Detection of plants infected with citrus tristeza virus using serological methods in northern and southern regions of Iran
Subject Areas : Journal of plant ecophysiology
1 - استادیار گروه گیاهپزشکی، دانشکده کشاورزی و علوم پایه، واحد رودهن ، دانشگاه آزاد اسلامی ، رودهن، ایران
Keywords: Citrus tristeza virus, Diba, Western blot, serological methods,
Abstract :
Citrus tristeza virus is economically one of the most important citrus diseases in Iran. To prevent the impact of the disease in citrus growing areas, the production of virus-free plants and the removal of infected plants are key methods. In line with these goals, it is very important to obtain simple and sensitive diagnostic tools. The main goal of the present study is to develop serological methods for efficient detection of Tristeza virus in citrus cultivation areas. For this purpose, the gene encoding the coat protein (CP25 KDa) of Citrus tristeza virus was expressed in bacteria and purified through affinity chromatography. For the production of specific antibody against Tristeza virus, purified recombinant envelope protein was used to immunize rabbits, and then purification of immunoglobulin was done using protein A column. Antibodies and conjugates were made and purified, suspected plants with symptoms of citrus tristeza virus infection were collected from different parts of Mazandaran and Kerman province, ELISA, western blot and DIBA serological tests were used to identify infected plants. The results showed that the produced antibodies can be effectively used to identify plants infected with citrus tristeza virus, and out of 51 samples suspected of virus contamination, fourteen plants were definitely diagnosed with a high percentage of infection.
1- Alavi , V., Khatabi, B,. And Salekdeh, G. H. ( 2005) Comparison of biologically distinct isolates of Citrus tristeza virus from Iran using major coat protein sequences. Australasian Plant Pathology, 34, 577–582.
2- Bar-Joseph M, Filatov V, Gofman R et al. (1997) Booster immuni zation with partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA. J Virol Methods 67:19–22.
3- Bar-Joseph M, Garnsey SM, Gonsalves D et al. (1979) The use of enzyme-linked immunosorbent assay for the detection of citrus tristeza virus. Phytopathology 69:190–194.
4- Bar-Joseph M, Lee RF. Citrus tristeza virus. Descriptions of Plant Viruses No. 353 (No. 33 revised), Kew, Surrey, UK, Common- wealth Mycological Institute, 1989, 7 pp.
5- Bar-Joseph M, Malkinson M. (1980) Hen yolk as a source of antiviral antibodies in the enzyme-linked immunosorbent assay (ELISA): a comparison of two plant viruses. J Virol Methods 1:1–5.
6- Bar-Joseph M., V. Filavot, R. Gofman, Y. Guang, A. Hadjinicolis, M. Mawassi, E. Gootwine, Y. Weisman and M. Malkinson.( 1997) Booster immunization with partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA. Journal of Virological Methods 67, 19–22.
7- Barzegar A., Sohi H.H. and H. Rahimian.2005. Comparative sequence analysis of coat protein gene of Iranian Citrus tristeza virus isolates. Journal of Phytopathology, 153: 457-463.
8- Cambra M, Gorris MT, Marroquın M et al. (2000) Incidence and epidemiology of citrus tristeza virus in the Valencian community of Spain. Virus Res 71:85–95.
9- Dewey, F.M. 1992. Detection of plant invading fungi by monoclonal antibodies. In: J. M. Duncan and L. Torrance, eds. Techniques for the Rapid Detection of Plant Pathogens.
10- Febres VJ, Ashoulin L, Mawassi M et al. (1996) The p27 protein is present at one end of citrus tristeza particles. Phytopathology86:1331–1335.
11- Garnsey SM, Bar-Joseph M, Lee RF. (1981) Applications of serolog ical indexing to develop control strategies for citrus tristeza virus. Proc Intern Soc Citriculture 1:448–452.
12- Garnsey SM, Permar TA, Cambra M, Henderson CT.( 1993) Direct tissue blot immunoassay (DTBIA) for detection of citrus tristeza virus(CTV). In: Moreno P, da Graca JV, Timmer LW (eds), Proc11th Conf Intern Organ Citrus Virol, CA, USA, Riverside, pp. 39–50.
13- Gonsalves D, Purcifull DE, Garnsey SM. (1978) Purification and serology of citrus tristeza virus. Phytopathology 68:553–559.
14- Gulati-Sakhujaa, A., Searsa, J., Nunez, A., Liua, H. 2009. Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis. Journal of Virological Methods.160:29-37.
15- Iracheta-Cardenas MM, Pena del Rıo MA, Rocha-Pena MA. (2005) Comparacion de antisueros comerciales para la deteccion del virus tristeza de los cıtricos. Rev Mex Fitopatol 23:323–328.
16- Kingsnorth, C. S., Asher, M. J. C., Keane, G. J. P., Chwarszczynska, D. M., Luterbacher, M. C. and Mutasa-Göttgens, E.S. 2003. Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening. Plant Pathology, 52: 673-680.
17- Laemmli, U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
18- Manjunath K.L., H.R. Pappu, R.F. Lee, C.L. Niblett and E.L. Civerolo(1993) Studies on the coat protein genes of four isolates of citrus tristeza closterovirus from In-dia: cloning, sequencing and expression. In: Proceedings of the 12th International Organization of Citrus Virology. (P. Moreno, J.V. da Graca and L.W Timmer ed.), IOCV, Riverside, CA, USA, 20–27.
19- Marco GM, Gumpf DJ.( 1991) A simple technique for the production of highly specific polyclonal antisera for citrus tristeza virus. In Brlansky RH, Lee RF, Timmer LW (eds), Proc 11th Conf Intern Organ Citrus Virol, CA, USA, Riverside, pp. 77–81.
20- Mathew DM, Riley K, Dodds JA. (1997) Comparison of detection methods for citrus tristeza virus in field trees during months of nonoptimal titer. Plant Dis 81:525–529.
21- Mutasa-Gottgens, E.S., Chwarszczynska, D.M., Halsey, K. and Asher, M.J.C. 2000. Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach. Plant Pathology, 49: 276-287.
22- Nakane, P.K.and A. Kawaoi, 1974. Peroxidase- labeled antibody. A new method of conjugation. J. Histochem., 22: 1048- 1091.
23- Niblett CL, Genc H, Cevik B, Halbert S, Brown L, et al. (2000) Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease. Virus Research 71, 97–106. doi: 10.1016/S0168-1702(00)00191-X
24- Nikolaeva O.V., A.V. Karasev, D.J. Gumpf, R.F. Lee and S.M. Garnsey (1995) Production of polyclonal antisera to the coat protein of citrus tristeza virus expressed in Escherichia coli: application for immunodiagnosis. Phy- topathology 85, 691–694.
25- Nikolaeva OV, Karasev AV, Powell CA, Gumpf DJ, Garnsey SM, Lee RF. (1996) Mapping of epitopes for citrus tristeza virus specific monoclonal antibodies using bacterially expressed coat protein fragments. Phytopathology 86, 974–979.
26- Ozturk S, Cirakoglu B. (2003) Production of a monoclonal antibody specific for Citrus tristeza virus. J Food Agri Immunol 15:65–73
27- Rocha-Pena MA, Lee RF, Lastra R et al. (1995a) Citrus tristeza virus and its aphid vector Toxoptera citricida. Threats to citrus production in Caribbean and Central and North America. Plant Dis 79:437–445
28- Rocha-Pena MA, Lee RF, Niblett CL. (1991b) Development of a dot-immunobinding assay for citrus tristeza virus. J Virol Methods34:297–309.
29- Rocha-Pena MA, Lee RF, Permar TA, Yokomi RK, Garnsey SM.( 1991a) Use of enzyme-linked immunosorbent and dot-immunobinding assays to evaluate two cross protection experiments after challenge with a severe citrus tristeza virus isolate. In: Brlansky RH, Lee RF, Timmer LW (eds), Proc 11th Conf Intern Organ Citrus Virol, CA, USA, Riverside, pp. 93–102.
30- Rocha-Pena MA, Lee RF. (1991) Serological techniques for detection of citrus tristeza virus. J Virol Methods 34:311.
31- Sequera, Z., and Nolasco, G.(2002) Bacterial expressed coat protein: development of a single antiserum for routine detection of Citrus tristeza virus. Phytopathol. Mediterr. 41, 55–62
32- Targon MLPN, Nikolaeva O, Manjunath KL, Lee RF, Muller GW Machado MA. (1997) Coat protein gene of a Brazilian isolate of the Citrus tristeza virus: cloning, expression in E. coli and production of polyclonal antiserum. Fitopatol Bras 22:99–102.
33- Thomas, S. and Balasundaran, M. 2001. Purification of sandal spike phytoplasma for the production of polyclonal antibody. Current Science, 80: 1489-1494.
34- Vela C, Cambra M, Cortes E et al. (1986) Production and characterization of monoclonal antibodies specific for citrus tristeza virus and their use in diagnosis. J Gen Virol 67:91–96.
35- Yinghai, X., Yuzhi, H., Yazhong, X., Wei, F., 2007. Preparation and Application of Polyclonal Antibody against a Recombinant Laccase,. Cellular and Molecular Immunology 4, 315-317.