Polyvalent detection of fruit tree tricho, capillo and foveaviruses by nested RT-PCR using degenerated and inosine containing primers (PDO RT-PCR)

A polyvalent nested RT-PCR test was developed to detect five well characterised filamentous fruit tree viruses, namely ACLSV, SCGRMV, ASPV, ASGV and CVA. Short stretches of homology located in the RNA polymerase encoding domain of the viral genomes were identified by multiple sequence alignments. The Polyvalent Degenerated Oligonucleotide nested RT-PCR (PDO nested RT-PCR) consisted as a first combined RT-PCR with a set of inosine containing degenerated primers, from which an aliquot was submitted to a nested PCR assay. The final 362 bp PCR product was successfully amplified from nucleic acid extracts obtained from leaf samples using two different silica-capture extraction procedures. All the viruses targeted as well as the Apricot latent foveavirus (ALV) could be detected. Sensitivity and polyvalence of the PDO test was evaluated by comparison to ELISA and to the homologous RT-PCR test previously developed for ACLSV. PDO RT-PCR was also used to screen a collection of Prunus graft-transmissible diseases of unknown aetiology maintained on GF305 peach seedlings. Positive amplification could be obtained from virus isolates or partially characterised new viruses associated with cherry and peach disorders. For identification of viral aetiologic agent candidates, PDO amplicons were sequenced allowing the identification of apparently new tricho-, fovea-and capilloviruses isolates or strains.