Assessing Frankia populations in plants and soil using molecular methods

In recent years, molecular approaches have increasingly supplemented nodulation‐dependent detection methods for studying Frankia populations in nature. The new methods are revealing much about the genetic diversity and distribution of Frankia , as well as refining and expanding knowledge about endophyte‐host specificities. PCR‐based approaches have been used to unravel the phylogenetic relationships of isolates, as well as of uncultured endophytes in root nodules of many actinorhizal plants from which no isolates have been obtained. A comparative sequence analysis of PCR‐amplified 16S ribosomal DNA led to the emendation of the family Frankiaceae to contain only the genus Frankia with four main subdivisions: (i) a large group mainly comprising Frankia alni and other typical nitrogen‐fixing strains belonging to the Alnus and the Casuarina host infection groups, respectively, (ii) uncultured endophytes of Dryas, Coriaria and Datisca species, (iii) strains of the Elaeagnus host infection group and (iv) atypical non‐nitrogen‐fixing strains. A considerable diversity among both cultured Frankia strains and uncultured endophytes in nodules was indicated using RFLP analyses of PCR‐amplified fragments of the 16S rRNA gene, the glutamine synthetase II ( gln II) gene, the intergenic spacer of the 16S‐23S rRNA operon or the intergenic spacer between the nitrogenase nif H and nif D ( nif H‐D) or the nif D and nif K ( nif D‐K) genes. The growing database of discriminative target sequences for frankiae is increasingly exploited for studies on the distribution of specific Frankia populations in the environment using PCR or in situ hybridization. Until recently, most studies have focused on the analysis of Frankia populations in root nodules, the natural locale of enrichment for this organism. These populations, however, represent only the fraction of physiologically active, infecting frankiae in soils rather than the total Frankia population. Future approaches to studies of Frankia populations should therefore incorporate the many opportunities for more than just phylogenetic analyses, the description of diversity and studies of Frankia populations in nodules. The molecular approaches open the door to more sophisticated studies of environmental influences on the dynamics of indigenous or introduced Frankia populations in plants and soil. These studies may lead to advancements in the management of actinorhizal plants and Frankia , provided specific Frankia populations can be attributed with silviculturally beneficial features. Such features include persistence and the growth in soil, competition with less efficient Frankia populations for nodule formation, prompt and efficient nodule formation and an ultimately superior nitrogen‐fixing capacity.