USE OF RT-PCR TECHNIQUES IN DETECTION OF VIRUSES OF PEPPER IN REPUBLIC OF MACEDONIA
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Published:
Dec 1, 2017
Keywords:
molecular detection, CMV, AMV, PVY
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Abstract
Plant viruses can be a limiting factor and cause economically significant damage in the agricultural production. That is why, early and precise detection is very important in prevention of significant yield losses. In this study, early detection of plant viruses was performed on pepper, as one of the economically most important crops in Republic of Macedonia. Pepper plants were tested for Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV) and Potato virus Y (PVY), most common RNA viruses in pepper production. Virus antigen accumulation was measured with the use of DAS-ELISA method on 259 plant samples. CMV was detected as most prevalent virus on pepper (51% in 2012, 34% in 2013 and 61% in 2014). In 2012, AMV was detected as second, while in 2013 and 2014 was PVY. Detection was performed using Reverse Transcription – Polymerase Chain Reaction (RT-PCR).
The RNA was extracted using TRIzol method from the collected leaf samples. RT-PCR was performed for each of the viruses separately due to the differences in the annealing temperatures of the used primers. In order to prevent RNA degradation of the plant viruses, each step of manipulation with the plant material was carefully optimized. The gained amplicons after RT-PCR, associated with different viruses were: for CMV the amplicon was 773 bp, for AMV was 669 bp, while for PVY 902 bp, respectively. The RT-PCR method exhibited high sensitivity, detecting the viruses in the early stages of infection, before observation of any visible symptoms on the pepper plants and measuring negative OD absorbance by DAS-ELISA.
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1. Alonso-Prados, J. L., Luis-Arteaga, M., Alvarez, J. M., Moriones, E., Batlle, A., Laviña, A., García- Arenal, F. and Fraile, A. (2003). Epidemics of aphid-transmitted viruses in melon crops in Spain. European Journal of Plant Pathology, 109: 129–138.
2. Bertolini, E., Olmos, A., López, M. M. and Cambra, M. (2003). Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology, 93:286-292.
3. Chatzivassiliou, E. K., Efthimiou, K., Drossos, E., Papadopoulou, A., Poimenidis, G. and Katis, N. I. (2004). A survey of tobacco viruses in tobacco crops and native flora in Greece. European Journal of Plant Pathology, 110: 1011–1023.
4. Chen, S., Gu, H., Wang, X., Chen, J. and Zhu, W. (2011). Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control. Acta Biochim Biophys Sin, 43: 465–471.
5. Choi, G.S., Kim, J.H., Lee, H.D., Kim, J.S., Ryu, K.H. (2005). Occurrence and distribution of viruses infecting pepper in Korea. Plant Pathol. J, 21(3): 258-261.
6. Clark, M.F. and Adams, A.N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of the plant viruses. J. Gen. Virology, 34 (3): 457-483.
7. Deyong, Z., Willingmann, P., Heinze, C., Adam, G., Pfunder, M., Frey, B., Frey, J. E. (2005). Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format. Journal of Virological Methods, 123: 101-108.
8. de Wijs, J. J., Suda-Bachmann, F. (1979). The long-term preservation of potato virus Y and watermelon mosaic virus in liquid nitrogen in comparison to other preservation methods. Neth. J. Pl. Path., 85: 23-29.
9. FAO. (2015). http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor
10. Fidan, H. and Güllü, M. (2014). Newly report of Cucumber mosaic virus (CMV) in caper bush, Capparis spinosa (L.) (Capparacaea) in Turkey. Scientific conference “Challenges in modern agricultural production”, Institute of Agriculture – Skopje, Skopje, R. Macedonia
11. Jankulovski, D. (1997). Pepper and tomato. NIP “BAS – TRADE”, Skopje, R. Macedonia.
12. Jaspars, E. M. J. and Bos, L. (1980). Alfalfa mosaic virus CMI/AAB description of plant viruses, No. 229. http://www.dpvweb.net/dpv/showdpv.php?dpvno=229.
13. Jovanchev, P., Pejchinovski, F., Jankulovski, D., Rusevski, R., Bandzo, S. and Popsimonova, G. (1996). Health status of pepper plants in the Republic Macedonia in 1995. Annual Book of Proceedings of Plant Protection, 7: 159-169.
14. Kim, M.S., Kim, M.J., Hong, J.S., Choi, J.K. and Ryu, K.H. (2010). Patterns in disease progress and the influence of single and multiple viral infections on pepper (Capsicum annuum L.) growth. Eur J Plant Pathol, 127: 53–61.
15. Lee, B. Y. and Ryu, K. H. (2009). Incidence of virus diseases and RT-PCR detection of Daphne-infecting viruses in Korea. Eur J Plant Pathol, 124: 127–132.
16. Llave, C., Martínez, B., Díaz-Ruíz, J. R. and López-Abella, D. (1999). Serological analysis and coat protein sequence determination of potato virus Y (PVY) pepper pathotypes and differentiation from other PVY strains. European Journal of Plant Pathology, 105: 847-857.
17. Miloševic, D. (2013). Diversity and characterization of pepper viruses in Serbia. Ph.D. Thesis. Faculty of Agriculture, University in Belgrade, Belgrade, Serbia.
18. Nair, R. M., Habili, N. and Randles, J. W. (2009). Infection of Cullen australasicum (syn. Psoralea australasica) with Alfalfa mosaic virus. Australasian Plant Disease Notes, 4: 46–48.
19. Nie, X. and 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.
20. Niimi, Y., Han, D.-S., Mori, S. and Kobayashi, H. (2003). Detection of cucumber mosaic virus, lily symptomless virus and lily mottle virus in Lilium species by RT-PCR technique. Scientia Horticulturae, 97 (1): 57–63.
21. Ormeño, J., Sepúlveda, P., Rojas, R. and Araya, J.E. (2006). Datura Genus Weeds as an Epidemiological Factor of Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), and Potato virus Y (PVY) on Solanaceus Crops. AgriculturaTécnica (Chile), 66 (4): 333-341.
22. Popovski, Z.T., Dimitrievska, B., Miskoska – Milevska, E. and Bandzo, K. (2013). PCR based methods – Manuel. University “Ss. Cyril and Methodius” – Skopje, Faculty of agricultural sciences and food, Skopje, R. Macedonia.
23. Raj, S. K., Srivastava, A., Chandra, G. and Singh, B. P. (2002). Characterization of cucumber mosaic virus isolate infecting Gladiolus cultivars and comparative evaluation of serological and molecular methods for sensitive diagnosis. Current Science, 83 (9): 1132-1137.
24. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Gjamovski, V. (2009). Virus status of pepper plants in the area around Kochani in 2008. Plant protection, Skopje, 20: 84-88.
25. Rusevski, R., Bandzo, K., Kuzmanovska, B., Sotirovski, K. and Risteski, M. (2013). Occurrence, distribution and dynamics of virus antigen accumulation in pepper cultivation on open fields in Republic of Macedonia during 2008-2009. African Journal of Agricultural Research, 8 (28): 3836-3841.
26. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Stanoevska, M. (2010). Virus status of pepper plants cultivated on open fields in R. Macedonia in 2009. Plant protection, Skopje, 21: 44-50.
27. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Stanoevska, M. (2011). Virus status of pepper plants cultivated on open fields. In: XVI International meeting on Biotechnology – Book of Proceedings, Čačak, Serbia, 2011: 429-434.
28. Rusevski, R. and Bandzo, S. (1998). Influence of some microclimate characteristics (temperature and humidity) on the aphid population and virus infections on pepper in green houses. Annual Book of Proceedings of Plant Protection, Skopje, 9: 141-159.
29. van Dongen, J. J., Macintyre, E. A., Gabert, J. A., Delabesse, E., Rossi, V., Saglio, G. Gottardi, E., Rambaldi, A., Dotti, G., Griesinger, F., Parreira, A., Gameiro, P., Diáz, M. G., Malec, M., Langerak, A. W., San Miguel, J. F. and Biondi, A. (1999). Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia, 12: 1901–1928
30. Vučurovic, A., Bulajic, A., Stankovic, I., Ristic, D., Berenji, J., Jovic, J. and Krstic, B. (2010). Occurrence, molecular detection and identification of Cucumber mosaic virus infecting pumpkin and squash in Serbia. In: 10th Meeting on plant protection, Zlatibor, 2010: 58-59.
31. Wang, P., Guo, Y., Cheng, J.-H., Dong, Q.-F., Ding, X.-F., Guo, J.-F. and Jiang, Y.-H. (2012). Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples. Eur J Plant Pathol, 132: 217–227.
32. Wernli, B. (1999). Bioreba AG., Xu, H. and Nie, J. (2006). Identification, characterization, and molecular detection of Alfalfa mosaic virus in potato. Phytopathology, 96 (11): 1237-1242.
34. Yu, C., Wu, J. and Zhou, X. (2005). Detection and subgrouping of Cucumber mosaic virus isolates by TAS–ELISA and immunocapture RT–PCR. Journal of Virological Methods, 123: 155–161.
35. Šutic, D. (1995). Plant viruses. Institute for plant protection and environment, Belgrade: 1-394.
2. Bertolini, E., Olmos, A., López, M. M. and Cambra, M. (2003). Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology, 93:286-292.
3. Chatzivassiliou, E. K., Efthimiou, K., Drossos, E., Papadopoulou, A., Poimenidis, G. and Katis, N. I. (2004). A survey of tobacco viruses in tobacco crops and native flora in Greece. European Journal of Plant Pathology, 110: 1011–1023.
4. Chen, S., Gu, H., Wang, X., Chen, J. and Zhu, W. (2011). Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control. Acta Biochim Biophys Sin, 43: 465–471.
5. Choi, G.S., Kim, J.H., Lee, H.D., Kim, J.S., Ryu, K.H. (2005). Occurrence and distribution of viruses infecting pepper in Korea. Plant Pathol. J, 21(3): 258-261.
6. Clark, M.F. and Adams, A.N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of the plant viruses. J. Gen. Virology, 34 (3): 457-483.
7. Deyong, Z., Willingmann, P., Heinze, C., Adam, G., Pfunder, M., Frey, B., Frey, J. E. (2005). Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format. Journal of Virological Methods, 123: 101-108.
8. de Wijs, J. J., Suda-Bachmann, F. (1979). The long-term preservation of potato virus Y and watermelon mosaic virus in liquid nitrogen in comparison to other preservation methods. Neth. J. Pl. Path., 85: 23-29.
9. FAO. (2015). http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor
10. Fidan, H. and Güllü, M. (2014). Newly report of Cucumber mosaic virus (CMV) in caper bush, Capparis spinosa (L.) (Capparacaea) in Turkey. Scientific conference “Challenges in modern agricultural production”, Institute of Agriculture – Skopje, Skopje, R. Macedonia
11. Jankulovski, D. (1997). Pepper and tomato. NIP “BAS – TRADE”, Skopje, R. Macedonia.
12. Jaspars, E. M. J. and Bos, L. (1980). Alfalfa mosaic virus CMI/AAB description of plant viruses, No. 229. http://www.dpvweb.net/dpv/showdpv.php?dpvno=229.
13. Jovanchev, P., Pejchinovski, F., Jankulovski, D., Rusevski, R., Bandzo, S. and Popsimonova, G. (1996). Health status of pepper plants in the Republic Macedonia in 1995. Annual Book of Proceedings of Plant Protection, 7: 159-169.
14. Kim, M.S., Kim, M.J., Hong, J.S., Choi, J.K. and Ryu, K.H. (2010). Patterns in disease progress and the influence of single and multiple viral infections on pepper (Capsicum annuum L.) growth. Eur J Plant Pathol, 127: 53–61.
15. Lee, B. Y. and Ryu, K. H. (2009). Incidence of virus diseases and RT-PCR detection of Daphne-infecting viruses in Korea. Eur J Plant Pathol, 124: 127–132.
16. Llave, C., Martínez, B., Díaz-Ruíz, J. R. and López-Abella, D. (1999). Serological analysis and coat protein sequence determination of potato virus Y (PVY) pepper pathotypes and differentiation from other PVY strains. European Journal of Plant Pathology, 105: 847-857.
17. Miloševic, D. (2013). Diversity and characterization of pepper viruses in Serbia. Ph.D. Thesis. Faculty of Agriculture, University in Belgrade, Belgrade, Serbia.
18. Nair, R. M., Habili, N. and Randles, J. W. (2009). Infection of Cullen australasicum (syn. Psoralea australasica) with Alfalfa mosaic virus. Australasian Plant Disease Notes, 4: 46–48.
19. Nie, X. and 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.
20. Niimi, Y., Han, D.-S., Mori, S. and Kobayashi, H. (2003). Detection of cucumber mosaic virus, lily symptomless virus and lily mottle virus in Lilium species by RT-PCR technique. Scientia Horticulturae, 97 (1): 57–63.
21. Ormeño, J., Sepúlveda, P., Rojas, R. and Araya, J.E. (2006). Datura Genus Weeds as an Epidemiological Factor of Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), and Potato virus Y (PVY) on Solanaceus Crops. AgriculturaTécnica (Chile), 66 (4): 333-341.
22. Popovski, Z.T., Dimitrievska, B., Miskoska – Milevska, E. and Bandzo, K. (2013). PCR based methods – Manuel. University “Ss. Cyril and Methodius” – Skopje, Faculty of agricultural sciences and food, Skopje, R. Macedonia.
23. Raj, S. K., Srivastava, A., Chandra, G. and Singh, B. P. (2002). Characterization of cucumber mosaic virus isolate infecting Gladiolus cultivars and comparative evaluation of serological and molecular methods for sensitive diagnosis. Current Science, 83 (9): 1132-1137.
24. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Gjamovski, V. (2009). Virus status of pepper plants in the area around Kochani in 2008. Plant protection, Skopje, 20: 84-88.
25. Rusevski, R., Bandzo, K., Kuzmanovska, B., Sotirovski, K. and Risteski, M. (2013). Occurrence, distribution and dynamics of virus antigen accumulation in pepper cultivation on open fields in Republic of Macedonia during 2008-2009. African Journal of Agricultural Research, 8 (28): 3836-3841.
26. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Stanoevska, M. (2010). Virus status of pepper plants cultivated on open fields in R. Macedonia in 2009. Plant protection, Skopje, 21: 44-50.
27. Rusevski, R., Bandzo, K., Kuzmanovska, B. and Stanoevska, M. (2011). Virus status of pepper plants cultivated on open fields. In: XVI International meeting on Biotechnology – Book of Proceedings, Čačak, Serbia, 2011: 429-434.
28. Rusevski, R. and Bandzo, S. (1998). Influence of some microclimate characteristics (temperature and humidity) on the aphid population and virus infections on pepper in green houses. Annual Book of Proceedings of Plant Protection, Skopje, 9: 141-159.
29. van Dongen, J. J., Macintyre, E. A., Gabert, J. A., Delabesse, E., Rossi, V., Saglio, G. Gottardi, E., Rambaldi, A., Dotti, G., Griesinger, F., Parreira, A., Gameiro, P., Diáz, M. G., Malec, M., Langerak, A. W., San Miguel, J. F. and Biondi, A. (1999). Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia, 12: 1901–1928
30. Vučurovic, A., Bulajic, A., Stankovic, I., Ristic, D., Berenji, J., Jovic, J. and Krstic, B. (2010). Occurrence, molecular detection and identification of Cucumber mosaic virus infecting pumpkin and squash in Serbia. In: 10th Meeting on plant protection, Zlatibor, 2010: 58-59.
31. Wang, P., Guo, Y., Cheng, J.-H., Dong, Q.-F., Ding, X.-F., Guo, J.-F. and Jiang, Y.-H. (2012). Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples. Eur J Plant Pathol, 132: 217–227.
32. Wernli, B. (1999). Bioreba AG., Xu, H. and Nie, J. (2006). Identification, characterization, and molecular detection of Alfalfa mosaic virus in potato. Phytopathology, 96 (11): 1237-1242.
34. Yu, C., Wu, J. and Zhou, X. (2005). Detection and subgrouping of Cucumber mosaic virus isolates by TAS–ELISA and immunocapture RT–PCR. Journal of Virological Methods, 123: 155–161.
35. Šutic, D. (1995). Plant viruses. Institute for plant protection and environment, Belgrade: 1-394.