A rapid and sensitive method for the detection of Didymella pinodella in Pisum sativum

BACKGROUND Ascochyta blight (AB) is a major disease that severely constrains crop yield and quality, capable of causing losses up to 80%. The pathogens responsible for AB are included in a species complex, with Didymella pinodella being one of the main causal agents. Current methods for detecting D. pinodella are based on morphological and molecular identification, both of them to be performed under laboratory conditions. To enable rapid on‐site diagnosis, we have developed a novel method that integrates recombinase aided amplification (RAA) with a lateral flow dipstick (LFD) for the specific detection of D. pinodella . RESULTS By aligning the whole genomes of D. pinodella and its closely related species, a specific gene fragment was screened and subsequently used as a target sequence for establishing a specific isothermal detection method for D. pinodella . The RAA‐LFD system exhibited high specificity, successfully distinguishing D. pinodella from closely related species. Both the sensitivity of real‐time PCR and the RAA‐LFD method for detecting D. pinodella was 2.13 × 10 −3 ng μL −1 . The RAA‐LFD assay successfully produced positive results from pea stems inoculated for 10 days and from artificially infested seed samples, which was consistent with the results obtained by real‐time PCR. The total time from DNA extraction to final detection was less than 60 min. CONCLUSION The method for rapid detection of plant pathogens involves magnetic bead‐based DNA extraction, isothermal nucleic acid amplification at 39 °C, and visual detection using a lateral flow dipstick at room temperature. RAA reagents and primer‐probe mixtures can be pre‐loaded into microcentrifuge tubes to streamline the on‐site operational workflow. This method requires minimal equipment and operational training, demonstrating significant potential for applications in on‐site pathogen screening, field surveillance, port quarantine, and controlling the spread of crop fungal pathogens.