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Oligonucleotides as Hybridization Probes to Localize Phytoplasmas in Host Plants and Insect Vectors

October 1999 , Volume 89 , Number  10
Pages  894 - 901

D. R. Webb , R. G. Bonfiglioli , L. Carraro , R. Osler , and R. H. Symons

First, second, and fifth authors: Department of Plant Science, Waite Institute, University of Adelaide, Glen Osmond, SA 5064, Australia; and third and fourth authors: Dipartimento Biologia Applicata alla Difesa delle Piante, Università di Udine, I-33100, Udine, Italy

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Accepted for publication 14 June 1999.

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.

Additional keywords: fluorescent in situ hybridization, 16S ribosomal RNA.

© 1999 The American Phytopathological Society