Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Nested PCR Detection of Pseudomonas syringae pv. actinidiae, the Causal Bacterium

Nested PCR을 통한 참다래 궤양병균 (Pseudomonas syringae pv. actinidiae)의 검출

  • Published : 2003.09.01
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Abstract

A PCR method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Pseudomonas syringae pv. actinidiae on kiwifruit leaves. A nested PCR was performed with primers designes from the coding sequence of the cfl gene, which is involved in production of the phytotoxin coronatine. The first and second primer sets efficiently amplified expected 665 and 310-bp fragments, respectively. With two successive amplifications, as few as 20 CFU/ml of P. syringae pv. actinidiae could be detected on ethidium bromide-stained agarose gel. Leaf samples were collected from 4 kiwifruit trees showing yellow halo spots on leaves and incubated in pepton-sucrose broth for 12 h at $16^{\circ}$C before PCR amplification. Positive detection was obtained with one sample, which was proved as a diseased plant in the next spring.

참다래 잎으로부터 궤양병균인 Pseudomnoas syringae pv. actinidiae를 배양하여 PCR을 통해 검출하는 방법을 개발하였다. Nested PCR을 위해 두 set의 primer를 식물 독소인 coronatine의 생합성에 관여하는 유전자 cfl의 염기서열로부터 설계하였다. 이 primer set를 사용하여 665rhk 310-bp의 절편이 증폭되었으며 nested PCR을 통한 궤양병균의 검출한계는 20 CFU/ml이었다. 4 그루의 참다래 나무로부터 노란색 무리로 나타나는 궤양병 초기 증상을 보이는 잎을 채취하여 pepton-sucrose 액체배지에 넣어 $16^{\circ}$C에서 12시간 배양 한뒤 PCR 을 시행한 결과 한 시료에서 예상했던 밴드가 증폭되었고 이듬해 봄 이 나무가 궤양병에 감염되었음을 확인하였다.

Keywords

References

  1. Audy, P., Braat, C. E., Saindon, G., Huang, H. C. and Laroche, A. 1996. A rapid and sensitive PCR-based assay for concurrent detection of bacteria causing common and halo blights in bean seed. Phytopathology 86: 361-366 https://doi.org/10.1094/Phyto-86-361
  2. Bender, C. L., Alarcon-Chaidez, F. and Gross, D. C. 1999. Pseudomonas syringae phytotoxins: Mode of action, regulation, and biosynthesis by peptide and polyketide synthetases. Microbiol. Mol. Rev. 63: 266-292
  3. Bender, C., Liyanage, H., Palmer, D., Ullrich, M., Young, S. and Mitchell, R. 1993. Characterization of the genes controlling biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid. Gene 133: 31-38 https://doi.org/10.1016/0378-1119(93)90221-N
  4. Bereswill, S., Bugert, P., Volksch, B., Ullrich, M., Bender, C. L. and Geider, K. 1994. Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products. Appl. Environ. Microbiol. 60: 2924-2930
  5. Bultreys, A. and Gheysen, I. 1999. Biological and molecular detection of toxic lipodepsipeptide-producing Pseudomonas syringae strains and PCR identification in plants. Appl. Environ. Microbiol. 65: 1904-1909
  6. Clerc, A., Manceau, A. and Nesme, X. 1998. Comparison of randomly amplified polymorphic DNA with amplified fragment length polymorphism to assess genetic diversity and genetic relatedness within genospecies III of Pseudomonas syringae. Appl. Environ. Microbiol. 64: 1180-1187
  7. Gardan, L., Shaff, H. and Grimont, P. A. 1997. DNA relatedness among pathovars of P. syringae and related bacteria. In : Pseudomonas syringae Pathovars and Related Pathogens, ed. by Rudolph, K., Burr, T. J., Mansfield, J. W., Stead, D., Vivian, A. and Von Kietzell, J., pp.445-448. Kluwer Academic Publishers, Dordrecht, The Netherlands
  8. Han, H. S., Koh, Y. J., Hur, J.-S. and Jung, J. S. 2003. Identification and characterization of coronatine-producing Pseudomonas syringae pv. actinidiae. J. Microbiol. Biotechnol. 13: 110-118
  9. Hildebrand, D. C., Schroth, M. N. and Sands, D. C., 1988. Pseudomonas, In : Laboratory Guide for Identification of Plant Pathogenic Bacteria, ed. by N. W. Schaad, pp.60-81. APS Press, St. Paul, Minn., USA
  10. 정재성, 한효심, 고영진. 2001. Pseudomonas syringae의 식물독소와 독소 생산 균주의 검출을 위한 PCR primer. 식물병연구 7: 123-133
  11. 고영진. 1995. 참다래의 주요 병. 식물병과 농업 1: 3-13
  12. 고영진, 박숙영, 이동현. 1996. 우리나라 참다래 궤양병 발생 특성 및 수간주입에 의한 방제. 한식병지 12: 324-330
  13. 고영진, 서정규, 이동현, 신종섭, 김승화. 1999. 참다래 궤양병의 약제 방제. 식물병과 농업 5: 95-99
  14. 고영진, 차병진, 정희정, 이동현. 1994. 참다래 궤양병의 격발 및 확산. 한식병지 10: 68-72
  15. Louws, F. J., Fulbright, D. w., Stephens, C. T. and deBruijn, F. J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl. Environ. Microbiol. 60: 2286-2295
  16. Lydon, J. and Patterson, C. D. 2001. Detection of tabtoxinproducing strains of Pseudomonas syringae by PCR. Lett. Appl. Microbiol. 32: 166-170 https://doi.org/10.1046/j.1472-765x.2001.00882.x
  17. Manceau, C. and Horvais, A. 1997. Assessment of genetic diversity among strains fo Pseudomonas syringae by PcR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P. syringae pv. tomato. Appl. Environ. Microbiol. 63: 498-505
  18. Mazarei, M. and Mostofipour, P. 1994. First report of bacterial canker of kiwifruit in Iran. Plant Pathology 43: 1055-1056 https://doi.org/10.1111/j.1365-3059.1994.tb01658.x
  19. Mitchell, R. E. 1976. Isolation and structure of a chlorosis inducing toxin of Pseudomonas phaseolicola. Phytochemistry 15: 1941-1947 https://doi.org/10.1016/S0031-9422(00)88851-2
  20. Prosen, D., Hatziloukas, E., Schaad, N. W. and Panopoulos, N. J. 1993. Specific detection of Pseudomonas syringae pv. phaseolicola DNA bean seed by polymerase chain reactionbased amplification of a phaseolotoxin gene region. Phytopathology 83: 965-970 https://doi.org/10.1094/Phyto-83-965
  21. Sawada, H., Takeuchi, T. and Matsuda, I. 1997. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences. Appl. Environ. Microbiol. 63: 282-288
  22. Schaad, N. W., Cheong, S. S., Tamaki, S., Hatziloukas, E. and Panopoulos, N. J. 1995. A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85: 243-248 https://doi.org/10.1094/Phyto-85-243
  23. Scortichini, M. 1994. Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy. Plant Pathology 43: 1035-1038 https://doi.org/10.1111/j.1365-3059.1994.tb01654.x
  24. Sorensen, K. N., Kim, K. H. and Takemoto, J. Y 1998. PCR detection of cyclic lipodepsipeptide-producing Pseudomonas syringae pv. syringae and similarity of strains. Appl. Environ. Microbiol. 64: 226-230
  25. Takikawa, Y, Serizawa, S., Ichikawa, T., Tsuyumu, S. and Goto, M. 1989. Pseudomonas syringae pv. actinidiae pv. nov.: The causal bacterium of canker of kiwirfuit in Japan. Ann. Phytopathol. Soc. Japan 55: 437-444 https://doi.org/10.3186/jjphytopath.55.437
  26. Tamura, K., Thkikawa, Y., Tsuyumu, S. and Goto, M. 1989. Characterization of the toxin produced by Pseudomonas syringae pv. actinidiae, the causal bacterium of kiwifruit canker Ann. Phytopathol. Soc. Japan 55: 512

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