Rapid Detection of a Downy Mildew Pathogen, , in Infected Onion Tissues and Soils by Loop-Mediated Isothermal Amplification.

Downy mildew of onion caused by a soil-inhabiting water mold, , is one of the most devastating diseases 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 for rapid detection of by visual inspection. The internal transcribed spacer 2 region of was used to design primer sets for LAMP reactions. The 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 femtogram levels of 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 for gDNA of other fungal pathogens that cause various diseases on onion and oomycetes, whereas the assay was positive for gDNA extracted from onion tissues showing the typical downy mildew symptoms. Finally, we examined the efficacy of the LAMP assay in detection of in soils. Soils collected from onion fields that had been contaminated with were solarized for 60 days. Whereas the LAMP assay was negative for the solarized soils, we were able to detect 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.