Abstract
AbstractIsolates of apple stem pitting (ASP) and pear vein yellows (PVY) were sap transmitted from apple and pear cultivars, respectively, to Nicotiana occidentalis 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 N. occidentalis 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. occidentalis, 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.
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