Multiplex RT-PCR Assay for Crinivirus Detection Using RNA Prepared from Three Extraction Methods on Tomato Plant

https://doi.org/10.22146/jpti.36558

Esti Prasetya Ningrum(1*), Sedyo Hartono(2), Sri Sulandari(3), Susamto Somowiyarjo(4)

(1) Agricultural Quarantine Centre of Tanjung Priok, Agricultural Quarantine Agency Jln. Enggano No.17, Tanjung Priok, North Jakarta 14310
(2) Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada Jln. Flora No. 1, Bulaksumur, Sleman,Yogyakarta 55281
(3) Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada Jln. Flora No. 1, Bulaksumur, Sleman,Yogyakarta 55281
(4) Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada Jln. Flora No. 1, Bulaksumur, Sleman,Yogyakarta 55281
(*) Corresponding Author

Abstract


Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) are members of the Crinivirus genus that induces yellowing symptoms in tomato plants. Detection of both viruses is generally carried out singly, thus it is necessary to develop a fast, accurate and efficient detection method to detect multiple viruses simultaneously in an effort to determine the suitable disease management strategies. This study was aimed to detect both viruses using the multiplex RT-PCR method and evaluate three methods of total RNA preparation used from tomato plants as RT-PCR templates. The methods evaluated were simple direct tube (SDT), simple dsRNA, and commercial kit (RNeasy Plant Mini Kit) as a comparison. The total source of RNA came from Crinivirus symptomatic tomato leaves from Kopeng, and Ketep (Central Java); Pakem (Yogyakarta); Malang (East Java); and Bogor (West Java). Single RT-PCR and multiplex RT-PCR using specific primers CPd I/CPd II and ToCV CF/ToCV CR with DNA band targets of 760 bp and 360 bp. The SDT and dsRNA methods have been successful in obtaining total RNA and viral RNA from tomato leaf samples. Total RNA RT-PCR with simple SDT and dsRNA methods followed by multiplex RT-PCR produces specific DNA band intensities that are comparable to Kit. RNA preparation with SDT and simple dsRNA methods is a simple, fast, easy and affordable method in providing templates for RT-PCR. Multiplex RT-PCR technique using two pairs of specific primers CPd I/CPd II and ToCV CF/ToCV CR is suitable for simultaneous testing of TICV and ToCV.


Keywords


dsRNA; multiplex RT-PCR; SDT; TICV; ToCV

Full Text:

PDF


References

Adiputra, J., S.H. Hidayat, & T.A. Damayanti. 2012. Evaluasi Tiga Metode Preparasi RNA Total untuk Deteksi Turnip mosaic potyvirus dari Benih Brassica rappa dengan Reverse Transcription-Polymerase Chain Reaction. Jurnal Fitopatologi Indonesia 8: 44-49.

Agrios, G.N. 2005. Plant Pathology. Fifth Edition. Elsevier Academic Press, New York. 922 p.

Amanda, U.D. & I.C, Cartealy. 2015. Isolasi RNA Total dari Mesokarp Buah Kelapa Sawit (Elaeis guineenssis Jacq. var. Tenera). Prosiding Semimar Nasional Masyarakat Biodiversitas Indonesia 1: 171-176.

Claros, M.G. & F.M. Canovas. 1999. RNA Isolation from Plant Tissues: A Practical Experience for Biological Undergraduates. Biochemical Education 27: 110-113.

Damayanti, T.A. & L.K. Putra. 2012. Preparasi RNA Virus Mosaik Bergaris dari Tanaman Tebu Menggunakan Metode Tabung PCR. Jurnal Fitopatologi Indonesia 8: 22-27.

Diningsih, E., G. Suastika, T.A. Damayanti & S. Susanto . 2017. Deteksi Cepat Carnation mottle virus pada Tanaman Anyelir (Dianthus caryophyllus L.). Jurnal Hortikultura 27: 95-104.

Dovas C.I., N.I. Katis, & A.D. Avgelis. 2002. Multiplex Detection of Crinivirus Associated with Epidemics of a Yellowing Disease of Tomato in Greece. Plant Disease 86: 1345– 349.

Duffus J.E., H.Y. Liu, & G.C. Wisler. 1996. Tomato infectious chlorosis virus-a new clostero-like virus transmitted by Trialeurodes vaporariorum. European Journal of Plant Pathology 102: 219–226.

Endarsih, W., S. Hartono, & S. Sulandari. 2017. Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan. Jurnal Perlindungan Tanaman Indonesia 21: 106-113.

[EPPO] European and Mediterranean Plant Protection Organization. 2005. Tomato chlorosis crinivirus. Bulletin EPPO 35: 439–441.

Fajarfika, R., S. Hartono, S. Sulandari, & S. Somowiyarjo. 2015. Deteksi Molekuler Penyebab penyakit Kuning (Tomato chlorosis virus dan Tomato infectious chlorosis virus) pada Tanaman Tomat. Jurnal Perlindungan Tanaman Indonesia 19: 80-88.

Fauquet, C.M., M.A Mayo, J. Maniloff, U. Desselberger & L.A Ball. 2005. Virus Taxonomy: Eight Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press. San Diego. CA. USA. 1162p.

Hartono, S., Natsuaki, T., Sayama, H., Atarashi, H. & Okuda, S. 2003. Yellowing Disease of Tomatoes Caused by Tomato infectious chlorosis virus Newly Recognized in Japan. Journal of General Plant Pathology 69: 61−64.

Hartono, S. & A. Wijonarko. 2007. Karakterisasi Biologi Molekuler Tomato infectious chlorosis virus Penyebab Penyakit Kuning pada Tanaman Tomat di Indonesia. Agricultural Science 9: 139−146.

Hirota, T., T. Natsuaki, T. Murai, H. Nishigawa, K. Niibori, K. Goto, S. Hartono, G. Suastika, & S. Okuda. 2010. Yellowing Disease of Tomato Caused by Tomato chlorosis virus Newly Recognized in Japan. Journal of General Plant Pathology 76: 168–171.

Jacquemond, M., A. Dalmont, F. Fabre, L. Guilbaud, & H. Lecoq. 2009. Comparative Whitefly Transmission of Tomato chlorosis virusand Tomato infectious chlorosis virus from Single or Mixed Infectious. Journal Plant Pathology 58: 221-227.

James, D. 1999. A Simple and Reliable Protocol for Detection of Apple Stem Grooving by RT-PCR and in a Multiplex PCR Assay. Journal of Virology Methods 83: 1-9.

Kusumaningrum, F., S. Hartono, & S. Sulandari. 2015. Infeksi Ganda Begomovirus dan Crinivirus pada Tanaman Tomat di Kabupaten Magelang, Jawa Tengah. Jurnal Perlindungan Tanaman Indonesia 19: 60-64.

Lefeurve, P., M. Hoareau, H. H. delatte, B. Reynaud, & J-M. Lett. 2007. A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of Tomato yellow leaf curl virus. Journal of Virological Method. 144 : 165-168.

Li, R.H., G.C. Wisler, H.-Y. Liu, & J.E. Duffus. 1998. Comparison of Diagnostic Techniques for Detecting Tomato infectious chlorosis virus. Plant Disease 82: 84–88.

Martelli, G.P., A.A. Agranovsky, & M. Bar-Joseph. 2002. The family Closteroviridae revised. Archives of Virology 147: 2039-2044.

Miftakhurohman. 2013. Identifikasi dan Deteksi Multiplex Reverse Transcripton Polymerase Chain Reaction Virus-Virus Penyebab Gejala Mosaik pada Nilam (Pogostemon cablin Benth.). Tesis. Insitut Pertanian Bogor, Bogor. 51 hlm.

Navas-Castillo J., R. Camero, M. Bueno, & E. Moriones. 2000. Severe Yellowing Outbreaks in Tomato in Spain Associated with Infections of Tomato chlorosis virus. Plant Disease 84: 835-837.

Nurulita. S. & G. Suastika. 2013. Identifikasi Tomato infectious chlorosis virus and Tomato chlorosis virus melalui Reverse Transcription Polymerase Chain Reaction dan Analisis Sikuen Nukleotida. Jurnal Fitopatologi Indonesia 9: 107-115.

Papayiannis, L.C., Iacovides, T.A., Katis, N.I., & Brown, J.K. 2010. Differentiation of Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus using realtime TaqMan® PCR. J. Virol. Methods. 165, 236–245.

Qiagen. 2012. RNeasy Mini Handbook. Sample and Assay Technologies. Germany. 78 p.

Suehiro, N., K. Matsuda, S. Okuda & T. Natsuaki. 2005. A Simplified Method for Obtaining Plant Viral RNA for RT-PCR. Journal of Virological Methods 125: 67-73.

Temaja, I.G.R.M., N.M.P. Puspawati, & N.N.A. Mayadewi. 2012. Utilization of SDT-RT- PCR for Plant Virus Detection. Journal of Agricultural Science and Biotechnology 1: 24-29.

Tian, T., V.A. Klaassen, J. Soong, G. Wisler, J.E. Duffus, & B.W. Falk. 1996. Generation of cDNAs Specific to Lettuce Infectious Yellows Closterovirus and Other Whitefly-Transmitted Viruses by RT-PCR and Degenerate Oligonucleotide Primers Corresponding to the Closterovirus Gene Encoding the Heat Shock Protein 70 Homolog. Phytopathology 86: 1167– 1173.

Vaira. A.M., G.P. Accotto, M. Vecchiati, & M. Bragaloni. 2002. Tomato infectious chlorosis virus Causes Lenurulitaaf Yellowing and Reddening of Tomato in Italy. Phytoparasitica 30: 290-294.

Viljoen, G.J., H. Nell & J.R Crowther. 2005. Molecular Diagnostic PCR Handbook. IAEA-
FAO, Springer, Dordrecht, The Netherlands. 325p.

Wintermantel, W.M. 2004. Emergence of Greenhouse Whitefly (Trialeurodes vaporariorum) Transmitted Criniviruses as Threats to Vegetable and Fruit Production in North America. APSnet feature. http://www.apsnet.org/online/feature/whitefly/, modified 2/2/2018.


Wintermantel, W.M., G. C. Wisler, A. G. Anchieta, H.-Y. Liu, A. V. Karasev, & I. E. Tzanetakis. 2005. The Complete Nucleotide Sequence and Genome Organization of Tomato chlorosis virus. Archives of Virology 150: 2287-2298.

Wintermantel, W.M. & G.C. Wisler. 2006. Vector Specificity, Host Range, and Genetic Diversity of Tomato chlorosis virus. Plant Disease 90: 814–819.

Wisler, G.C., H.-Y. Liu, V.A. Klassen, J.E. Duffus dan B.W. Falk. 1996. Tomato Infectious Chlorosis Virus Has a Bipartite Genome and Induce Phloem-Limited Inclusions Characteristic of the Closteroviruses. Phytopathology 86: 623-626.

Yang L, Wang C., Wang L., Xu C., & Chen K. 2013. An Efficient Multiplex PCR Assay for Early Detection of Agrobacterium tumefaciens in Transgenic Plant Material. Turkish Journal of Agriculture and Forestry 37: 157-162.

Zou,Y., M.G Mason,Y. Wang, E. Wee, C. Turni, & P.J Blackall. 2017. Nucleic acid purification from plants, animals and microbes in under 30 seconds. PLoS Biol. 15 (11): e2003916.https://doi. org/10.1371/journal.pbio.2003916.



DOI: https://doi.org/10.22146/jpti.36558

Article Metrics

Abstract views : 644 | views : 373

Refbacks

  • There are currently no refbacks.




Copyright (c) 2019 Jurnal Perlindungan Tanaman Indonesia

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

 height=auto

Jurnal Perlindungan Tanaman Indonesia ISSN 1410-1637 (print)ISSN 2548-4788 (online) Accredited Journal, Based on Decree of the Minister of Research, Technology and Higher Education, Republic of Indonesia Number 30/E/KPT/2018, Vol. 21 No. 1 the Year 2017 - Vol. 25 No. 1 the Year 2021Indexing & Abstracting , 

View My Stats