Deciphering the passion fruit woodiness disease associated viruses in India, and development of real-time turbidimetric RT-LAMP assay and multiplex PCR for their detection

Passion fruit woodiness disease is a major constraint to passion fruit production in India, causing substantial reductions in yield and fruit quality. The disease is often associated with mixed viral infections, complicating diagnosis and disease management. This study aimed to characterize viruses associated with passion fruit woodiness disease in major passion fruit-growing regions of Karnataka, Kerala, and Tamil Nadu using high-throughput sequencing and molecular diagnostic approaches. Disease incidence varied among surveyed locations and was generally higher in warmer production regions. High-throughput sequencing enabled the reconstruction of complete or near-complete genomes of three RNA viruses: passiflora latent virus ( Carlavirus latenspassiflorae ) (PLV), potato virus S ( Carlavirus sigmasolani ) (PVS), and cucumber mosaic virus ( Cucumovirus CMV ) (CMV). Phylogenetic analyses revealed close relationships between the detected isolates and previously reported strains, while recombination and single nucleotide polymorphism analyses indicated substantial genetic diversity within viral populations. Real-time turbidometric reverse-transcription loop-mediated isothermal amplification assays successfully detected all three viruses, with PLV exhibiting the fastest amplification and highest viral load, followed by PVS and CMV. Passiflora latent virus was detected in 12 locations, CMV in 10 locations, and PVS in 4 of the 16 samples analyzed. Mixed infections involving the three viruses were identified in four locations. Virus-specific primers were further validated through singleplex, duplex, and multiplex polymerase chain reaction assays, enabling simultaneous detection of all three viruses in a single reaction. The results were consistent with reverse-transcription loop-mediated isothermal amplification assays and were confirmed by Sanger sequencing. This study provides the first report of passiflora latent virus and cucumber mosaic virus infecting passion fruit in India and the first global evidence of potato virus S infecting this host. The findings demonstrate the widespread occurrence of mixed viral infections associated with passion fruit woodiness disease and establish sensitive, reliable, and scalable diagnostic tools for virus detection. These results contribute to improved disease surveillance, certification programs, and integrated management strategies for sustainable passion fruit production.