Sensitive detection of apple stem grooving and apple stem pitting viruses from infected apple trees by RT-PCR

Apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) occur worldwide in both apple and pear. The reverse transcription - polymerase chain reaction (RT-PCR) techniques became recently available for diagnosis of these fruit tree viruses. These techniques appear more effective than enzyme linked immunosorbent assay (ELISA) when no good quality antisera are available (ASPV), when the virus is present in low concentration, or the detection possible only from specific tissue or at specific time periods from their woody tissue hosts (ASGV). Using degenerate primers designed from the comparison of published sequences for apple chlorotic leaf spot virus (ACLSV), ASGV, and ASPV, amplification products have been obtained from herbaceous host plants and apple trees infected with different isolates of ASGV and ASPV. Their sequencing allowed the design of specific primers for the detection of these viruses in apple trees all year long from leaves or bark of annual shoots. With a first generation of specific primers. the quality of template viral RNA preparations fi om these woody hosts has been the limiting factor for a reliable detection of these viruses, in these conditions ds-RNA preparations proved to be the best target for sensitive and reproducible detection of virus infection. With a second generation of specific primers (for ASGV) exhibiting better characteristics and used in combination with more stable enzymes for the reverse transcription step. and with more stringent conditions for amplification (higher cycling temperature), reliable results can be obtained directly from diluted crude extracts from the same woody material.