Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta
- 15 Downloads
Tomato bacterial speck, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most devastating bacterial diseases in tomato worldwide. To establish a rapid amplification system for the detection of Pst, a loop-mediated isothermal amplification (LAMP) method, which includes two external primers (F3/B3), two internal primers (FIP/BIP) and one backward loop primer (B-Loop), was designed based on the hrpZ gene. The specificity of the LAMP primer set was widely validated on Pst and non-target strains. The conditions for LAMP detection of Pst were optimized to complete in 60 min at 63 °C. The amplification was confirmed through gel electrophoresis or visual inspection using calcein. In the sensitivity test, the detection limit of the LAMP assay was 1.61 × 10 fg μL−1 for genomic DNA and 1.05 × 103 CFU mL−1 for bacterial suspension without DNA extraction. The novel method was also applied for the detection of Pst in artificially and naturally infected tomato leaf and stem tissue samples, and even the early onset of disease could be detected by the assay. This method can rapidly detect Pst-infected tissues without strain enrichment and complex DNA extraction, and hence, it is suitable for quarantine and field detection.
KeywordsTomato bacterial speck Pseudomonas syringae pv. tomato Loop-mediated isothermal amplification (LAMP) Visualization
This work was supported by the National key research & development (R&D) plan (No. 2017YFD0200300), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (No. CAAS-ASTIP-IVFCAAS), and Modern Agro-industry Technology Research System in China (No. CARS-25).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This study does not contain any studies with human participants or animals performed by any of the authors.
- Braun-Kiewnick, A., & Sands, D. C. (2001). Pseudomonas. In N. W. Schaad, J. B. Jones, & W. Chun (Eds.), Laboratory guide for identification of plant pathogenic Bacteria (3rd ed., pp. 84–120). St. Paul: APS Press.Google Scholar
- Bryan, M. K. (1933). Bacterial speck of tomatoes. Phytopathology, 23, 897–904.Google Scholar
- Bühlmann, A., Pothier, J. F., Rezzonico, F., Smits, T. H. M., Andreou, M., Boonham, N., Duffy, B., & Frey, J. E. (2013a). Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight. Journal of Microbiological Methods, 92, 332–339.CrossRefGoogle Scholar
- Bühlmann, A., Pothier, J. F., Tomlinson, J. A., Frey, J. E., Boonham, N., Smits, T. H. M., & Duffy, B. (2013b). Genomics-informed design of loop-mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level. Plant Pathology, 62, 475–484.CrossRefGoogle Scholar
- Hodgetts, J., Hall, J., Karamura, G., Grant, D. J., Studholme, D. J., Boonham, N., Karamura, E., & Smith, J. J. (2015a). Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification. Journal of Applied Microbiology, 119, 1651–1658.CrossRefGoogle Scholar
- Hodgetts, J., Karamura, G., Johnson, G., Hall, J., Perkins, K., Beed, F., Nakato, V., Grant, M., Studholme, D. J., Boonham, N., & Smith, J. (2015b). Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt. Plant Pathology, 64, 559–567.CrossRefGoogle Scholar
- Kil, E. J., Kim, S., Lee, Y. J., Kang, E. H., Lee, M., Cho, S. H., Kim, M. K., Lee, K. Y., Heo, N. Y., Choi, H. S., Kwon, S. T., & Lee, S. (2015). Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination. Journal of Virological Methods, 213, 68–74.CrossRefGoogle Scholar
- Meng, X. L., Xie, X. W., Shi, Y. X., Chai, A. L., Ma, Z. H., & Li, B. J. (2016). Evaluation of a loop-mediated isothermal amplification assay based on hrpZ gene for rapid detection and identification of Pseudomonas syringae pv. lachrymans in cucumber leaves. Journal of Applied Microbiology, 122, 441–449.CrossRefGoogle Scholar
- Tamura, K., & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10, 512–526.Google Scholar
- Young, J. M., Saddler, G. S., Takikawa, Y., de Boer, S. H., Vauterin, L., Gvozdyak, R. I., & Stead, D. E. (1996). Names of plant pathogenic bacteria 1863-1995-the ISPP accepted list of bacterial names. Review Plant Pathology, 75, 721–763.Google Scholar
- Zhang, S. Y., Dai, D. J., Wang, H. D., & Zhang, C. Q. (2019). One-step loop-mediated isothermal amplifcation (LAMP) for the rapid and sensitive detection of Fusarium fujikuroi in bakanae disease through NRPS31, an important gene in the gibberellic acid bio-synthesis. Scientific Reports, 9, 3726.CrossRefGoogle Scholar
- Zhao, M. M., Shi, Y. H., Wu, L., Guo, L. C., Liu, W., Xiong, C., Yan, S., Sun, W., & Chen, S. L. (2016). Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence. Scientific Reports, 6, 25370.CrossRefGoogle Scholar