Molecular Evidence of the Relationship Between a Virus Associated with Flat Apple Disease and Cherry rasp leaf virus as Determined by RT-PCR.

Abstract

Flat apple disease-associated virus (FAV) was mechanically transmitted to the propagation host Chenopodium quinoa and double-stranded (ds)RNA recovered using CFII chromatography. Purified dsRNA was used to generate cDNA clones which were sequenced and the information used to design oligonucleotide primers for reverse transcription-polymerase chain reaction (RT-PCR) and tube capture (TC)/RT-PCR analyses. Oligonucleotide primers for RT-PCR analysis and dot blot hybridization using digoxigenin-labeled cDNA clones were used for the detection of FAV and Cherry rasp leaf virus (CRLV) in C. quinoa, in leaf and bud wood tissue of apple, or both. Primers JQ3D33FF/FR amplified a virus-specific 429-bp fragment and reliably detected all isolates of FAV and CRLV tested by RT-PCR and TC/RT-PCR. Primers JQ2C1FF/FR amplified a 370-bp fragment and detected FAV and some isolates of CRLV. Comparison of amino acid residues derived from the 429-bp fragments of FAV and CRLV gave 95% identity. The RT-PCR assays provided strong evidence of a relationship between FAV and CRLV. These assays were also used to confirm virus elimination in apple plants after heat therapy. Western blot analysis of FAV revealed capsid protein subunits of approximately 22 and 24 kDa. Our data support biological and serological evidence that FAV and CRLV are isolates of the same virus. Searches of the database produced sequence matches only with RNA2 of Apple latent spherical virus (ALSV), a new member of the family Comoviridae. This suggests that both primer pairs presumably target regions on RNA2 of FAV/CRLV and that these viruses may be more closely related to ALSV than to other members of this family.