Evaluation of a 23S rRNA insertion as target for the analysis of uncultured Frankia populations in root nodules of alders by whole cell hybridization

An actinomycetes-specific insertion in domain III of the 23S rRNA was used as target for the analysis of uncultured Frankia populations in nodule homogenates of alders by whole cell hybridization. Fluorescent, Cy3-labeled oligonucleotide probes enabled detection of filaments and vesicles without permeabilization whereas detection of spores required a previous permeabilization with lysozyme. The signal intensity obtained on spores, however, remained quite low and their detection was not quantitative. Digoxigenin-labeled probes also allowed the detection of filaments, vesicles and spores of Frankia. However, for all cell types a previous permeabilization with SDS/DTT and an additional incubation with lysozyme was necessary Hybrid detection with antibody/alkaline phosphatase conjugates and NBT/BCIP as substrate resulted in clear images of filaments, vesicles and spores and in cell numbers comparable to those obtained by fluorescent probes. The analysis of Frankia populations in nodule homogenates of different alders (Alnus glutinosa, A. incana, A. viridis and A. nepalensis) could be performed on subgroup-level with both oligonucleotide as well as in vitro transcript probes. The analysis revealed the presence of only one Frankia population in every nodule homogenate. Filaments and vesicles in nodules of the spore (-) type as well as filaments, vesicles and spores in nodules of the spore (+) type always belonged to the same group. Populations in nodules of the sport (-) type were usually identified as Frankia population belonging to group IV of the Alnus host infection group, with the exception of the Frankia population in nodules of A. nepalensis which belonged to group IIIa. Nodules of the spore (+) type contained Frankia populations either belonging to group IIIa or to group IV.