Discovery of a divergent apple stem pitting virus variant and development of an RT-PCR assay for improved detection in South African pear orchards

Abstract In this study, the prevalence and genome diversity of apple stem pitting virus (ASPV) in commercial pear orchards in South Africa were investigated. Leaves were collected from trees in two geographically distinct regions, representing six different pear cultivars covering 12 orchards. Total RNA was extracted and assayed for ASPV using RT-PCR. A subset of 18 samples was subjected to high-throughput sequencing (HTS) for a more detailed analysis. Bioinformatic analyses of the HTS data, led to the identification and sequencing of a divergent ASPV variant with 85.10% nucleotide identity to the closest ASPV match in GenBank (isolate 13TF179E, MZ148065.1). Additionally, the study reports the development and validation of an end-point multiplex RT-PCR assay capable of detecting all known pear ASPV variants in South Africa. The multiplex produces three distinct amplicons: one targeting actin for an internal control, one to broadly detect foveaviruses, and one specific to ASPV. The sequence analyses of known ASPV variants confirmed high sequence diversity within this species and emphasized the need to further investigate the relationship between ASPV isolates.