Whole-Cell Hybridization of Frankia Strains with Fluorescence- or Digoxigenin-Labeled, 16S rRNA-Targeted Oligonucleotide Probes

Abstract

Whole-cell hybridization with non-radioactively labeled oligonucleotide probes was used to detect and identify Frankia strains in pure cultures and in nodules. Digoxigenin-labeled probes, which were detected with antibody-alkaline phosphatase conjugates, were more suitable for in situ detection of Frankia strains than fluorescent probes since the sensitivity of the former was higher and problems arising from the autofluorescence of cells and plant material were avoided. Successful detection of Frankia strains in paraformaldehyde-fixed cell material with digoxigenin-labeled oligonucleotide probes depended on pretreatments to permeabilize the cells. Specific hybridization signals on vesicles were obtained after lysozyme pretreatment (1 mg ml ^-1 for 30 min at 20°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. Identification of Frankia vesicles in nodule homogenates was possible only after the removal of the polysaccharide capsule surrounding the vesicles. Incubation with H ₂ O ₂ (15% in water for 1 h at room temperature) before lysozyme and detergent treatments was found to facilitate specific hybridization. No filaments or spores could be detected in nodule homogenates. 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.