Evaluation of reverse transcription – polymerase chain reaction assays for detecting Apple chlorotic leaf spot virus in certification and quarantine programs

Abstract

Apple chlorotic leaf spot virus (ACLSV), the type member of the genus Trichovirus, family Flexiviridae, occurs worldwide in a wide range of rosaceous hosts and is an important pathogen targeted in certification and quarantine programs. Detection of ACLSV in infected hosts has been based on inoculation by grafting (indexing), enzyme-linked immunosorbent assays (ELISA), and more recently, on reverse transcription – polymerase chain reaction (RT–PCR). The latter has the potential to become a reliable molecular indexing tool. However, its suitability for routine diagnosis needs to be verified for a broad spectrum of virus isolates in a wide range of hosts. In this study, oligonucleotide primers targeting the coat-protein (CP) region and the replicase region of ACLSV were compared in RT–PCR assays and applied to detection of virus isolates from natural hosts originating from domestic and imported sources of plant germplasm. Primers CLS6860 and CLS7536 amplifying a 676-bp (base pair) fragment in the CP-coding region of ACLSV were the most reliable, detecting all isolates, in all tested hosts. The forward/reverse primer pair 4F/4R, which amplifies a 390-bp fragment in the replicase-coding region, detected most of the tested ACLSV isolates, but generated some nonspecific amplifications with several other fruit-tree viruses in the family Flexiviridae. Alignment of the associated nucleotide sequences of the respective viruses indicated high levels of identity with the targeted replicase region, supporting the use of this region for broad-spectrum detection of distantly related viruses. However, this region is less useful for accurate virus identification. Comparison of the RT–PCR data with the ELISA and indexing results highlighted the advantages of a reliable molecular assay for problematic hosts.