Advancing nematode identification on potato: an isothermal recombinase polymerase amplification assay for Stubby Root nematode, Paratrichodorus allius

Authors: GORAYA, MANKANWAL - North Dakota State University; YAN, GUIPING - North Dakota State University; Whitworth, Jonathan; Swisher Grimm, Kylie

Submitted to: American Journal of Potato Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2023
Publication Date: 12/29/2023
Citation: Goraya, M., Yan, G., Whitworth, J.L., Swisher Grimm, K.D. 2023. Advancing nematode identification on potato: an isothermal recombinase polymerase amplification assay for Stubby Root nematode, Paratrichodorus allius. American Journal of Potato Research. 101:52-64. https://doi.org/10.1007/s12230-023-09940-4.
DOI: https://doi.org/10.1007/s12230-023-09940-4

Interpretive Summary: The stubby root nematode (Paratrichodorus allius) transmits Tobacco rattle virus in potatoes and can cause necrotic tuber symptoms called Corky ringspot. Identification of P. allius under a microscope is time consuming and requires knowledge of species morphological characteristics. Previously developed molecular techniques require a well-equipped laboratory. A new recombinase polymerase amplification (RPA) assay was developed, which requires minimal sample preparation, low temperature (37-42 0°C), and short time duration (20-40 minutes) for detection. RPA primers target the internal transcribed spacer (ITS) region of ribosomal DNA allowing identification of P. allius. RPA assay conditions were optimized to amplify DNA at 40 0°C in 20 minutes. RPA tests indicating P. allius presence can be seen following agarose gel electrophoresis and SYBR Green I dye which results in a green color for a positive test. Diverse stubby root nematode species available in GenBank were tested to confirm the test 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 SYBR Green I-based assay. The test was validated with P. allius nematodes extracted from 19 fields soil samples from three states. The developed RPA assay can serve as an efficient tool for rapidly detecting P. allius and could serve as a management method for this nematode.

Technical 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.