Abstract
Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Konjac mosaic virus (KoMV) and Zantedeschia mild mosaic virus (ZaMMV) are important potyviruses previously identified in calla lily plants in Taiwan. In order to save time and cost of virus detection, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were designed based on the sequences of 3′ terminal region of respective viruses. To prevent false negative results, a primer pair specific to plant mitochondrial nad5 mRNA was used to produce a 185-bp fragment as an internal control of RT-PCR. The specificities of primers were confirmed by means of simplex and multiplex PCR assays. Optimal primer concentration ratio was identified by multiplex PCR assay. Total RNAs purified from virus-infected plants were used directly or mixed in different combinations, and then tested by multiplex RT-PCR. The result indicated that the expected RT-PCR products could be specifically amplified and identified on the basis of their molecular sizes. The detection sensitivity of multiplex RT-PCR was 25–625 times higher than that of indirect-ELISA (I-ELISA) depending on the virus. When applied to field surveys, multiplex RT-PCR could detect more single as well as mixed infection samples than I-ELISA. Accordingly, our multiplex RT-PCR assay provides a simple, rapid and reliable method for multiple potyvirus detection in calla lily.
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References
Bertolini, E., Olmos, A., Martinez, M. C., Gorris, M. T., & Cambra, M. (2001). Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees. Journal of Virological Methods, 92, 33–41.
Chang, Y.-C., Chen, Y.-L., & Chung, F.-C. (2001). Mosaic disease of calla lily caused by a new potyvirus in Taiwan. Plant Disease, 85, 1289.
Chen, Y.-L., Chung, F.-C., & Chang, Y.-C. (1998). Molecular cloning and characterization of Dasheen mosaic potyvirus from aroid plants in Taiwan. Plant Pathology Bulletin, 7, 226–227.
Chen, C.-C., Chao, C.-H., Chen, C.-C., Yeh, S.-D., Tsai, H.-T., & Chang, C.-A. (2003). Identification of Turnip mosaic virus isolates causing yellow stripe and spot on calla lily. Plant Disease, 87, 901–905.
Chen, C.-C., Chang, C.-A., Tsai, H.-T., & Hsu, H. T. (2004). Identification of a potyvirus causing latent infection in calla lilies. Plant Disease, 88, 1046.
Du, Z., Chen, J., & Hiruki, C. (2006). Optimization and application of a multiplex RT-PCR system for simultaneous detection of five potato virus using18S rRNA as an internal control. Plant Disease, 90, 185–189.
Hsu, Y.-C., Yeh, T.-J., & Chang, Y.-C. (2005). A new combination of RT-PCR and reverse dot blot hybridization for rapid detection and identification of potyviruses. Journal of Virological Methods, 128, 54–60.
Hu, W.-C., Lin, W.-F., Huang, W.-Z., Huang, C.-H., & Chang, Y.-C. (2007). The production and application of the antiserum against Zantedeschia mosaic virus recombinant coat protein. Plant Pathology Bulletin, 16, 31–40.
Huang, C.-H., & Chang, Y.-C. (2005). Identification and molecular characterization of Zantedeschia mild mosaic virus, a new calla lily-infecting potyvirus. Archives of Virology, 150, 1221–1230.
Huang, W.-Z., Chung, F.-C., Chen, Y.-L., Huang, C.-H., & Chang, Y.-C. (2005). Development and comparison of three detection methods for calla lily-infecting Dasheen mosaic virus. Plant Pathology Bulletin, 14, 239–250.
Huang, C.-H., Hu, W.-C., Yang, T.-C., & Chang, Y.-C. (2007). Zantedeschia mild mosaic virus, a new widespread virus in calla lily, detected by ELISA, dot-blot hybridization and IC-RT-PCR. Plant Pathology, 56, 183–189.
Kuehny, J. S. (2000). Crop reports: Calla history and culture. HortTechnology, 10, 267–274.
Kwon, S. B., Ha, J. H., Yoon, J. Y., & Ryu, K. H. (2002). Zantedeschia mosaic virus causing leaf mosaic symptom in calla lily is a new potyvirus. Archives of Virology, 147, 2281–2289.
Lee, S.-C., & Chang, Y.-C. (2006). Multiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA. Plant Pathology Bulletin, 15, 187–196.
Lee, S.-C., & Chang, Y.-C. (2008). Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus. European Journal of Plant Pathology, 122, 297–306.
Menzel, W., Jelkmann, W., & Maiss, E. (2002). Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. Journal of Virological Methods, 99, 81–92.
Nie, X., & Singh, R. P. (2000). Detection of multiple potato viruses using an oligo(dT) as a common cDNA primer in multiplex RT-PCR. Journal of Virological Methods, 86, 179–185.
Nishiguchi, M., Yamasaki, S., Lu, X.-Z., Shimoyama, A., Hanada, K., Sonoda, S., et al. (2006). Konjak mosaic virus: the complete nucleotide sequence of the genomic RNA and its comparison with other potyviruses. Archives of Virology, 151, 1643–1650.
Pham, K., Langeveld, S. A., Lemmers, M. E. C., & Derks, A. F. L. M. (2002). Detection and identification of potyviruses in Zantedeschia. Acta Horticulturae, 568, 143–148.
Rana, G. L., Vovlas, C., & Zettler, F. W. (1983). Manual transmission of Dasheen mosaic virus from Richardia to nonaraceous host. Plant Disease, 67, 1121–1122.
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.
Thompson, J. R., Wetzel, S., Klerks, M. M., Vaskova, D., Schoen, C. D., Spak, 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. Journal of Virological Methods, 111, 85–93.
Zettler, F. W., & Hartman, R. D. (1987). Dasheen mosaic virus as a pathogen of cultivated aroids and control of the virus by tissue culture. Plant Disease, 71, 958–963.
Acknowledgements
This study was supported by a grant from the National Science Council (NSC96-2317-B-002-002), Executive Yuan, Taiwan, R. O. C. We thank Mr. Ming-Chung Liu (TSIPS) for kind provision of calla lily samples, and Mr. Ying-Lien Chen for preparation of DsMV cDNA clone and specific primer design.
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Hu, WC., Huang, CH., Lee, SC. et al. Detection of four calla potyviruses by multiplex RT-PCR using nad5 mRNA as an internal control. Eur J Plant Pathol 126, 43–52 (2010). https://doi.org/10.1007/s10658-009-9519-y
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DOI: https://doi.org/10.1007/s10658-009-9519-y