Abstract
ABSTRACTPotatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily byDickeyaandPectobacterium, results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detectingD. dadantii, a common soft rot causing bacteria. TheD. dadantii-specific LAMP primers were designed targeting a highly conserved genomic region withinD. dadantii, the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains ofD. dadantii) and exclusivity panels (85 strains, including allDickeyaspecies, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by performing assays with 10-fold serially diluted pure gDNA ofD. dadantiiand gDNA spiked with host crude extract; the assay detected the target pathogen down to 1 pg (188 copies) without being adversely affected by the host crude extract. The developed LAMP assay specifically detected the target pathogen in infected plant materials. Additional multi-operator blind and multi-instrument tests were conducted to assess the assay’s robustness and applicability, consistently yielding accurate results without false positives or negatives. These findings demonstrate the assay’s potential utility for biosecurity, routine diagnostics, and epidemiological studies.
Publisher
Cold Spring Harbor Laboratory