Detection and characterization of European apple stem pitting virus sources from apple and pear by PCR and partial sequence analysis

Apple stem pitting, pear vein yellows, pear stony pit and quince sooty ringspot isolates collected in Europe were propagated in the field in woody indicators. RNA template preparation from woody plant tissue was performed by immunocapture using antiserum 647 that had been obtained from the ASPV coat protein fusion protein of isolate PA66. The genomic RNA was subjected to RT-PCR without a separate nucleic acid extraction step. A primer combination from the 3'-terminal region of the virus resulting in an amplification product of 264 bp was used for routine detection of all virus sources. All ASPV diseased isolates that induced symptoms on woody indicators two years after infection were positive by IC-RT-PCR, indicating the high sensitivity of the test under field conditions. Selected isolates of apple stem pitting virus from apple and pear were analyzed by a primer combination encompassing the coat protein gene. Amplification products of different electrophoretic mobility were observed, suggesting variations among the coat proteins of different sources. The amplification products of five isolates were cloned and sequenced revealing sequence variations and deletions.