WHOLE-CELL HYBRIDIZATION OF FRANKIA STRAINS WITH FLUORESCENCE-LABELEDOR DIGOXIGENIN-LABELED, 16S RIBOSOMAL-RNA-TARGETED OLIGONUCLEOTIDE PROBES

Whole-cell hybridization with non-radioactively labeled oligonucleotid e probes was used to detect and identify Frankia strains in pure cultu res and in nodules. Digoxigenin-labeled probes, which were detected wi th antibody-alkaline phosphatase conjugates, were more suitable for in situ detection of Frankia strains than fluorescent probes since the s ensitivity of the former was higher and problems arising from the auto fluorescence of cells and plant material were avoided. Successful dete ction of Frankia strains in paraformaldehyde-fixed cell material with digoxigenin-labeled oligonucleotide probes depended on pretreatments t o permeabilize the cells. Specific hybridization signals on vesicles w ere obtained after lysozyme pretreatment (1 mg ml-1 for 30 min at 20-d egrees-C). Reliable penetration of the antibody-enzyme conjugate into hyphae required additional washing with the detergent Nonidet P-40 (0. 1%) and toluene (1% in ethanol) after lysozyme treatment. Identificati on of Frankia vesicles in nodule homogenates was possible only after t he removal of the polysaccharide capsule surrounding the vesicles. Inc ubation with H2O2 (15% in water for 1 h at room temperature) before ly sozyme and detergent treatments was found to facilitate specific hybri dization. No filaments or spores could be detected in nodule homogenat es. This technique should be a powerful tool in the identification of Frankia isolates, in the characterization of as-yet-uncultured nodule populations, and in the confirmation of the origin of unusual Frankia isolates.