Characterization of uncultured Frankia strains by 16S rRNA sequence analysis

Studies on the root nodules of <em>Coriaria nepalensis</em> using light and electron microscope revealed that the nodule structure and morphology of the endophyte is unique among the actinorhizal plants and similar only to those of <em>Datisca cannabina.</em> Plant cells containing the endophyte were relatively enlarged as compared to the non-infected cells. Transverse sections of the nodules revealed that the infected cells form a compact kidney-shaped zone around the stele which is located on one side of the nodule lobe. Inside the host cell, the hyphae of the endophyte were located in the peripheral region of the cytoplasm whilst the elongate vesicles were present near the central vacuole. Host cell mitochondria were abundant and mostly found in close vicinity of the hyphal/vesicular junction of the endophyte. <em>In situ</em> hybridization studies showed localization of the <em>nifH</em> mRNA within the elongate vesicles of the endophyte. The results indicate that the elongate vesicles of the <em>Coriaria</em> nodule endophyte are functionally identical to the spherical vesicles of the <em>Alnus</em> endophyte.Acetylene reduction (C ₂ H ₂ reduction) and uptake hydrogenase activity of the root nodules of <em>Coriaria nepalensis</em> and <em>Datisca cannabina</em> were measured to study seasonal fluctuations in the enzyme activities. Nitrogenase activity of the nodules of both plants showed biphasic curves with peaks in spring and late summer. Less than 15% of the enzyme activity was retained by the nodules in winter. Results showed that maximum nitrogenase activity coincides with the period of vigorous plant growth in both hosts. Uptake hydrogenase activity was detected in nodules of both plants throughout the year. The peak enzyme activity in both <em>Coriaria</em> and <em>Datisca</em> nodules was recorded in May. Vesicle cluster fraction of the crushed nodule suspensions showed the highest uptake hydrogenase activity, indicating that the enzyme is associated with the endophyte.Nine <em>Frankia-like a</em> ctinomycetes were isolated from root nodules of <em>Coriaria nepalensis.</em> All isolates formed hyphae and sporangia typical of <em>Frankia,</em> but failed to induce nodules on <em>Coriaria</em> seedlings, or reduce acetylene in pure culture. The relationship of these strains to atypical and typical <em>Frankia</em> strains isolated from other actinorhizal plants and various other actinomycetes was investigated by comparing fatty acid patterns. All <em>Frankia</em> strains, including atypical isolates, showed fatty acid profiles distinct from those of <em>Actinomyces</em> , <em>Geodermatophilus</em> , <em>Nocardia</em> , <em>Mycobacterium</em> and <em>Streptomyces.</em> For<em>Frankia</em> strains a characteristic pattern of five fatty acids was found that comprised 75% or more of the total content. Three subgroups were identified among 30 <em>Frankia</em> strains compared. Atypical strains isolated from <em>Coriaria</em> were found in the largest subgroup which contained most <em>Frankia</em> strains from other hosts, while ineffective strains from other hosts were distributed in all three subgroups.Non-infective, atypical strains isolated from <em>Coriaria</em> nodules were characterized further by partial 16S rRNA sequence analysis. Ribosomal RNA isolated from all atypical strains hybridized strongly with a <em>Frankia</em> genus probe and indicated that the isolates did belong to the genus <em>Frankia.</em> This was confirmed for two of the isolates (Cn3 and Cn7) from <em>Coriaria</em> nodules by comparison of partial 16S rRNA sequences of the cloned amplification products. Both isolates showed about 95% sequence homology to those of the confirmed <em>Frankia</em> strains from other hosts. Similar high homology values were obtained when 16S rRNA sequences of Cn3 and Cn7 were compared with those of the amplification products obtained directly from nodules of <em>Coriaria nepalensis.</em> In two variable regions compared, none of the isolates showed identical sequences to those obtained from nodules. These results suggest that the isolates are members of the genus <em>Frankia</em> , <em></em> but different from the strain identified within the root nodules of <em>Coriaria</em> by 16S rRNA sequence analysis.The endophyte of the root nodules of <em>Datisca cannabina</em> was identified as <em>Frankia</em> by sequence analysis of the partial 16S rRNA gene, amplified directly from the nodules by polymerase chain reaction. Moreover, 16S rRNA sequence analysis indicated that the endophyte of <em>Datisca</em> nodules is closely related to that of the <em>Coriaria</em> nodule endophyte, to which it also resembles morphologically in the symbiotic state. Relatedness of the endophytes of <em>Coriaria</em> and <em>Datisca</em> nodules was further proved by the closely related <em>nifH</em> sequences obtained from the nodules by PCR. 16S rRNA sequence analysis of the non-infective atypical strain Dc2 obtained from the <em>Datisca</em> nodules revealed its close relationship to the genus <em>Frankia.</em>Nodulation in <em>Datisca</em> was achieved with crushed nodule suspensions from both <em>Coriaria</em> and <em>Datisca</em> , <em></em> whereas various pure <em>Frankia</em> strains failed to induce nodulation. The origin of the endophyte in <em>Datisca</em> nodules induced by crushed nodules of <em>Coriaria</em> collected from Murree (Pakistan) was investigated by comparing partial 16S rRNA sequences with those of the endophytes of both plants. The sequences in the variable region were found to be identical to those of <em>Coriaria</em> nodule endophyte, confirming that the endophyte of <em>Coriaria</em> can cross-nodulate <em>Datisca</em> plants. Acetylene reduction by the root nodules indicated effectiveness of the nodules. <em>Coriaria</em> seedlings could only be nodulated by crushed nodule suspensions of <em>Coriaria nepalensis.</em> All pure cultures of <em>Frankia</em> including atypical strains used as single inoculum or in combination with the nodule filtrate, failed to induce nodulation on <em>Coriaria</em> seedlings.Sequence information obtained from the root nodules of <em>Coriaria</em> and <em>Datisca</em> by PCR amplification of partial 16S rRNA gene of the endophyte, was used to develop an oligonucleotide probe. The probe was designed against the V6 variable region of the 16S rRNA that was identical in the 16S rRNA of the endophytes of <em>Coriaria</em> and <em>Datisca.</em> The probe did not cross-react with RNA of typical and atypical <em>Frankia</em> strains isolated from <em>Alnus</em> , <em>Casuarina</em> , <em>Colletia</em> , <em>Comptonia</em> , <em>Elaeagnus</em> and <em>Hippophae.</em>To investigate the presence of <em>Frankia</em> strains infective on <em>Coriaria</em> and <em>Datisca</em> , soil samples were collected from different areas of Pakistan and used as inoculum. Three types of soil samples i.e. rhizosphere soil, non-rhizosphere soil and soil from sites effected by erosion were used to inoculate seedlings of <em>Coriaria</em> and <em>Datisca.</em> Abundance of compatible <em>Frankia</em> strains in most areas was indicated by profuse nodulation of the host plants, while soil samples from Athmokam and Puddar failed to induce nodulation on <em>Coriaria</em> seedlings. Exceptionally good nodulation of <em>Coriaria</em> and <em>Datisca</em> with soil samples collected from Abbottabad and Kaghan respectively, reflects abundance of compatible <em>Frankia</em> strains in the soils. This also indicates that these soils can be used effectively as an inoculum for large scale inoculation programmes instead of nodules that are difficult to obtain throughout the year.Successful nodulation of plants inoculated with soil suspensions can confirm the existence of compatible <em>Frankia</em> cells, but the possibility of the presence of more than one infective strain cannot be ruled out. Genetic diversity among the <em>Frankia</em> strains in <em>Datisca</em> nodules originaly induced by soil inoculum collected from different areas of Pakistan, was determined by sequence analysis of 16S rRNA. Part of the 16S rRNA gene was amplified from nodules and the cloned PCR products were screened initially with a <em>Frankia</em> genus probe and then with the specific probe. Three categories of the clones were identified i.e. those reacting with both probes and those hybridizing with only the <em>Frankia</em> genus probe, or only with the probe specific for the <em>Datisca</em> endophyte. Four types of <em>Frankia</em> sequences and one non <em>-Frankia</em> sequence were identified by sequence analysis of the cloned PCR products. The results suggest the presence of more than one <em>Datisca-</em> compatible <em>Frankia</em> strains in the soils collected from different areas.