Molecular characterization of Frankia microsymbionts from spore-positive and spore-negative nodules in a natural alder stand.

Among the Frankia strains capable of establishing a nitrogen-fixing symbiosis with actinorrhizal plants, in planta sporangial formation is not universal and has led to the distinction between spore-positive (Sp+) and spore-negative (Sp-) nodules. Numerous Frankia strains have been isolated in pure culture from Sp+ nodules of different host plants, but, although they were able to reinfect their respective host plant, none of them was able to differentiate endophytic sporangia under laboratory conditions. The first step of this study was to demonstrate, at the molecular level, the existence of specific Sp+ strain genotypes differing from Sp- strain genotypes in a single alder stand. In a second step, by way of PCR amplification and sequencing of the PCR products, we have characterized oligonucleotide primers specific for the genus Frankia and for each of the two types of Frankia microsymbionts able, or not, to differentiate sporangia inside natural green alder nodules. These primers applied in PCRs with DNA extracted from nodules confirmed the morphological identification and revealed the presence of nodules colonized by both types of actinomycetes. Finally, a preliminary PCR study was conducted with DNA extracted directly from soil samples which permitted checking the rhizosphere of Sp+ and Sp- nodules for the presence of the corresponding strains.