A loop-mediated isothermal amplification assay for the plant-parasitic nematode Aphelenchoides besseyi in rice seedlings

Abstract

Aphelenchoides besseyi is a seed-borne plant-parasitic nematode pathogen that causes rice white-tip disease worldwide. For quarantine purposes at custom border inspection and in field surveillance, a rapid, highly specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for the nematode detection was developed. The assay targets the sequence variable region of the mitochondrial cytochrome oxidase subunit I (COI) gene of the nematode. It is capable of distinguishing the A. besseyi rice pathotype from the closely related A. besseyi nest fern pathotype. The assay is highly sensitive, with the detection minimum limit at 103 copy numbers/µl cloned target plasmid or a single nematode crude DNA. A minimum of 35min is needed for the assay to complete, and the detection results can be rapidly determined by the color change of hydroxynaphathol blue dye or by the indication bar on a specific lateral flow dipstick. The application potential of this assay on rice seeding was demonstrated at the sensitivity level of five nematodes in single rice seedling. This is the first LAMP assay for A. besseyi rice race detection using a mitochondria gene as marker. This assay is well-suited for phytosanitary examination and seedling screening purposes in nurseries.