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Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta

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Abstract

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.

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Acknowledgements

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).

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  1. Zhen-Dong Chen and Hua-Jun Kang contributed equally to this work.

Authors and Affiliations

  1. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Zhen-Dong Chen, Hua-Jun Kang, A-Li Chai, Yan-Xia Shi, Xue-Wen Xie, Lei Li & Bao-Ju Li

  2. Vegetable Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, 530007, China

    Zhen-Dong Chen

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  1. Zhen-Dong Chen
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  2. Hua-Jun Kang
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  3. A-Li Chai
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  4. Yan-Xia Shi
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  5. Xue-Wen Xie
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  6. Lei Li
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Correspondence to A-Li Chai or Bao-Ju Li.

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Chen, ZD., Kang, HJ., Chai, AL. et al. Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta. Eur J Plant Pathol 156, 739–750 (2020). https://doi.org/10.1007/s10658-019-01923-8

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  • DOI: https://doi.org/10.1007/s10658-019-01923-8

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