Diversity of frankiae in soils from five continents.

Clone libraries of nifH gene fragments specific for the nitrogen-fixing actinomycete Frankia were generated from six soils obtained from five continents using a nested PCR. Comparative sequence analyses of all libraries (n=247 clones) using 96 to 97% similarity thresholds revealed the presence of three and four clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively. Diversity of frankiae was represented by fewer clusters (i.e., up to four in total) within individual libraries, with one cluster generally harboring the vast majority of sequences. Meta-analysis including sequences previously published for cultures (n=48) and for uncultured frankiae in root nodules of Morella pensylvanica formed in bioassays with the respective soils (n=121) revealed a higher overall diversity with four and six clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively, and displayed large differences in cluster assignments between sequences retrieved from clone libraries and those obtained from nodules, with assignments to the same cluster only rarely encountered for individual soils. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules indicating the inadequacy of bioassays for the analysis of frankiae in soil and the role of plants in the selection of frankiae from soil for root nodule formation.

Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents.

Bioassays with Morella pensylvanica as capture plant and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed were used to investigate the diversity of Frankia in soils over a broad geographic range, i.e., from sites in five continents (Africa, Europe, Asia, North America, and South America). Phylogenetic analyses of 522-bp nifH gene fragments of 100 uncultured frankiae from root nodules of M. pensylvanica and of 58 Frankia strains resulted in a clear differentiation between frankiae of the Elaeagnus and the Alnus host infection groups, with sequences from each group found in all soils and the assignment of all sequences to four and five clusters within these groups, respectively. All clusters were formed or dominated by frankiae obtained from one or two soils with single sequences occasionally present from frankiae of other soils. Variation within a cluster was generally low for sequences representing frankiae in nodules induced by the same soil, but large between sequences of frankiae originating from different soils. Three clusters, one within the Elaeagnus and two within the Alnus host infection groups, were represented entirely by uncultured frankiae with no sequences from cultured relatives available. These results demonstrate large differences in nodule-forming frankiae in five soils from a broad geographic range, but low diversity of nodule-forming Frankia populations within any of these soils.