Development of a loop-mediated isothermal amplification (LAMP) method to detect the potato zebra chip pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) and differentiate haplotypes A and B

Authors: JIANG, JUNYE - Alberta Agriculture And Rural Development; FEINDEL, WILL - Alberta Agriculture And Rural Development; Swisher Grimm, Kylie; HARDING, MICHAEL - 8a Hay And Cattle Co; FEINDEL, DAVID - Alberta Agriculture And Rural Development; BAJEMA, STACEY - Alberta Agriculture And Rural Development; FENG, JIE - Alberta Agriculture And Rural Development

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/6/2022
Publication Date: 12/8/2022
Citation: Jiang, J., Feindel, W., Swisher Grimm, K.D., Harding, M., Feindel, D., Bajema, S., Feng, J. 2022. Development of a loop-mediated isothermal amplification (LAMP) method to detect the potato zebra chip pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) and differentiate haplotypes A and B. Plant Disease. 107(6):1697-1702. https://doi.org/10.1094/PDIS-09-22-2258-SR.
DOI: https://doi.org/10.1094/PDIS-09-22-2258-SR

Interpretive Summary: The bacterium, ‘Candidatus Liberibacter solanacearum,’ causes devastation to solanaceous crops including potato and tomato in the United States, Mexico, and Central America. Researchers with the Potato Growers of Alberta and Alberta Plant Health Lab, Alberta Agriculture, Forestry and Rural Economic Development, in collaboration with an USDA-ARS scientist in Prosser, WA, generated a new low-cost, timely assay to distinguish between two genetic variants of the bacterium. The tool, known as loop-mediated isothermal amplification or LAMP, relies on a molecular assay that produces visible results without the use of fancy reagents or machines. Results were validated on samples of the two predominant ‘Ca. L. solanacearum’ genetic variants that have previously shown differences in virulency in both potato and tomato. This tool will provide rapid pathogen identification that will aid growers and fieldman in their disease management programs.

Technical Abstract: Candidatus Liberibacter solanacearum (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signal from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 non-target plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA, for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks, for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately and directly differentiate haplotypes A and B. This tool will provide rapid pathogen identification that will aid growers and fieldman in their disease management programs.