PCR-RFLP direct fingerprinting of uncultured Frankia microsymbiont from Dryas drummondii nodules

The total nucleic acids directly obtained by a mini-extraction protocol from root nodules of Dryas drummondii were pure enough to be amplified by the Polymerase Chain Reaction (PCR). When nif primers targeted at the D-K intergenic region were used, single ge nomic fragments of variable size were obtained from Dryas and Alnus nodules, and from Frankia reference strains isolated from Alnus, Casuarina, Elaeagnus, Hippophae and Shepherdia. After RsaI and HaeIII endonucleases restriction of such amplified fragments (PCR-RFPL), very discriminant fingerprints were obtained. The microsymbiont of D. drummondii appeared very distant from the two known phenotypic- and 16S rRNA-based groups: (i) the Alnus-Casuarina-infective group, and (ii) the Elaeagnus-infective group. Direct PCR-RFLP fingerprinting of uncultured natural Frankia communities will serve to enrich existing databases and make the identification and the evaluation of Frankia biodiversity easier.