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Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

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Abstract

Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

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References

  • Acheche, H., Fattouch, S., Mhirsi, S., Marzouki, N., & Marrackchi, M. (1999). Use of optimized PCR methods for the detection of GLRaV3: a Closterovirus associated with grapevine leafroll in Tunisian grapevine plants. Plant Molecular Biology Reporter, 17, 31–42.

    Article  CAS  Google Scholar 

  • Adsule, P. G., Upadhyay, A., & Sawant, I. S. (2011). Vision 2030. National Research Centre for Grapes (Indian Council for Agricultural Research), Pune, Maharashtra phttp://nrcgrapes.nic.in/zipfiles/NRCG-Vision-2030.pdf.

  • Anfoka, G. H., Shahrour, W., & Nakhla, M. K. (2004). Detection and molecular characterization of Grapevine fanleaf virus and Grapevine leafroll-associated virus 3 in Jordan. Journal of Plant Pathology, 86, 203–207.

    CAS  Google Scholar 

  • Engel, E. A., Girardi, C., Escobar, P. F., Arredondo, V., Dominguez, C., Perez-Acle, T., et al. (2008). Genome analysis and detection of a Chilean isolate of Grapevine leafroll associated virus-3. Virus Genes, 37, 110–118.

    Article  CAS  PubMed  Google Scholar 

  • Gouveia, P., Dandlen, S., Costa, A., Marques, N., & Nolasco, G. (2012). Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3. European Journal of Plant Pathology, 133, 237–245.

    Article  CAS  Google Scholar 

  • Gugerli, P. (2009). 25 years of serological identification of grapevine leafroll-associated viruses: antiserum and monoclonal antibodies to GLRaV-1 to GLRaV-9. Extended Abstracts of 16th Meeting of ICVG (Dijon, France, pp. 24-28).

  • Habili, N., Fazeli, C., & Rezaian, M. (1997). Identification of a cDNA clone specific to Grapevine leafroll-associated virus 1, and occurrence of the virus in Australia. European Journal of Plant Pathology, 46, 516–522.

    Article  CAS  Google Scholar 

  • Jooste, A. E. C., Maree, H. J., Bellstedt, D., Goszczynski, D. E., Pietersen, G., & Burger, J. T. (2010). Three genetic Grapevine leafroll-associated virus-3 (GLRaV-3) variants identified from South African vineyards show high variability in their 5’UTR. Archives of Virology, 155, 1997–2006.

    Article  CAS  PubMed  Google Scholar 

  • Kumar, S., Baranwal, V. K., Singh, P., Jain, R. K., Sawant, S. D., & Singh, S. K. (2012). Characterization of a Grapevine leafroll-associated virus 3 from India showing incongruence in its phylogeny. Virus Genes, 45, 195–200.

    Article  CAS  PubMed  Google Scholar 

  • Kumar, S., Singh, L., Ferretti, L., Barba, M., Zaidi, A. A., & Hallan, V. (2013). Evidence of Grapevine leafroll associated virus-1–3, Grapevine fleck virus and Grapevine virus B occurring in Himachal Pradesh, India. Indian Journal of Virology, 24, 66–69.

    Article  PubMed Central  PubMed  Google Scholar 

  • Ling, K. S., Zhu, H. Y., Jiang, Z. Y., & Gonsalves, D. (2000). Effective application of DAS-ELISA for detection of Grapevine leafroll associated closterovirus-3 using a polyclonal antiserum developed from recombinant coat protein. European Journal of Plant Pathology, 106, 301–309.

    Article  CAS  Google Scholar 

  • Ling, K. S., Zhu, H. Y., Petrovic, N., & Gonsalves, D. (2001). Comparative effectiveness of ELISA and RT-PCR for detecting Grapevine leafroll-associated Closterovirus-3 in field samples. American Journal of Enology and Viticulture, 52, 21–27.

    CAS  Google Scholar 

  • Mackenzie, D. J., McLean, M. A., Mukerji, S., & Green, M. (1997). Improved RNA extraction from woody plants for the detection of viral pathogens by reverse transcription-polymerase chain reaction. Plant Disease, 81, 222–226.

    Article  CAS  Google Scholar 

  • Martelli, G. P., AbouGhanem-Sabanadzovic, N., Agranovsky, A. A., Al Rwahnih, M., Dolja, V. V., Dovas, C. I., et al. (2012). Taxonomic revision of the family Closteroviridae with special reference to the grapevine leafroll-associated members of the genus Ampleovirus and the putative species unassigned to the family. Journal of Plant Pathology, 94, 7–19.

    Google Scholar 

  • Minafra, A., & Hadidi, A. (1994). Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification. Journal of Virological Methods, 47, 175–187.

    Article  CAS  PubMed  Google Scholar 

  • Mulholland, V. (2009). Immunocapture-PCR for plant virus detection. Methods in Molecular Biology, 508, 183–192.

    CAS  PubMed  Google Scholar 

  • Rayapati, A. N., O’Neil, S., & Walsh, D. (2008). Grapevine leafroll disease. Washington State University Extension Bulletin EB2027E.p:1-20. http://cru.cahe.wsu.edu/CEPublications/eb2027e/eb2027e.pdf

  • Rowhani, A., Biardi, L., Johnson, R., Saldarelli, P., Zhang, Y. P., Chin, J., et al. (2000). Simplified sample preparation method and one-tube RT-PCR for grapevine viruses. Extended Abstracts of 13 th Meeting of ICVG (Adelaide, Australia, p.148).

  • Sefc, K. M., Leonhardt, W., & Steinkellner, H. (2000). Partial sequence identification of grapevine-leafroll-associated virus-1 and development of a highly sensitive IC-RT-PCR detection method. Journal of Virological Methods, 86, 101–106.

    Article  CAS  PubMed  Google Scholar 

  • Shaughnessy, B., (2012). Grape disease management. http://www.grapeheaven.com/learn/Grape-Disease-Management-i129.aspx

  • Viswanathan, R., Ganesh Kumar, V., Karuppaiah, R., Scindiya, M., & Chinnaraja, C. (2013). Development of Duplex-Immunocapture (Duplex-IC) RT-PCR for the detection of Sugercane streak mosaic virus and Sugercane mosaic virus in sugarcane. Sugar Technology, 15, 399–405.

    Article  CAS  Google Scholar 

  • Wetzel, T., Candress, T., Macquaire, G., Ravelonandro, M., & Dunez, J. (1992). A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection. Journal of Virological Methods, 39, 27–37.

    Article  CAS  PubMed  Google Scholar 

  • Zein, H. S., Hussein, M. H., Hussein, H. A., & Miyatake, K. (2008). The utilization of monoclonal antibodies in immunocapture RT-PCR and dot blotting immunobinding assays for the detection of Cucumber mosaic virus. Arab Journal of Biotechnology, 46, 95–106.

    Google Scholar 

  • Zimmermann, D., Bass, P., Legin, R., & Walter, B. (1990). Characterization and serological detection of four closterovirus-like particles associated with leafroll disease on grapevine. Journal of Phytopathology, 130, 205–218.

    Article  Google Scholar 

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Acknowledgment

Sandeep Kumar would like to acknowledge the assistance of the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India, for providing an INSPIRE (Innovation in Science Pursuit for Inspired Research) fellowship for his Ph.D. program.

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Author notes

  1. Sandeep Kumar

    Present address: All India Coordinated Research Project on MULLaRP, Centre for Pulses Research, Berhampur, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India

Authors and Affiliations

  1. Plant Virology Unit, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Sandeep Kumar, Richa Rai & V. K. Baranwal

Authors
  1. Sandeep Kumar
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  2. Richa Rai
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  3. V. K. Baranwal
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Correspondence to V. K. Baranwal.

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Kumar, S., Rai, R. & Baranwal, V.K. Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3). Phytoparasitica 43, 311–316 (2015). https://doi.org/10.1007/s12600-014-0445-y

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