ABSTRACT 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.