ISHS Acta Horticulturae 309: XV International Symposium on Fruit Tree Diseases
CDNA CLONING OF DSRNA ASSOCIATED WITH APPLE STEM PITTING DISEASE AND EVIDENCE FOR THE RELATIONSHIP OF THE VIRUS-LIKE AGENTS ASSOCIATED WITH APPLE STEM PITTING AND PEAR VEIN YELLOWS |
| Authors: | W. Jelkmann, L. Kunze, H.-J. Vetten, D.-E. Lesemann |
| Abstract:
From apple stem pitting (ASP)-affected apple an agent was sap transmitted to Nicotiana occidentalis '37 B'. Plant tissue of both hosts was used for double-stranded RNA extractions. Analogous experiments were performed with pear vein yellows (PVY)-diseased pear plants. DsRNA of ASPV was denatured, reverse transcribed into cDNA, blunt end annealed to Bluescript M13+, and Escherichia coli DH5  transformed.
Specificity of selected cDNA clones was confirmed in slot blot- and Northern blot hybridizations, using plasmid pASPV206 carrying the putative viral coat protein gene and oligo-labelled with [-32P]dATP. Positive reactions were obtained with samples from ASP- and PVY-affected tissue as well as with those from N. occidentalis. The viral coat protein gene was subcloned into a suitable plasmid, coat protein expressed in E. coli, and used to immunize rabbits.
The resulting antisera enabled us to detect virus-like particles in tissue extracts of woody hosts affected by ASP and PVY using IEM techniques.
Decoration of PVY-associated particles with antiserum to coat protein of ASPV and results of the hybridization experiments provide evidence for a viral etiology of the two diseases and for a close relationship of the two entities associated with ASP and PVY.
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