Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors

Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, or iginally designed as polymerase chain reaction primers, as hybridization pr obes for the nonradioactive detection of Italian clover phyllody (ICPh) phy toplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius var iegatus) 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 prob es that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigenin-labeled probes were det ected in both plant and insect tissue using a chromogenic alkaline phosphat ase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate re action. An enhancement of a signal was observed in plant tissue when a tyra mide signal-amplification procedure was incorporated into the biotin or dig oxigenin detection systems. The results obtained using these techniques wit h 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 diffe rent phytoplasmas present in multiple infections, this technique should pro vide a powerful new tool for epidemiological and vector ecology studies.