Neofusicoccum laricinum, an important pathogenic species, causes shoot blight of larch. In China, large areas of Larix principis-rupprechtii forests are threatened by this pathogen. Currently, this pathogen is on the list of quarantine pests in Chinese. Due to the widespread and severe damage caused by N. laricinum, a reliable and accurate diagnostic tool is urgently needed. In this study, we first identified a Nlar12009 as a N. laricinum-specific gene through genomic sequence data and bioinformatic analysis. Specific primer pairs and DNA probes were designed to detect the target pathogen using a novel recombinase polymerase amplification assay with a lateral flow dipstick (RPA-LFD) method. We optimized the RPA-LFD assay to ensure high specificity to N. laricinum. Our results showed that the assay exclusively detected N. laricinium isolates with no cross-reaction with other isolates of fungaland oomycete species and nematodes. Furthermore, our detection technique exhibited a 10-fold higher sensitivity (10 fg/mL) than conventional polymerase chain reaction (PCR) for N. laricinum detection. Our developed RPA-LFD assay is proved to be a highly specific, sensitive, time-saving, and convenient method for the diagnosis of N. laricinum and shows great potential in field application.
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