Molecular Detection and Sequence Analysis of Grapevine Rupestris Stem Pitting-Associated Virus in Vineyards

Abstract

Grapevine Rupestris stem pitting-associated virus (GRSPaV) is an economically important latent virus affecting grapevine productivity and plant vigor in vineyard ecosystems. Field surveys were conducted in major grape-growing regions to determine the presence, distribution, and molecular diversity of GRSPaV in commercial vineyards. Leaf and woody tissue samples were collected from both symptomatic and asymptomatic vines and subjected to molecular detection using reverse transcription polymerase chain reaction (RT-PCR) with virus-specific primers targeting conserved genomic regions. Amplification of expected fragments confirmed the presence of GRSPaV in multiple vineyards, including plants without visible symptoms, indicating widespread latent infection. Sequencing of RT-PCR products revealed high nucleotide identity with reference GRSPaV isolates reported from diverse viticultural regions worldwide. Phylogenetic analysis grouped field isolates into established GRSPaV lineages, while also revealing moderate sequence variability among isolates from different geographic locations and grape cultivars. The observed genetic diversity suggests ongoing viral evolution potentially influenced by vegetative propagation practices and long-term vineyard establishment. Disease incidence was higher in vineyards relying on uncertified planting material and continuous clonal propagation systems. Although infections were largely latent, subtle reductions in vine vigor, shoot growth, and yield potential were observed over successive seasons. The integration of RT-PCR detection and sequence analysis provided reliable diagnosis and improved understanding of virus epidemiology and distribution. The findings highlight the importance of certified virus-free planting material and routine molecular screening to prevent virus accumulation in vineyard systems.