Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 7, 2010
Publication Date: January 3, 2011
Citation: Liming, L., Li, R., Mock, R.G., Kinard, G.R. 2011. Development of a polyprobe to detect six viroids of pome and stone fruits. Journal of Virological Methods. 171:91-97.
Interpretive Summary: Viroids are small segments of genetic code (RNA) that are not covered by a protein coat or shell. They can infect and cause a wide range of diseases in plants. Many types of fruit trees including apples, pears, peaches, plums, and cherries can be infected by one or more of six different viroids. If present in trees, it is difficult to detect these viroids before tree cuttings are shipped to countries around the world. Here we report an improved method to test for and detect these viroids that is faster, more reliable and less expensive than methods that are currently used. A probe was constructed that will bind to any one of these six viroids in a lab test, as evidenced by an emission of light that can be detected by a sensitive camera. This test has the potential for being used routinely in programs that test and ship fruit tree material worldwide.
A simple and sensitive dot blot hybridization assay using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous detection of six viroids that infect pome and stone fruit trees. The polyprobe was constructed by sequentially cloning partial sequences of each viroid into a single vector with run-off transcription driven by the T7 promoter. All six viroids were detectable within a dilution range of 5-3 to 5-4 in total nucleic acid extracts from infected trees. Individual trees were co-inoculated to create mixed infections and all four pome fruit viroids and both stone fruit viroids could be detected in pear and peach trees, respectively, using the polyprobe. The results of the assays using the polyprobe were comparable to those using single probes. The methods were validated by testing geographically diverse isolates of viroids, as well as field samples from several collections in the U.S. The assay offers a rapid, reliable and cost-effective approach to the simultaneous detection of six fruit trees viroids and has the potential for routine use in quarantine, certification, and genebank curation programs where numerous samples are tested and distributed worldwide.