Rapid and quantitative detection of Pythium inflatum by real-time fluorescence loop-mediated isothermal amplification assay

Abstract

Pythium inflatum is the causal agent of Pythium maize stalk rot, which is one of the most devastating diseases of maize (Zea mays L.). P. inflatum is currently a major concern in global maize production. To the best of our knowledge, no effective resistance to P. inflatum is known in maize, and no effective measures have been reported for the control of this pathogen once maize plants have been infected. Early and accurate detection of P. inflatum is essential to guide maize planting and to protect maize production. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid quantitative detection of P. inflatum in soil. The detection limit of the RealAmp assay was approximately 0.1 pg/μl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificially infested soil. No cross-reactions with other related pathogens were observed. Results of the RealAmp assay for quantifying the genomic DNA of P. inflatum were confirmed by testing with artificially and naturally infested samples. The quantification of the soil-borne pathogen DNA of P. inflatum in naturally infested samples was not significantly different compared with classic real-time PCR (P < 0.05). Additionally, the RealAmp assay could be detected via an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification. Consequently, the inhibitory effects of the stain on DNA amplification were avoided. Therefore, the assay could be used more conveniently in the field as a simple, rapid, and effective technique and has the potential to become an alternative tool for detecting and monitoring P. inflatum in the field.