The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Development of an Improved Loop-Mediated Isothermal Amplification Assay for On-Site Diagnosis of Fire Blight in Apple and Pear

  • Shin, Doo-San (Major in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University) ;
  • Heo, Gwang-Il (Major in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University) ;
  • Son, Soo-Hyeong (Major in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University) ;
  • Oh, Chang-Sik (Department of Horticultural Biotechnology, Kyung Hee University) ;
  • Lee, Young-Kee (Department of Agro-food Safety and Crop Protection, National Institute of Agriculture Sciences, Rural Development Administration) ;
  • Cha, Jae-Soon (Major in Plant Medicine, School of Applied Plant Science & Biotechnology, Chungbuk National University)
  • Received : 2017.03.27
  • Accepted : 2018.05.17
  • Published : 2018.06.01
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Abstract

Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to $10^4$ cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The $B{\ddot{u}}hlmann$-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.

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References

  1. Acimovic, S. G., Zeng, Q., McGhee, G. C., Sundin, G. W. and Wise, J. C. 2015. Control of fre blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes. Front. Plant Sci. 6:16.
  2. Braun-Kiewnick, A., Altenbach, D., Oberhansli, T., Bitterlin, W. and Duffy, B. 2011. A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fre blight diagnosis. J. Microbiol. Methods 87:1-9. https://doi.org/10.1016/j.mimet.2011.06.015
  3. Buhlmann, A., Pothier, J. F., Rezzonico, F., Smits, T. H. M., Andreou, M., Boonham, N., Duffy, B. and Frey, J. E. 2013. Erwinia amylovora loop-mediated isothermal amplifcation (LAMP) assay for rapid pathogen detection and on-site diagnosis of fre blight. J. Microbiol. Methods 92:332-339. https://doi.org/10.1016/j.mimet.2012.12.017
  4. Drenova, N. V., Isin, M. M., Dzhaimurzina, A. A., Zharmukhamedova, G. A. and Aitkulov, A. K. 2012. Bacterial fre blight in the Republic of Kazakhstan. Plant Health Research and Practice 1:44-48.
  5. Fang, X., Chen, H., Yu, S., Jiang, X. and Kong, J. 2010a. Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplifcation chip. Anal. Chem. 83:690-695.
  6. Fang, X., Liu, Y., Kong, J. and Jiang, X. 2010b. Loop-mediated isothermal amplifcation integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Anal. Chem. 82:3002-3006. https://doi.org/10.1021/ac1000652
  7. Gorris, M. T., Cambra, E., Paulin, J. P., Chartier, R., Cambra, M. and Lopez, M. M. 1996. Production and characterization of monoclonal antibodies specific for Erwinia amylovora and their use in different serological techniques. Acta Hortic. 411:47-52.
  8. Heo, G. I., Shin, D. S., Son, S. H., Oh, C. S., Park, D. H., Lee, Y. K. and Cha, J. S. 2017. On-site diagnosis of fre blight with antibody-based diagnostic strips. Res. Plant Dis. 23:306-313.
  9. Kiddle, G., Hardinge, P., Buttigieg, N., Gandelman, O., Pereira, C., McElgunn, C. J., Rizzoli, M., Jackson, R., Appleton, N., Moore, C., Tisi, L. C. and Murray, J. A. H. 2012, GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplifcation (LAMP) suitable for feld use. BMC Biotechnol. 12:15. https://doi.org/10.1186/1472-6750-12-15
  10. Kim, W. S., Gardan, L., Rhim, S. L. and Geider, K. 1999. Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai). Int. J. Syst. Bacteriol. 49:899-906 https://doi.org/10.1099/00207713-49-2-899
  11. Moradi, A., Nasiri, J., Abdollahi, H. and Almasi, M. 2012. Development and evaluation of a loop-mediated isothermal amplifcation assay for detection of Erwinia amylovora based on chromosomal DNA. Eur. J. Plant Pathol. 133:609-620. https://doi.org/10.1007/s10658-012-9939-y
  12. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. 2000. Loop-mediated isothermal amplifcation of DNA, Nucleic Acids Res. 28:E63. https://doi.org/10.1093/nar/28.12.e63
  13. Park, D. H., Lee, Y. G., Kim, J. S., Cha, J. S. and Oh, C. S. 2017. Current status of fre blight caused by Erwinia amylovora and action for its management in Korea. J. Plant Pathol. 99:59-63.
  14. Powney, R., Beer, S., Plummer, K., Luck, J. and Rodoni, B. 2011. The specifcity of PCR-based protocols for detection of Erwinia amylovora. Australas. Plant Pathol. 40:87-97. https://doi.org/10.1007/s13313-010-0017-7
  15. Rhim, S. L., Voelkschb, B., Gardanc, L., Paulinc, J. P., Langlotzd, C., Kimd, W. S. and Geider, K. 1999. Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees. Plant Pathol. 48:514-520. https://doi.org/10.1046/j.1365-3059.1999.00376.x
  16. Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a laboratory manual. CSHL Press, NY, USA.
  17. Shrestha, R., Koo, J. H., Park, D. H., Hwang, I., Hur, J. H. and Lim, C. K. 2003. Erwinia pyrifoliae, a causal endemic pathogen of shoot blight of Asian pear tree in Korea. Plant Pathol. J. 19:294-300. https://doi.org/10.5423/PPJ.2003.19.6.294
  18. Shrestha, R., Lee, S. H., Kim, J. E., Wilson, C., Choi, S. G., Park, D. H., Wang, M. H., Hur, J. H. and Lim, C. K. 2007. Diversity and detection of Korean Erwinia pyrifoliae strains as determined by plasmid profiling, phylogenetic analysis and PCR. Plant Pathol. 56:1023-1031. https://doi.org/10.1111/j.1365-3059.2007.01679.x
  19. Smits, T. H. M., Rezzonico, F. and Duffy, B. 2011. Evolutionary insights from Erwinia amylovora genomics. J. Biotechnol. 155:34-39. https://doi.org/10.1016/j.jbiotec.2010.10.075
  20. Temple, T. N. and Johnson, K. B. 2011. Evaluation of loop-mediated isothermal amplifcation for rapid detection of Erwinia amylovora on pear and apple fruit flowers. Plant Dis. 95:423-430. https://doi.org/10.1094/PDIS-09-10-0636
  21. Van der Zwet, T., Orolaza-Halbrent, N. and Zeller, W. 2012. Fire Blight, History, Biology, and Management. APS Press, MN, USA.