A 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, resulting in significant yield losses worldwide. Accurate detection of this pathogen is critical for effective disease management. In this study, we developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the internal transcribed spacer (ITS) region of P. sojae DNA. Conventional PCR detected the pathogen in only 18.7% of infected soybean seedlings using a 1 μL DNA sample, while nested PCR detected 71.9%. In contrast, the ITS-LAMP assay achieved 100% detection with the same input volume, demonstrating superior sensitivity. Notably, nested PCR required a fivefold increase in template volume (5 μL) to reach comparable detection levels. The ITS-LAMP assay exhibited a detection limit of 1 pg μL⁻¹, which is two orders of magnitude lower than that of the previously reported PS-PYpt1-LAMP assay. Visual detection was enabled through both colorimetric change and agarose gel electrophoresis. Primer specificity was confirmed through blind testing across a wide range of oomycete and fungal species that also infect soybean. With 100% specificity, sensitivity, and accuracy and robust resistance to inhibitors, the LAMP assay provides a valuable tool for rapid and accurate detection of P. sojae, enhancing Phytophthora root and stem rot management.