Detection and localization of viruses in orchids by tissue-print hybridization

A tissue-print hybridization assay for the detection and localization of viruses in orchids is described. Orchid leaves were cut transversely and the cut surfaces were printed on to nitrocellulose paper. Because orchid leaves are often succulent, there is excellent transfer of plant sap to the membrane, resulting in clear tissue prints. The nucleic acid on the nitrocellulose membrane was then hybridized to radio-labelled cDNAs of cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV), and the viruses were detected by autoradiography. The assay was able to detect as little as 20 pg of purified virus, which is 2–3 orders of magnitude more sensitive than the enzyme-linked immunosorbent assay (ELISA) for CyMV. When tissue prints derived from different parts of the plants were used for hybridization, the method was able to show the distribution of viruses within the plants. On the basis of its sensitivity and ease of use, this method should find wide application in the detection and localization of viruses in orchids as well as in other succulent plants.