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 A', 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.

The high molecular weight dsRNA associated with little cherry disease (LCD) was extracted from infected plant tissue and cloned as cDNA. The sequence of the 3' 8337 nucleotides was determined. Computer assisted translation of the sequence portion identified six open reading frames potentially encoding proteins (from 5' to 3') with molecular masses of 70 kDa, 61 kDa, 46 kDa, 76 kDa, 21 kDa and 27 kDa respectively. A 3'-terminal non-translated region of 210 nucleotides was present. The 70 kDa protein represents a homolog of the cellular HSP70 heat shock proteins, and the 61 kDa protein showed homology to the similarly encoded products of beet yellows (BYV), citrus tristeza (CTV) and lettuce infectious yellows (LIYV) closteroviruses. The putative coat protein (CP) was found to be of 46 kDa and its diverged copy of 76 kDa. The potential coding capacity of these notably large closterovirus proteins was confirmed by their expression in vitro and immunoblotting. No proteins with significant similarity to the two C-terminal proteins were identified, but they are related in molecular mass and location to BYV. The gene arrangement as well as the alignments of the closteroviruses CPs and their diverged copies suggest that the mealybug transmissible virus associated with LCD takes an intermediate evolutionary position between the aphid- and whitefly transmissible closteroviruses.

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