Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.
Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5' end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.