Dr. Webb et al., Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors, PHYTOPATHOL, 89(10), 1999, pp. 894-901
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.