Effector gene-based diagnostic assay for the precise detection of yellow rust infection in wheat

Stripe rust caused by Puccinia striiformis f. sp. tritici ( Pst ) remains a major constraint to wheat productivity, necessitating rapid and accurate detection tools for effective disease management. In the present study, an effector-mediated diagnostic approach was developed targeting the Puccinia NPR1 interactor (PNPi) gene for precise detection of stripe rust infection in wheat. Specific primers were designed to amplify a 146 bp fragment of the single-copy PNPi effector gene. The assay demonstrated high specificity, successfully amplifying all tested Indian stripe rust pathotypes while showing no cross-reactivity with other rust pathogens or 42 non-target fungal species. The developed PCR assay reliably detected the pathogen in wheat leaf samples as early as 5 days post-inoculation, confirming its utility for early diagnosis. A SYBR Green-based quantitative PCR (qPCR) assay was further optimized, exhibiting a strong linear relationship (R 2 > 0.989) between cycle threshold (Cq) values and pathogen DNA concentration, with a detection limit as low as 0.007 ng μL −1 . Field validation revealed a strong correlation between pathogen load and disease severity, with Cq values inversely related to infection levels. Overall, the effector-based qPCR assay provides a rapid, sensitive, and highly specific tool for early detection and quantification of stripe rust, enabling improved disease surveillance and management strategies.