SPECIFIC IDENTIFICATION OF COCONUT TINANGAJA VIROID FOR DIFFERENTIAL FIELD DIAGNOSIS OF VIROIDS IN COCONUT PALM

Tinangaja is a widespread lethal disease of putative viroid etiology a ffecting coconut palm on the island of Guam. Determination of its dist ribution and mode of spread requires a simple and reliable diagnostic procedure that is specific for the associated coconut tinangaja viroid (CTiVd). A method of extracting tissue followed by analytical agarose gel electrophoresis for CTiVd detection has been developed and used t o identify the viroid in leaf samples of suspect symptomatic palms gro wing in the field. Two-dimensional polyacrylamide gel electrophoresis showed that the viroid band contained circular molecules that are typi cal for viroids. Confirmation of the identity of CTiVd and detection o f law levels of viroid below the threshold of detection by agarose gel electrophoresis was achieved either by diagnostic oligonucleotide-pro be (DOP) hybridization assay or by reverse-transcription polymerase ch ain reaction (RT-PCR) with the oligonucleotide probe as one of the two PCR primers. RT-PCR was not substantially more sensitive than DOP-hyb ridization assay. This procedure also was applicable to coconut cadang -cadang viroid (CCCVd), and oligonucleotide probes designed to be spec ific for either CTiVd or CCCVd distinguished between these two viroids in coconut leaf extracts. This strategy provides a rapid and specific indexing procedure for the two characterized viroids of coconut palm and will be applicable to further studies on the viroid-like sequences previously reported in tropical monocotyledons.