Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for Detection of the Potato Powdery Scab Pathogen Spongospora subterranea.

ABSTRACT

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.