Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacteria and Viruses in Pepper and Tomato Seeds

고추와 토마토 종자에서 종자전염 세균 및 바이러스의 동시 검출을 위한 One-step Multiplex RT-PCR 방법

  • Jeong, Kyu-Sik (Variety Testing Division, Korea Seed and Variety Service) ;
  • Soh, Eun-Hee (Variety Testing Division, Korea Seed and Variety Service)
  • 정규식 (국립종자원 재배시험과) ;
  • 소은희 (국립종자원 재배시험과)
  • Received : 2010.12.31
  • Accepted : 2011.03.29
  • Published : 2011.04.30
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Abstract

The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc), Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV) in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R) of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

우리나라에서 주로 재배되는 가지과작물의 종자 전염 병원균 중에서 세균성 병원균 Xanthomonns campestris pv. vesicatoria(Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc)와 바이러스 병원균 Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV)를 종자로부터 동시검출하기 위한 One-step Multiplex RT-PCR을 개발하였다. 각각의 병원균을 특이적으로 증폭시키는 병원균 검출용 프라이머 15세트를 primer-blast 프로그램을 이용하여 제작하였고 각각의 병원균을 특이적으로 검출할 수 있는 5세트의 프라이머 세트(Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMVF/R)를 선발하였다. 이들 프라이머 세트는 프라이머간의 또는 병원균 cDNA간의 간섭없이 타겟 병원균만을 검출하였다. 가지과 작물 중에서 유통 중인 고추종자 20품종과 토마토종자 10품종에 대한 종자 감염 병원균 검출을 위한 PCR을 수행한 결과, 2개 품종의 토마토종자에서 Ecc가 검출되었고 4개 품종의 고추종자에서도 Ecc가 검출되었다. 특히 고추 종자에선 Ecc가 검출된 4개 품종 외에 1개 품종에서 PMMoV, TMGMV가 동시 검출되었다. 이로 인해 개발된 One-step multiplex RT-PCR은 서로 간섭 현상 없이 병원균을 효율적으로 검출할 수 있음을 보여 주었다.

Keywords

References

  1. 식물병리학 교재연구모임. 2006. 식물병리학(제5판). 월드싸이언스. pp. 633-667.
  2. Berg, T., Tesoriero, L. and Hailstones, D. L. 2006. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas. Lett. Appl. Microbiol. 42: 624-630.
  3. Cetinkaya-Yildiz, R. and Aysan, Y. 2007. A new seed-borne pathogen on tomato: Erwinia carotovora subsp. carotovora and seed treatments. ISHA Acta Horticulturae 729: III Balkan Symposium on Vegetables and Potatoes (Abstract).
  4. Cho, J. D., Kim, J. S., Lee, S. H. and Chung, B. N. 2007. Triplex Virion Capture (VC)/RT-PCR for Three Seed Transmissible Tobamoviruses of CGMMV, ZGMMV and KGMMV Occurring on Cucurbitaceae. Res. Plant Dis 13: 82-87. https://doi.org/10.5423/RPD.2007.13.2.082
  5. de Leon, L., Siverio, F. and Rodriguez, A. 2006. Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation. J. Microbiol. Methods 67: 141-149. https://doi.org/10.1016/j.mimet.2006.03.007
  6. Jones, J. B., Stall, R. E. and Jones, J. 1998. Diversity among Xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol 36: 41-58. https://doi.org/10.1146/annurev.phyto.36.1.41
  7. Kang, H. W., Kwon, S. W. and Go, S. J. 2003. PCR-based specific and sensitive detection of Pectobacterium carotovora subsp. carotovora by primers generated from a URP-PCR fingerprinting-derived polymorphic band. Plant Pathol. 52:127-133. https://doi.org/10.1046/j.1365-3059.2003.00822.x
  8. Kim, H. G. 2009. Sensitive and specific detection of Xanthomonas campestris pv. vesicatoria by PCR using pathovar-specific primers based on rhs family gene sequences. Microbiological Research 164: 36-42. https://doi.org/10.1016/j.micres.2006.11.005
  9. Kim, J. G., Taylor, K. W., Hotson, A., Keegan, M., Schmelz, E. A.and Mudgett, M. B. 2008. XopD SUMO Protease Affects Host Transcription, Promotes Pathogen Growth, and Delays Symptom Development in Xanthomonas-Infected Tomato Leaves. The Plant Cell 20: 1915-1929. https://doi.org/10.1105/tpc.108.058529
  10. Kim, J. H., Choi, G. S., Kim, J. S., Lee, S. H., Choi, J. K. and Ryu, K. H. 2006. Development of single-tube multiplex immunocapture RT-PCR Assay for simultaneous detection of two pepper Tobamoviruses. Plant Pathol. J. 22: 164-167. https://doi.org/10.5423/PPJ.2006.22.2.164
  11. Lee, S. D., Yoon, C. M., Lee, Y. K., Choi, Y. C. and Cho, Y. S. 1999. Occurrence and distribution of bacterial canker of red pepper caused by Clavibacter michiganensis subsp. michiganensis. Plant Dis. Agric. 5: 105-110.
  12. Lee, S. H. 1981. Studies on virus disease occurring in various crops in Korea. Res. Report RDA 23: 62-74.
  13. Menzel, W., Jelkmann, W. and Maiss, E. 2002. Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. J. Virol. Methods 99: 81-92. https://doi.org/10.1016/S0166-0934(01)00381-0
  14. Nolasco, G., deBlas, C., Torres, V. and Ponz, F. 1993. A method conbining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subvirial pathogenes. J. Viro. Methods 45: 201-218. https://doi.org/10.1016/0166-0934(93)90104-Y
  15. Park, D. S., Shim, J. K., Kim, J. S., Lim, C. K., Shrestha, R., Hahn, J. H., Parrella, G., Verdin, E., Gognalons, P. and Marchoux, G. 2006. Detection and characterization of tobacco mild green mosaic virus (TMGMV) large type isolate from trailing petunia in france. Commun Agric. Appl. Biol. Sci. 71: 1237-1244.
  16. Sharman, M., Thomas, J. E. and Dietzgen, R. G. 2000. Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J. Virol. Methods 89: 75-88. https://doi.org/10.1016/S0166-0934(00)00204-4
  17. Suehiro, N., Matsuda, K., Okuda, S. and Natsuaki, T. 2005. A simplified method for obtaining plant viral RNA for RT-PCR. J. Virol. Methods 125: 67-73. https://doi.org/10.1016/j.jviromet.2005.01.002