Abstract :
Isolates of apple stem pitting (ASP) and pear vein yellows
(PVY) were sap transmitted frotn apple and pear cuitivars,
respectively, to Nieotiana oeeidentalis ssp. obliqua.
Total nucleic acid extraction, including a proteinase Kdigestion
of leaf homogenates prior to phenol/
chloroform extraction, was of limited use for RNA
template preparation. Amplification products were
obtained from Aʹ, oeeidentalis and from apple and pear
leaf tissue. Three primer combinations near the viral 3 -
terminus resulted in amplification products of 264 bp,
610bp and 1548bp. The specificity of the products was
confirmed by restriction enzyme digestion. To improve
RNA template preparation and the reliability of PCR
tests from woody plant tissue, a fusion protein-specific
antiserum 647 was prepared to in vitro expressed viral
coat protein. This allowed us effective immunocapture
RT-PCR assays from woody tissue infected with either
ASPV and PVYV. Antiserum 647 reacted in an indirect
plate-trapped ELISA with ASPV isolates from N, oeeidentalis,
whereas two antisera prepared previously to a
different coat protein fusion construct were not successful
in any of the ELISA types investigated. In addition a
positive test result was obtained from PVY infected pear
but not from apple
