Abstract
Plant positive-strand RNA viruses exhibit high mutation rates that drive genetic diversity and adaptation to different hosts. While herbaceous perennials potentially serve as reservoirs for viral diversification through persistent infections, the mechanisms underlying this process remain largely unexplored. We investigated the temporal dynamics of viral load and genomic diversity of Plantago asiatica mosaic virus (PlAMV) during long-term infection in its natural herbaceous perennial host, Plantago asiatica. Mechanical inoculation of P. asiatica with PlAMV isolate Vi resulted in systemic infection that persisted through aboveground senescence (withering) and regeneration cycles. RT-qPCR analysis revealed that PlAMV migrated from inoculated leaves to uninoculated upper leaves and roots, maintaining infection during overwintering by accumulating in roots. Population diversity analysis of the triple gene block protein (TGBp) 1-3 region showed significantly increased mutation frequencies after the aboveground senescence and regeneration (172 days post-inoculation) compared to those observed before senescence (21 and 45 days post-inoculation) in three of four infected plants. Chimeric viruses constructed with mutations acquired during persistent infection exhibited altered pathogenicity and accumulation patterns in P. asiatica, Nicotiana benthamiana, and Chenopodium quinoa. Host range analysis of natural PlAMV isolates representing the major phylogenetic clades revealed clade-dependent and even isolate-specific differences in infectivity across 17 plant species. These findings provide the first experimental evidence that this perennial species acts as an amplifier of viral genetic diversity, contributing to a deeper understanding of how persistent infection in such a host can shape virus evolution and potentially facilitating the emergence of variants with expanded host ranges.