Rapid Loop-Mediated Isothermal Amplification Assay for the Detection of Root Rot in Soybean Caused by Phytophthora sojae

Abstract

Soybean (Glycine max) production is severely impacted by Phytophthora sojae, the causal agent of Phytophthora root and stem rot, which leads to substantial yield losses worldwide. Rapid and accurate detection of this pathogen is critical for disease management and prevention. In this study, we developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the internal transcribed spacer (ITS) region of P. sojae DNA. While conventional PCR detected P. sojae in only 18.75% of infected soybean seedlings, nested PCR achieved 71.88% detection using 1 μL of template DNA and 100% detection when 5 μL was used. In contrast, the ITS-LAMP assay achieved 100% detection using just 1 μL of template, highlighting its superior sensitivity. The ITS-LAMP assay has a detection limit of 1 pg and enabled straightforward visual detection through colorimetric change or agarose gel electrophoresis. It was more sensitive than the previously reported PS-PYpt1-LAMP assay, which had a detection limit of 100 pg. Primer specificity was confirmed through blind testing across a wide range of oomycete and fungal species. With high specificity, sensitivity, and resistance to inhibitors, this LAMP assay provides a valuable tool for rapid and accurate P. sojae detection, enhancing Phytophthora root and stem rot management.