Rapid and sensitive detection of Apple stem pitting virus in apple trees through RNA amplification and probing with fluorescent molecular beacons

Currently, detection of Apple stein pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in 'Golden Delicious' and 'Gravenstein' apple trees was compared throughout the year with mechanical inoculation onto Nicotiana occidentalis 37B, a candidate indicator for ASPV. This revealed that only AmpliDet RNA consistently detected the virus in bark tissue, irrespective of the season. Season-specific tissues such as buds, petals, and fruits, but not leaves, also were reliable sources for detection of ASPV by the AmpliDet RNA system.