Integrated Molecular and Biological Assays for Rapid Detection of Apple Scar Skin Viroid in Pear

Abstract

A viroid in oriental pears (Pyrus hybrids) was associated with dappling and cracking fruit symptoms in graft-inoculated Malus pumila cv. Lord Lambourne. The viroid hybridized with a cRNA probe to apple scar skin viroid (ASSVd). Detection of the viroid by hybridization assays of tissue blots gave erratic results when used with some pear cultivars. Two methods for improving detection of the pear viroid were compared. In the first, pear bark chips were graft-inoculated into a viroid host, which was then tested by tissue blot hybridization. Digoxigenin-labeled cRNA probe and an antidigoxigenin antibody conjugate were used with a chemiluminescent substrate for detection of viroid in the tissue blots. Greenhouse-grown Pyrus communis cv. Nouveau Poiteau and Malus pumila cv. Virginia Crab amplified ASSVd to levels that were detectable in tissue blots of shoots and leaf petioles, even though the leaves and shoots were symptomless. In the second method, pears were indexed on the ASSVd indicator plants Stark's Earliest and Sugar Crab apple grown in growth chambers under a 24-h photoperiod. ASSVd-infected apple and the infected pears induced similar symptoms of leaf epinasty in the indicators in 5 to 8 weeks. Although infection in pear was rapidly and reliably detected by both methods, tissue blot hybridization was faster. Inoculation of an amplification host was useful when numerous cultivars or genetically diverse plants were tested and genetically similar positive and negative control plants were unavailable.