Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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PCR Detection Method for Rapid Diagnosis of Bacterial Canker Caused by Clavibacter michiganensis on Tomato

토마토 궤양병 신속 진단을 위한 Clavibacter michiganensis의 PCR 검출법

  • Park, Mi-Jeong (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science) ;
  • Back, Chang-Gi (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science) ;
  • Park, Jong-Han (Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science)
  • 박미정 (농촌진흥청국립원예특작과학원원예특작환경과) ;
  • 백창기 (농촌진흥청국립원예특작과학원원예특작환경과) ;
  • 박종한 (농촌진흥청국립원예특작과학원원예특작환경과)
  • Received : 2018.10.19
  • Accepted : 2018.12.06
  • Published : 2018.12.31
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Abstract

Bacterial canker caused by Clavibacter michiganensis is considered to be one of the most serious diseases, leading to economic damage to tomato worldwide. Diagnosis of the bacterial canker on tomato is known to be difficult because the causal pathogen is slow-growing on artificial media as well as causes latent infection in tomato. In this study, as a less time-consuming method, a specific primer set was newly designed for rapid detection of C. michiganensis. The method presented here is so simple, easy, and fast that it can be useful and practical in direct detection of the bacterial canker pathogen from tomato plants.

Clavibacter michiganensis는 토마토에 궤양병을 일으키는 식물병원성 세균으로 인공배지에서 자라는 속도가 매우 느리기 때문에 감염조직으로부터 병원균을 분리 배양하는 방법을 통해서는 진단하기가 쉽지 않다. 또한 토마토 궤양병균은 식물체 내에서 오랜 잠복기를 거친 후에 병징을 나타내기 때문에 방제하기 어려운 세균병 중에 하나이므로 발병 시 신속한 진단을 통해 빠른 방제가 이루어져야 한다. 본 연구에서는 토마토 궤양병균의 검출을 위한 특이 프라이머를 제작함으로써 감염 식물체의 direct PCR을 통해 토마토 궤양병에 대한 빠르고 정확한 진단이 가능하도록 하였다. 새로 개발된 CmmF와 CmmR 프라이머 세트로 PCR을 수행했을 때, 토마토 궤양병균의 16-23S ribosomal RNA intergenic spacer 영역에서 약 165 bp의 단일 밴드가 특이적으로 증폭되었다. 반면에 토마토 궤양병균과 유연관계에 있는 고추 궤양병균이나 다른 Clavibacter 종 세균에서는 전혀 증폭되지 않는 것을 확인할 수 있었다. 이 방법은 감염 식물체로부터 DNA를 추출하지 않더라도 감염조직의 즙액에서 바로 토마토 궤양병균의 DNA 증폭이 가능하고 총 진단시간을 줄일 수 있다는 이점이 있기 때문에 토마토 궤양병의 진단에 유용하게 사용될 수 있을 것으로 판단된다.

Keywords

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Fig. 1. Clavibacter michiganensis-specific PCR using the CmmF/CmmR primer pair. PCR was carried out under condition of 95oC, 3 min predenaturation / 95 oC, 5 sec; 66 oC, 10 sec; 72 oC, 10 sec (35 cycles) / 72 oC, 5 min final extension. Ten out of ten strains of C. michiganensis (lane 1 to lane 10) were amplified. Lane M: 100 bp plus DNA ladder, Lane 1: C. michiganensis 12-019, Lane 2: C. michiganensis 12-031, Lane 3: C. michiganensis 13-065, Lane 4: C. michiganensis 14-132, Lane 5: C. michiganensis 14-135, Lane 6: C. michiganensis 14-310, Lane 7: C. michiganensis 15-079, Lane 8: C. michiganensis 16-043, Lane 9: C. michiganensis 17-156, Lane 10: C. michiganensis 17-338, Lane 11: C. tessellarius NCPPB 3664, Lane 12: C. nebraskensis NCPPB 2581, Lane 13: C. insidiosus NCPPB 1109, Lane 14: C. capsici KACC 18448, Lane 15: Tomato DNA, Lane 16: Tomato leaf sap.

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Fig. 2. Species-specific PCR detection of Clavibacter michiganensis using the CmmF/CmmR primer pair. No amplification was detected except C. michiganensis (lane 1, positive control). Lane M: 100 bp plus DNA ladder, Lane 1: Clavibacter michiganensis, Lane 2: Acidovorax citrulli, Lane 3: Acidovorax konjaci, Lane 4: Agrobacterium tumefaciens, Lane 5: Pectobacterium carotovorum, Lane 6: Pseudomonas corrugata, Lane 7: Pseudomonas syringae, Lane 8: Pseudomonas viridiflava, Lane 9: Xanthomonas arboricola, Lane 10: Xanthomonas perforans.

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Fig. 3. Sensitivity of PCR detection from bacterial cells of Clavibacter michiganensis using the CmmF/CmmR primer pair in a 10-fold dilution series. Amplified products were obtained from lane 1 (109 cfu/ ml) to lane 6 (104 cfu/ml). Lane 6 showed a weak PCR product band. Lane M: 100bp plus DNA ladder, Lane 1: 109 cfu/ml, Lane 2: 108 cfu/ ml, Lane 3: 107 cfu/ml, Lane 4: 106 cfu/ml, Lane 5: 105 cfu/ml, Lane 6: 104 cfu/ml, Lane 7: 103 cfu/ml, Lane 8: 102 cfu/ml, Lane 9: 101 cfu/ml, Lane 10: 100 cfu/ml, Lane 11: 10-1 cfu/ml, Lane 12: distilled water.

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Fig. 4. Specific detection of Clavibacter michiganensis from different parts of artificially infected plant (A) using the CmmF/CmmR primer pair by direct PCR (B). Lane 1 to lane 9 showed PCR amplification of genomic DNA of C. michiganensis directly from various parts of the diseased plant. Lane M: 100bp plus DNA ladder, Lane C: uninfected control plant, Lane 1: leaf showing wilting symptom, Lane 2: leaf showing wilting symptom, Lane 3: petiole of leaf showing wilting symptom, Lane 4: shoots with no symptoms, Lane 5: petiole of leaf showing wilting symptom, Lane 6: middle part of stem, Lane 7: lower part of stem, Lane 8: root, Lane 9: discolored xylem vessel.

Table 1. List of bacterial strains used in this study

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Table 2. Primer sequences of PCR-detection for Clavibacter michiganensis

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Table 3. Detection of Clavibacter michiganensis in the artificially inoculated tomato seedling by plating undiluted (1/1) and serially diluted (1/10-1/106) leaf extract on selective BCT medium and PCR assay

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References

  1. Bach, H. J., Jessen, I., Schloter, M. and Munch, J. C. 2003. A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies. J. Microbiol. Methods 52: 85-91. https://doi.org/10.1016/S0167-7012(02)00152-5
  2. Choi, J. E., Yoo, S. J. and Kim, H. G. 1997. Factors affecting development of bacterial canker and disease control strategy. Plant Dis. Agric. 3: 1-4. URL http://ext.newnonmun.com/indexDownloadOpen.php?type=view&a=393701# [13 December 2018]. (In Korean)
  3. de Leon, L., Siverio, F., Lopez, M. M. and Rodriguez, A. 2011. Clavibacter michiganensis subsp. michiganensis, a seedborne tomato pathogen: healthy seeds are still the goal. Plant Dis. 95: 1328-1338. https://doi.org/10.1094/PDIS-02-11-0091
  4. Dreier, J., Bermpohl, A. and Eichenlaub, R. 1995. Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopahology 85: 462-468. https://doi.org/10.1094/Phyto-85-462
  5. Fatmi, M. and Schaad, N. W. 1988. Semiselective agar medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato seed. Phytopathology 78: 121-126. https://doi.org/10.1094/Phyto-78-121
  6. Ftayeh, R. M., von Tiedemann, A. and Rudolph, K. W. E. 2011. A new selective medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato plants and seed. Phytopathology 101: 1355-1364. https://doi.org/10.1094/PHYTO-02-11-0045
  7. Gitaitis, R. D., Beaver, R. W. and Voloudakis, A. E. 1991. Detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato. Plant Dis. 75: 834-838. https://doi.org/10.1094/PD-75-0834
  8. Hadas, R., Kritzman, G., Klietman, F., Gefen, T. and Manulis, S. 2005. Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds. Plant Pathol. 54: 643-649. https://doi.org/10.1111/j.1365-3059.2005.01230.x
  9. Kaneshiro, W. S., Mizumoto, C. Y. and Alvarez, A. M. 2006. Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophyte using ELISA, Biolog and 16S rDNA sequencing. Eur. J. Plant Pathol. 116: 45-56. https://doi.org/10.1007/s10658-006-9036-1
  10. Kokoskova, B., Mraz, I. and Fousek, J. 2010. Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis. Folia Microbiol. 55: 239-244. https://doi.org/10.1007/s12223-010-0035-4
  11. Louws, F. J., Rademaker, J. L. W. and de Bruijn, F. J. 1999. The three Ds of PCR-based genomic analysis of phytobacteria: Diversity, detection, and disease diagnosis. Annu. Rev. Phytopathol. 37: 81-125. https://doi.org/10.1146/annurev.phyto.37.1.81
  12. Li, X., Tambong, J., Yuan, K., Chen, W., Xu, H., Levesque, C. A. et al. 2018. Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses. Int. J. Syst. Evol. Microbiol. 68: 234-240. https://doi.org/10.1099/ijsem.0.002492
  13. Luo, L. X., Walters, C., Bolkan, H., Liu, X. L. and Li, J. Q. 2008. Quantification of visible cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay. Plant Pathol. 57: 332-337. https://doi.org/10.1111/j.1365-3059.2007.01736.x
  14. Myung, I. S., Kim, D. G., An, S. H., Kee, Y. K. and Kim, W.G. 2008. First report of bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis in Korea. Plant Dis. 92: 1472.
  15. Pastrik, K. H. and Rainey, F. A. 1999. Identification of Clavibacter michiganensis subspecies by polymerase chain reaction-based techniques. J. Phytopathol. 147: 687-693. https://doi.org/10.1046/j.1439-0434.1999.00442.x
  16. Santos, M. S., Cruz, L., Norskov, P. and Rasmussen, O. F. 1997. A rapid and sensitive detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds by polymerase chain reaction. Seed Sci. Technol. 25: 581-584.
  17. Smith, E. F. 1910. A new tomato disease of economic importance. Science 31: 794-796.
  18. Yasuhara-Bell, J., de Silva, A., Heuchelin, S. A., Chaky, J. L. and Alvarez, A. M. 2016. Detection of Goss's wilt pathogen Clavibacter michiganensis subsp. nebraskensis in maize by loop-mediated amplification. Phytopathology 106: 226-235. https://doi.org/10.1094/PHYTO-10-15-0249-R
  19. Zhao, W. J., Chen, H. Y. Zhu, S. F., Xia, M. X. and Tan, T. W. 2007. Onestep detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato seeds using a TaqMan probe. J. Plant Pathol. 89: 349-351.