence variability and its specific detection by multiplex fluorescent RT-PCR in the presence of Apple scar skin viroid. Journal of Plant Pathology, 84, 27–34.Google Scholar
  6. Diener, T. O. (1971). Potato spindle tuber “virus”: IV. A replicating, low molecular weight RNA. Virology, 45, 411–428.PubMedCrossRefGoogle Scholar
  7. Faggioli, F., Ragozzino, E., & Barba, M. (2001). Simultaneous detection of stone or pome fruit viroids by single tube RT-PCR. Acta Horticulturae, 550, 59–63.Google Scholar
  8. Fekih Hassen, I., Roussel, S., Kummert, J., Fakhfakh, H., Marrakchi, M., & Jijakli, M. H. (2006). Development of a rapid RT-PCR test for the detection of peach latent mosaic viroid, pear blister canker viroid, hop stunt viroid and apple scar skin viroid in fruit trees from Tunisia. Journal of Phytopathology, 154, 217–223.CrossRefGoogle Scholar
  9. Flores, R., Hernández, C., Martínez de Alba, A. E., Daròs, J. A., & Di Serio, F. (2005). Viroids and viroid-host interactions. Annual Review of Phytopathology, 43, 117–139.PubMedCrossRefGoogle Scholar
  10. Flores, R., Randles, J. W., Owens, R. A., Bar-Joseph, M., & Diener, T. O. (2005b). Viroids. In C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, & A. L. Ball (Eds.), Virus taxonomy, eight report of the international committee on taxonomy of viruses (pp. 1145–1159). London: Elsevier/Academic.Google Scholar
  11. Gambino, G., & Gribaudo, I. (2006). Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Virology, 96, 1223–1229.Google Scholar
  12. Hadidi, A., Flores, R., Randles, J. W., & Semancik, J. S. Editors. (2003). Viroids. Collingwood, Victoria, Australia: CSIRO Publishing.Google Scholar
  13. Joyce, P. A., Constable, F. E., Crosslin, J., Eastwell, K., Howell, W. E., & Rodoni, B. C. (2006). Characterisation of Pear blister canker viroid isolates from Australian pome fruit orchards. Australasian Plant Pathology, 35, 465–471.CrossRefGoogle Scholar
  14. Kaponi, M. S., Luigi, M., Barba, M., & Kyriakopoulou, P. E. (2010a). Pospiviroidae viroids in naturally infected stone and pome fruits in Greece. Julius-Kühn-Archiv, 427, 353–356.Google Scholar
  15. Kaponi, M. S., Luigi, M., Barba, M., Sano, T., & Kyriakopoulou, P. E. (2010b). First report and molecular analysis of Apple scar skin viroid in sweet cherry. Julius-Kühn-Archiv, 427, 361–365.Google Scholar
  16. Kyriakopoulou, P. E., Giunchedi, L., & Hadidi, A. (2001). Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: implications on possible origin of these viroids in the Mediterranean region. Journal of Plant Pathology, 83, 51–62.Google Scholar
  17. Li, R., Mock, R., Huang, Q., Abad, J., Hartung, J., & Kinard, G. (2008). A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods, 154, 48–55.PubMedCrossRefGoogle Scholar
  18. 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
  19. Lolic, B., Afechtal, M., Matic, S., Myrta, A., & Di Serio, F. (2007). Detection by tissue-printing of pome fruit viroids and characterization of pear blister canker viroid in Bosnia and Herzegovina. Journal of Plant Pathology, 89, 369–375.Google Scholar
  20. Nakaune, R., & Nakano, M. (2008). Identification of a new Apscaviroid from Japanese persimmon. Archives of Virology, 153, 969–972.PubMedCrossRefGoogle Scholar
  21. Pokharel, R., Mock, R., Li, R., Kinard, G., & Larsen, H. (2010). Association of multiple virus infections with apple disease in western Colorado. Phytopathology, 100, S101.Google Scholar
  22. Roy, A., Fayad, A., Barthe, G., & Brlansky, R. H. (2005). A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees. Journal of Virological Methods, 129, 47–55.PubMedCrossRefGoogle Scholar
  23. Sano, T., Isono, S., Matsuki, K., Kawaguchi-Ito, Y., Tanaka, K., Kondo, K., Iijima, A., & Bar-Joseph, M. (2008). Vegetative propagation and its possible role as a genetic bottleneck in the shaping of the apple fruit crinkle viroid populations in apple and hop plants. Virus Genes, 37, 298–303.PubMedCrossRefGoogle Scholar
  24. Shamloul, A. M., Faggioli, F., Keith, J. M., & Hadidi, A. (2002). A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. Journal of Virological Methods, 105, 115–121.PubMedCrossRefGoogle Scholar
  25. Uga, H., & Tsuda, S. (2005). A one-step reverse transcription-polymerase chain reaction system for the simultaneous detection and identification of multiple Tospovirus infection. Phytopathology, 95, 166–171.PubMedCrossRefGoogle Scholar
  26. Walia, Y., Dhir, S., Bhadoria, S., Hallan, V., & Zaidi, A. A. (2011). Molecular characterization of apple scar skin viroid from Himalayan wild cherry. Forest Pathology (in press). doi  10.1111/j.1439-0329.2011.00723.x.
  27. Zhao, Y., & Niu, J. (2008). Apricot is a new host of Apple scar skin viroid. Australasian Plant Disease Notes, 3, 98–100.CrossRefGoogle Scholar

Copyright information

© KNPV 2012

Authors and Affiliations

  1. 1.National Germplasm Resources LaboratoryUSDA-ARSBeltsvilleUSA
  2. 2.Institute of Plant VirologyFujian Agriculture and Forestry UniversityFujianChina

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