Rapid detection of a downy mildew pathogen, Peronospora destructor, in infected onion tissues and soils by loop-mediated isothermal amplification.

Abstract

Downy mildew of onion caused by a soil-inhabiting water mold, Peronospora destructor is one of the most devastating disease that can destroy entire onion fields in a matter of days. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay that allows rapid detection of P. destructor by visual inspection. The internal transcribed spacer 2 (ITS2) region of P. destructor was used to design primer sets for LAMP reactions. Optimal temperature and incubation time were determined for the most efficient primer set. In the optimized condition, the LAMP assay exhibited at least 100 times more sensitivity than conventional PCR, detecting fentogram levels of P. destructor genomic DNA (gDNA). Detection of the pathogen from a small number of spores without gDNA extraction further confirmed the high sensitivity of the assay. For specificity, the LAMP assay was negative to gDNA of other fungal pathogens that cause various diseases on onion and oomycetes, while the assay was positive to gDNA extracted from onion tissues showing the typical downy mildew symptoms. Finally, we examined the efficacy of the LAMP assay in detection of P. destructor in soils. Soils collected from onion fields that had been contaminated with P. destructor were solarized for 60 days. While the LAMP assay was negative to the solarized soils, we were able to detect P. destructor that oversummers in fields. The LAMP assay developed in this study enables rapid detection and diagnosis of downy mildew of onion in infected tissues and in soil.