Development of RT-RPA assays for rapid detection of mango-infecting viruses using high-throughput sequencing-derived...

Plant viral diseases pose a major threat to perennial fruit crops, where infections often remain latent and persist over long periods, facilitating unnoticed spread through planting material. Recent advances in high-throughput sequencing (HTS) have greatly expanded knowledge of plant viral diversity and plant-virus interactions, including the identification of emerging and re-emerging viruses. However, translating virome-level discoveries into practical disease management tools remains a significant challenge, particularly for field-level surveillance and sustainable crop protection. Mango (Mangifera indica), a globally important fruit crop, exemplifies this gap, as virus infections are poorly correlated with visible symptoms and reliable on-site diagnostic tools are limited. In this study, HTS was employed to reconfirm the presence of mangifera indica latent virus (MiLV) and mangifera virus 1 (MaV-1) in mango plants and to generate validated genomic information for diagnostic assay development. Building on these data, reverse transcription-recombinase polymerase amplification (RT-RPA) assays were developed and optimized for rapid virus detection. The assays operated efficiently under isothermal conditions (40 °C) within 25 min. The MiLV RT-RPA assay enabled direct detection from crude leaf extracts without the need of RNA purification, whereas MaV-1 detection required purified RNA as template. Sensitivity analysis showed that the MiLV RT-RPA assay detected viral RNA up to 0.01 fg µl⁻¹ (equivalent to a 10⁻¹⁰ dilution of 100 ng µl⁻¹ RNA) and MaV-1 up to 0.1 ng µl⁻¹ showed complete concordance with RT-PCR (Cohen's K = 1.00). Validation using field-collected symptomatic and asymptomatic samples demonstrated complete concordance with conventional RT-PCR. The detection of both viruses in young grafted plants showed the potential role of vegetative propagation in virus dissemination and emphasizes the importance of screening both scion and rootstock materials. This study demonstrates how HTS-based insights into plant viral diversity can be effectively translated into rapid, field-deployable molecular diagnostics. The developed RT-RPA assays provide a practical tool for routine virus surveillance, certification of virus-free planting material, thereby contributing to improved stress resilience and sustainable mango production.