Rapid detection of the phytopathogenic oomycete Phytopythium helicoides with a visualized loop-mediated isothermal amplification assay.

Abstract

The phytopathogenic oomycete Phytopythium helicoides, previously known as Pythium helicoides, has emerged as a new threat to the Shatangju citrus cultivar (Citrus reticulata cv. Shatangju; '' in Chinese) in southern China. To enable rapid diagnosis and control of the leaf blight and stem rot caused by P. helicoides, we developed a visualized loop-mediated isothermal amplification (LAMP) assay targeting the β-tubulin gene of the pathogen. Using a carefully selected set of primers and optimized reaction conditions at 60 °C for 60 min, the LAMP assay can specifically detect the pathogen among various citrus-associated phytopathogens. The assay demonstrated high sensitivity, detecting as little as 30 pg of the pathogen DNA, comparable to the sensitivity of quantitative polymerase chain reaction (qPCR). We validated the practical application of this LAMP assay for diagnosing infections in citrus leaves and strawberry crowns. This newly developed LAMP assay offers a specific, sensitive, and rapid detection tool for P. helicoides, aiding in the control of Shatangju citrus leaf blight and stem rot, as well as other related crop diseases like strawberry crown rot.