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A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China

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Abstract

Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate the occurrence and distribution of citrus viroids in China.

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References

  • Amy, M. B., Igor, A. Y., & Steven, H. S. (2004). Validating internal controls for quantitative plant gene expression studies. BMC Plant Biology, 4, 14. doi:10.1186/1471-2229-4-14.

    Article  Google Scholar 

  • Ashulin, L., Lachman, O., Hadas, O., & Bar-Joseph, M. (1991). Nucleotide sequence of a new viroid species, Citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Research, 19, 4767. doi:10.1093/nar/19.17.4767.

    Article  PubMed  CAS  Google Scholar 

  • Cohen, O., Batuman, O., Stanbekova, G., Sano, T., Mawassi, W., & Bar-Joseph, M. (2006). Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees. Virus Genes, 33, 287–292.

    PubMed  CAS  Google Scholar 

  • Diener, T. O. (2001). The viroid: biological oddity or evolutionary fossil. Advances in Virus Research, 57, 137–184. doi:10.1016/S0065-3527(01)57003-7.

    Article  PubMed  CAS  Google Scholar 

  • Duran-Vila, N., Pina, J. A., & Navarro, L. (1993). Improved indexing of citrus viroids. Proceedings of the 10th Conference of the International Organization of Citrus Virologists, Riverside, California, 10, pp. 202–211.

  • Duran-Vila, N., Roistacher, C. N., Rivera-Bustamente, R., & Semancik, J. S. (1988). A definition of citrus viroid groups and their relationship to the exocortis disease. The Journal of General Virology, 69, 3069–3080. doi:10.1099/0022-1317-69-12-3069.

    Article  Google Scholar 

  • Duran-Vila, N., Semancik, J. S., & Broadbent, P. (2000). Viroid diseases, cachexia, and exocortis. In L. W. Timmer, S. M. Garnsey, & J. H. Graham (Eds.), Compendium of citrus diseases (pp. 51–54, 2nd ed.). St. Paul, MN: American Phytopathology Society.

    Google Scholar 

  • Flores, R., Hernandez, C., Martinez de Alba, A. E., Daros, J. A., & Di Serio, F. (2005). Viroids and viroid–host interactions. Annual Review of Phytopathology, 43, 117–139. doi:10.1146/annurev.phyto.43.040204.140243.

    Article  PubMed  CAS  Google Scholar 

  • Gross, H. J., Krupp, G., Domdey, H., Raba, M., Jank, P., Lossow, C., et al. (1982). Nucleotide sequence and secondary structure of citrus exocortis and Chrysanthemum stunt viroid. European Journal of Biochemistry, 121, 249–257. doi:10.1111/j.1432-1033.1982.tb05779.x.

    Article  PubMed  CAS  Google Scholar 

  • Hutton, R. J., Broadbent, P., & Bevington, K. B. (2000). Viroid dwarfing for high density plantings. Horticultural Reviews, 24, 277–317.

    Google Scholar 

  • Ito, T., Ieki, H., & Ozaki, K. (2002). Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. Journal of Virological Methods, 106, 235–239. doi:10.1016/S0166-0934(02)00147-7.

    Article  PubMed  CAS  Google Scholar 

  • Ito, T., Ieki, H., Ozaki, K., & Ito, T. (2001). Characterization of a new citrus viroid species tentatively termed Citrus viroid OS. Archives of Virology, 146, 975–982. doi:10.1007/s007050170129.

    Article  PubMed  CAS  Google Scholar 

  • Levy, L., Hadidi, A., & Garnsey, S. M. (1992). Reverse transcription-polymerase chain reaction assays for the rapid detection of citrus viroids using multiplex primer sets. Proceeding of the International Society of Citriculture, 2, 800–803.

    Google Scholar 

  • Puchta, H., Ramm, K., Luckinger, R., Hadas, R., Bar-Joseph, M., & Sanger, H. L. (1991). Primary and secondary structure of Citrus viroid IV (CVd IV), a new chimeric viroid present in dwarfed grapefruit in Israel. Nucleic Acids Research, 19, 6640. doi:10.1093/nar/19.23.6640.

    Article  PubMed  CAS  Google Scholar 

  • Roistacher, C. N., Calavan, E. C., Blue, R. L., Navarro, L., & Gonzales, R. (1977). A new more sensitive citron indicator for the detection of mild isolates of Citrus exocortis viroid (CEVd). Plant Disease Reporter, 61, l35–l139.

    Google Scholar 

  • Sano, T., Hataya, T., & Shikata, E. (1988). Complete nucleotide sequence of a viroid isolated from Etrog citron, a new member of Hop stunt viroid group. Nucleic Acids Research, 16, 347. doi:10.1093/nar/16.1.347.

    Article  PubMed  CAS  Google Scholar 

  • Semancik, J. S., Rakowski, A. G., Bash, J. A., & Gumpf, D. J. (1997). Application of selected viroids for dwarfing and enhancement of production of ‘Valencia’ orange. Journal of Horticultural Science, 72, 563–570.

    Google Scholar 

  • Semancik, J. S., Roistacher, C. N., & Duran-Vila, N. (1988). A new viroid is the causal agent of the citrus cachexia disease. Proceedings of the 10th Conference of the International Organization of Citrus Virologists, Riverside, California, 10, pp. 125–135.

  • Semancik, J. S., & Weathers, L. G. (1972). Exocortis virus: an infectious free-nucleic acid plant virus with unusual properties. Virology, 46, 456–466. doi:10.1016/0042-6822(72)90281-4.

    Article  Google Scholar 

  • Serra, P., Barbosa, C. J., Daros, J. A., Flores, R., & Duran-Vila, N. (2008). Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid. Virology, 370, 102–112. doi:10.1016/j.virol.2007.07.033.

    Article  PubMed  CAS  Google Scholar 

  • Sieburth, P. J., Irey, M., Garusey, S. M., Semancik, J. S., Levy, L., & Hilf, M. E. (2002). The Use of RT-PCR in the Florida citrus viroid indexing program. Proceedings of the Fifteenth Conference on International Organization of Citrus Virologists, Riverside, California, 15, pp. 230–238.

  • Tessitori, M., La Rosa, R., Albanese, G., & Catara, A. (1996). PCR diagnosis of citrus viroids in field samples. Proceedings of the Thirteenth Conference on International Organization of Citrus Virologists, Riverside, California, 13, pp. 230–235.

  • Yang, X., Hadidi, A., & Garnsey, S. M. (1992). Enzymatic cDNA amplification of Citrus exocortis and Cachexia viroids from infected citrus hosts. Phytopathology, 82, 279–285. doi:10.1094/Phyto-82-279.

    Article  CAS  Google Scholar 

  • Zhou, C. Y., Hailstones, D., Connor, R., Barkley, P., & Bowyer, J. (2001). A method for micro and rapid extraction of Citrus tristeza virus (CTV) nucleic acid applied to RT-PCR amplification. Journal of Fujian Agricultural University, 30, 200, in Chinese.

    Google Scholar 

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Acknowledgements

We thank Drs. Deborah Hailstones and Francesco Faggioli for the valuable comments and critical reading of the manuscript. This work was supported by the National Science R&D Programme (2007BAD47B03, 2007BAD61B04) and Natural Science Foundation Project of CQ CSTC (2006BB1351).

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Authors and Affiliations

  1. National Citrus Virus Exclusion Center, Citrus Research Institute, Southwest University, Chongqing, 400712, People’s Republic of China

    Xuefeng Wang, Changyong Zhou, Kezhi Tang, Yan Zhou & Zhongan Li

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  1. Xuefeng Wang
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  2. Changyong Zhou
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  3. Kezhi Tang
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  4. Yan Zhou
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  5. Zhongan Li
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Correspondence to Changyong Zhou.

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Wang, X., Zhou, C., Tang, K. et al. 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 (2009). https://doi.org/10.1007/s10658-008-9386-y

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  • DOI: https://doi.org/10.1007/s10658-008-9386-y

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