Development of a lateral flow immunoassay for rapid detection of piper yellow mottle and cucumber mosaic viruses in black pepper

Piper yellow mottle virus (PYMoV) and cucumber mosaic virus (CMV) are major viral pathogens causing stunt disease in black pepper and related Piper species. As no chemical control is available, early and accurate detection is essential to prevent virus spread through vegetative propagation. Existing PCR-based assays are effective but often time-consuming, expensive, and reliant on specialized equipment. This study reports the development of a simple, rapid, and cost-effective lateral flow immunoassay (LFIA) for on-site detection of PYMoV and CMV infecting black pepper. Polyclonal antiserum was produced by immunizing rabbits with in vitro expressed coat proteins of both viruses, and its specificity was confirmed by western blotting. Purified immunoglobulin G (IgG) was used to prepare gold nanoparticle (GNP)-IgG conjugates. LFIA strips were assembled by optimizing sample pads, conjugate release pads, nitrocellulose membranes (10 µm pore size), test lines (0.75 mg/ml anti-PYMoV and anti-CMV IgG), control line (1 mg/ml goat anti-rabbit IgG), and absorbent pads. The optimized LFIA accurately detected PYMoV and CMV in virus-infected black pepper plants. The limits of detection for anti-PYMoV and anti-CMV LFIA were 100 ng and 50 ng of their respective homologous antigens. The assays were further validated using field samples from multiple black pepper varieties and other Piper species, including P. betle and P. longum. This LFIA offers a practical tool for rapid, on-site diagnosis to support the management of viral diseases in black pepper cultivation.