UBC Theses and Dissertations
Purification and serology of apple chlorotic leaf spot virus isolated from Prunus glandulosa Thompson, Daniel Andrew
Two isolates (A and B) of apple chlorotic leafspot virus (ACLSV) associated with line pattern symptoms in Prunus, glandulosa were isolated in Chenopodium quinoa and purified by a simple method using bentonite clarification, polyethylene glycol precipitation, and caesium sulphate isopycnic centrifugation. Symptoms on herbaceous hosts were as previously reported for ACLSV. No symptoms were observed on a range of ACLSV woody host indicators. Isolates A and B had a coat protein MW of 26 and 24.5 kilodaltons respectively, ssRNA of MW 2.6 x 10⁶ daltons, and dsRNA of MW 5.6 x 10⁶, 4.9 x 10⁶, and 4.5 x 10⁶ daltons. The particle widths were 12 nm and lengths were 782 nm (A) and 732 nm (B). Buoyant density in caesium sulphate was 1.27 g/cc³. In thin sections of C. quinoa, flexuous rods were seen in the cytoplasm and nucleus of young sieve-tube members. Polyclonal antisera prepared against the A and B isolates had high background reactions and required cross-adsorption with host sap. Three monoclonal antibodies (MAB) against isolate A detected Prunus strains while a fourth detected both Malus and Prunus strains in C. quinoa with low background reactions. The broad spectrum MAB could not be used as a trapping antibody but could be directly conjugated with alkaline phosphatase or used in an indirect triple-antibody sandwich ELISA. Tobacco mosaic virus (TMV) recovered from P. glandulosa was identical in serological and physical properties to the TMV-U1 type strain.
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