Advancing Nematode Identification on Potato: An Isothermal Recombinase Polymerase Amplification Assay for Stubby Root Nematode, Paratrichodorus allius

Abstract

The stubby root nematode, Paratrichodorus allius, is an important plant-parasitic nematode that feeds on plant roots and transmits Tobacco rattle virus to potato. Identification of P. allius based on morphometric measurements requires taxonomic knowledge, while previously developed molecular techniques require a well-equipped laboratory. A new recombinase polymerase amplification (RPA) assay was developed in this study, which requires minimal sample preparation, low temperature (37–42 °C), and short time duration (20–40 min) for detection of P. allius. RPA primers were designed targeting the internal transcribed spacer (ITS) region, and conditions were optimized to amplify DNA at 40 °C in 20 min. RPA products were visualized using agarose gel electrophoresis and SYBR Green I dye. In-silico analysis was conducted to predict the primer specificity. The RPA assay was specific to P. allius only. Detection sensitivity of the agarose gel electrophoresis-based RPA assay was 1/4th portion of a single nematode, and a single nematode by the SYBR Green I-based assay. This assay was validated with P. allius infested potato fields. The developed RPA assay can serve as an efficient tool for rapidly detecting P. allius from infested potato fields to help growers with their management decisions.