Quantitative detection of four pome fruit viruses in apple trees throughout the year

Abstract

A one-step real-time RT-PCR assay (RT-qPCR) with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed to detect and quantify RNA targets from Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple chlorotic leaf spot virus (ACLSV) in infected apple trees. Single PCR products of 87 bp (ApMV), 70 bp (ASGV), 104 bp (ASPV) and 148 bp (ACLSV) were obtained, and melting curve analyses revealed distinct melting temperature peaks for each virus. A dilution series using in vitro synthesized transcripts containing the target sequences as standards yielded a reproducible quantitative assay, with a wide dynamic range of detection and low coefficients of variance. The content of selected viruses in apple plant tissues was stable throughout the year, and their accumulation did not significantly change between different plant tissues. The only minor exceptions were for ApMV and ACLSV, in which noticeable differences in their concentrations in various biological material were observed within the year. This divergence did not influence their year-round detectability. This one-step RT-qPCR assay is a valuable tool for year-round diagnostics, and molecular studies of the biology of ApMV, ASGV, ASPV and ACLSV.