Developing PCR-Based Assays for Detecting Pea Seed-borne Mosaic Virus (PSbMV) in Plants, Seeds, and Its Aphid Vector, <i>Acyrthosiphon pisum</i>

Pea seed-borne mosaic virus (PSbMV) poses a major threat to global pulse production. This virus, transmitted through seeds, can spread within fields via insect vectors, especially pea aphids (<i>Acyrthosiphon pisum</i>), in a nonpersistent manner. To mitigate the risks associated with PSbMV, it is crucial to plant virus-free seeds, detect the virus at an early stage, and implement effective control measures for the vectors, given that most commercial pulse cultivars are vulnerable to the virus. This study designed and assessed multiple primers for PCR-based virus detection and demonstrated their capability to identify PSbMV isolates in infected plant tissues. The primers successfully detected PSbMV in dried plant tissues and in aphids collected from infected plants, even after being stored at room temperature for up to 3 months. Furthermore, a hydrolysis probe-based assay was developed, and its effectiveness for quantitative PCR (qPCR), digital PCR (dPCR), and droplet digital PCR (ddPCR) was evaluated. Our results showed high sensitivity and linearity of the assay, capable of detecting PSbMV at concentrations as low as 22 copies per reaction mix using digital PCRs. Our findings underscore the effectiveness of the developed primers and assay for the rapid and sensitive detection of PSbMV isolates in a variety of plant tissues, aphids, and seed samples, offering improved tools for disease monitoring and management in agricultural settings.