Localization of typical and atypical Frankia isolates from Casuarina sp. in nodules formed on Casuarina equisetifolia

Aims Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia . Methods Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored. Results The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. q PCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only. Conclusions These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia , but has likely been isolated as a surface contaminant from Casuarina nodules.