DNA barcode‐targeted triplex droplet digital PCR for three high‐risk soybean fungal pathogens in soybean seeds

BACKGROUND Soybean crops face significant threats from the fungal pathogens Diaporthe aspalathi , Diaporthe caulivora , and Cadophora gregata , which cause stem canker and brown stem rot leading to substantial yield losses. Current detection methods rely on time‐consuming isolation and culturing, underscoring the need for rapid, sensitive, and direct diagnostic tools. This study aimed to develop a triplex droplet digital polymerase chain reaction (ddPCR) assay for simultaneous detection of these three pathogens without requiring fungal cultivation. RESULTS A triplex ddPCR assay was designed using specific regions of the translation elongation factor 1‐α ( EF‐1α ) and β‐tubulin ( TUB2 ) genes, which were identified via comparative DNA barcode analysis of fungi infecting soybean plants. The assay achieved high sensitivity with absolute quantification limits of 0.20 copies/μL for D. aspalathi , 0.34 copies/μL for D. caulivora , and 2.58 copies/μL for C. gregata . Recovery rates from fungal‐mycelium‐spiked soybean seed samples ranged from 58.8% to 91.7%. When applied to field samples, the assay detected the target pathogens in 60% of imported soybean seeds. CONCLUSION The triplex ddPCR method enables accurate, sensitive, and rapid simultaneous detection of three high‐risk fungal pathogens in soybean seeds. This assay supports large‐scale screening of imported commodities, facilitates early disease management, and has the potential to reduce significant economic losses in soybean production.