RAPID DIFFERENTIATION OF CLOSELY-RELATED ISOLATES OF 2 PLANT-VIRUSES BY POLYMERASE CHAIN-REACTION AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

Immunocapture reverse transcriptase-polymerase chain reaction (RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the product has been shown to be an effective procedure for discrim inating serologically indistinguishable isolates of two plant viruses, raspberry bushy dwarf (RBDV) and zucchini yellow mosaic (ZYMV). For b oth viruses, only limited sequence information was available at the ti me of primer design, but most of the isolates which were tested could be amplified (the one exception being a serologically quite distinct i solate of ZYMV). Restriction endonucleases revealing diagnostic RFLPs were readily identified. Each of two isolates of ZYMV could be detecte d in the presence of the other and the relative proportions approximat ely quantified by visual estimation of the relative intensity of the a ppropriate bands. A range of isolates of different RBDV pathotypes wer e compared; isolates were grouped in ways that accorded with their kno wn history. Computer analysis of the published sequence from which the primers had been derived showed the sequenced isolate to be identical with an isolate imported from the USSR. The PCR/RFLP procedure is rap id (it can be completed in less than 2 days), effective and will proba bly be generally applicable to distinguishing closely related virus is olates, even where little sequence information is available.