CHEMILUMINESCENT DETECTION OF POTATO AND POME FRUIT VIROIDS BY DIGOXIGENIN-LABELED DOT-BLOT AND TISSUE BLOT HYBRIDIZATION

A chemiluminescent molecular hybridization protocol was compared to P- 32 autoradiography for detecting potato spindle tuber viroid (PSTVd) a nd apple scar skin group viroids (ASSVd). Labeled cRNA probes for PSTV d and ASSVd were synthesized by SP6 RNA polymerase transcription using digoxigenin-11-UTP or alpha-[P-32]UTP. Dot blot hybridization of puri fied viroids and sap extracts from infected plants showed that chemilu minescent detection using digoxigenin-labeled probes was as sensitive as autoradiography using P-32 probes. A minimum of 2.0-2.5 pg purified viroid was detected. ASSVd could be detected in as little as 0.4 ng o f total nucleic acid extract from infected tissue or in sap extracts d iluted to 10(-3) with healthy extracts. Tissue blots of PSTVd-infected potato tubers and tomato roots, stems and leaves and ASSVd-infected a pple fruit, stems and petioles, gave positive reactions when hybridize d with the digoxigenin probe. No reaction with similar tissues from he althy plants was observed.