A rapid detection method for the plant pathogen Sclerotinia sclerotiorum based on loop-mediated isothermal amplification (LAMP)

Abstract

The ascomycete Sclerotinia sclerotiorum is a devastating plant pathogen with a very broad host range. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeting the Ssos5 sequence for visual detection of S. sclerotiorum. The LAMP reaction was optimal at 63 °C for 45 min. When hydroxynaphthol blue (HNB) was added before amplification, samples with S. sclerotiorum DNA developed a characteristic sky blue colour but those without DNA or with the DNA of nine other plant-pathogenic fungi did not. Results obtained with LAMP and HNB were confirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for S. sclerotiorum was 0.1 fg μL−1 of genomic DNA per reaction, while that of conventional PCR was 100 fg μL−1. Detection results were identical when 13 samples of rapeseed tissue infected with S. sclerotiorum were subjected to LAMP, conventional PCR, and conventional isolation. Because the LAMP assay reported here is simple, rapid, sensitive, and specific, it should be valuable for the detection of S. sclerotiorum in quarantine efforts and field surveys.