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.     

Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups.     

Variation in Frankia Populations of the Elaeagnus Host Infection Group in Nodules of Six Host Plant Species after Inoculation with Soil

The potential role of host plant species in the selection of symbiotic, nitrogen-fixing Frankia strains belonging to the Elaeagnus host infection group was assessed in bioassays with two Morella, three Elaeagnus, and one Shepherdia species as capture plants, inoculated with soil slurries made with soil collected from a mixed pine/grassland area in central Wisconsin, USA. Comparative sequence analysis of nifH gene fragments amplified from homogenates of at least 20 individual lobes of root nodules harvested from capture plants of each species confirmed the more promiscuous character of Morella cerifera and Morella pensylvanica that formed nodules with frankiae of the Alnus and the Elaeagnus host infection groups, while frankiae in nodules formed on Elaeagnus umbellata, Elaeagnus angustifolia, Elaeagnus commutata, and Shepherdia argentea generally belonged to the Elaeagnus host infection group. Diversity of frankiae of the Elaeagnus host infection groups was larger in nodules on both Morella species than in nodules formed on the other plant species. None of the plants, however, captured the entire diversity of nodule-forming frankiae. The distribution of clusters of Frankia populations and their abundance in nodules was unique for each of the plant species, with only one cluster being ubiquitous and most abundant while the remaining clusters were only present in nodules of one (six clusters) or two (two clusters) host plant species. These results demonstrate large effects of the host plant species in the selection of Frankia strains from soil for potential nodule formation and thus the significant effect of the choice of capture plant species in bioassays on diversity estimates in soil.     

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n?=?10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var. americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environmental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil.     

Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

Genomospecies identification and phylogenomic relevance of AFLP analysis of isolated and non-isolated strains of Frankia spp

Amplified fragment length polymorphism (AFLP) was tested as an alternative to the DNA-DNA hybridization technique (DDH) to delineate genomospecies and the phylogenetic structure within the genus Frankia. Forty Frankia strains, including representatives of seven DDH genomospecies, were typed in order to infer current genome mispairing (CGM) and evolutionary genomic distance (EGD). The constructed phylogeny revealed the presence of three main clusters corresponding to the previously identified host-infecting groups. In all instances, strains previously assigned to the same genomospecies were grouped in coherent clusters. A highly significant correlation was found between DDH values and CGM computed from AFLP data. The species definition threshold was found to range from 0.071 to 0.098 mismatches per site, according to host-infecting groups, presumably as a result of large genome size differences. Genomic distances allowed new Frankia strains to be assigned to nine genomospecies previously determined by DDH. The applicability of AFLP for the characterization of uncultured endophytic strains was tested on experimentally inoculated plants and then applied to Alnus incana and A. viridis field nodules hosting culture refractory spore-positive (Sp+, that sporulate in planta) strains. Only 1.3% of all AFLP fragments were shown to be generated by the contaminant plant DNA and did not interfere with accurate genomospecies identification of strains. When applied to field nodules, the procedure revealed that Alnus Sp+ strains were bona fide members of the Alnus-Myrica host infecting group. They displayed significant genomic divergence from genomospecies G1 of Alnus infecting strains (i.e. Frankia alni) and thus may belong to another subspecies or genomospecies.     

Natural Diversity of Frankia Strains in Actinorhizal Root Nodules from Promiscuous Hosts in the Family Myricaceae

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N₂-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

The improvement and utilization in forestry of nitrogen fixation by actinorhizal plants with special reference toAlnus in Scotland

Summary Alnus species are used widely in Britain for land reclamation, forestry and other purposes. Rapid juvenile growth of the AmericanAlnus rubra makes it an attractive species for planting on N-deficient soils, particularly those of low organic content. In small plot trials, this species is nodulated by indigenous soil frankiae as effectively asAlnus glutinosa. Over a three year period both species return similar amounts of N to the ecosystem, estimated at up to 10–12 kg N ha^–1. Several strains ofFrankia have been isolated from local (Lennox Forest)A. rubra nodules. These differ morphologically and in their growth on different culture media, both from each other and fromA. glutinosa nodule isolates. AllAlnus isolates, however, have a total cellular fatty acid composition qualitatively similar to some other Group B frankiae. Glasshouse tests in N free culture suggest thatA. rubra nodules formed after inoculation of seedlings with American spore (–) isolates are three times more effective in N fixation than those inoculated with LennoxA. rubra spore (+) nodule homogenates. By contrast, the early growth of seedlings inoculated with spore (–)Frankia strains suggests at best a 35% improvement in N fixing activity over seedlings inoculated with LennoxA. rubra nodule isolates. Nevertheless, this improvement in activity, together with the better performance of seedlings inoculated with isolates compared with those treated with crushed nodule preparations, suggest that it would be worthwhile commercially to inoculate nursery stock with a spore (–)Frankia strain.     

Conservation of nif sequences in Frankia

Summary Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneunoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.     

Frankia bacteria in Alnus rubra forests: genetic diversity and determinants of assemblage structure

To quantify the genetic diversity of Frankia bacteria associated with Alnus rubra in natural settings and to examine the relative importance of site age, management, and geographic location in structuring Frankia assemblages in A. rubra forests, root nodules from four A. rubra sites in the Pacific Northwest, USA were sampled. Frankia genetic diversity at each site was compared using sequence-based analyses of a 606 bp fragment of the nifH gene. At a 3% sequence similarity cutoff, a total of 5 Frankia genotypes were identified from 317 successfully sequenced nodules. Sites varied in the total number of genotypes present, but were typically dominated by only one or two genotypes. Phylogenetic analyses showed that all of the A. rubra-Frankia genotypes grouped with other Alnus-infective Frankia. Analysis of similarity (ANOSIM) and chi-square analyses indicated that Frankia assemblages were more strongly influenced by site age/management than geographic location. This study demonstrates that the Frankia assemblages in A. rubra forests have low genotype diversity, but that genotype abundance can differ significantly in forests of different age/management history.     

Identification of Frankia Strains by Two-Dimensional Polyacrylamide Gel Electrophoresis

Fifteen Frankia strains from five different plant species were analyzed by two-dimensional polyacryl-amide gel electrophoresis to determine their relatedness by comparing the polypeptide patterns obtained. Three major subgroups (A, C, and D) were found in the Alnus-Comptonia-Myrica cross-inoculation group. An isolate from Purshia tridentata had a unique protein pattern and represents a distinct group of frankiae. Members of group A were isolated from root nodules of Alnus incana subsp. rugosa and Alnus viridis subsp. crispa. Group C organisms were from A. incana subsp. rugosa and Comptonia peregrina nodules, and group D organisms were from A. incana subsp. rugosa, A. viridis subsp. cripsa, and Myrica pensylvanica root nodules. Isolates from each gel group were obtained at several widely separated geographical locations. The results indicate that two-dimensional polyacrylamide gel electrophoresis is useful for identifying Frankia isolates.     

Natural diversity of nodular microsymbionts of Myrica rubra

Myrica is often considered a promiscuous actinorhizal genus. However, there are large differences in diversity among Myrica spp., and M. gale does not exhibit such promiscuity in its natural environment. In order to understand the diversity of nodular microsymbionts of M. rubra in natural environments and whether or not the M. rubra is a `promiscuous' host, we studied the natural diversity of nodular microsymbionts of different cultivars of M. rubra. 15 nodules from nine horticultural cultivars of M. rubra were collected in 7 sites of eastern, southeastern, central and northern part of Zhejiang province, China. Unisolated strains were compared by sequence analyses of their nifD-nifK intergenic spacers and PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing sequences of their nifD-nifK intergenic spacers and reference strains. There is a high degree of diversity among nodular Frankia symbionts of M. rubra. Frankia strains from cluster I and cluster III were found in nodules from many different cultivars of M. rubra. Furthermore, there were sometimes two strains which belong to different infective clusters of Frankia in the same nodule, and Frankia strains of cluster I were often dominant strains when there were two strains. M. rubra can thus be considered to be promiscuous in nature. Identical sequences in nodules from different plants at widely separated sites were commonly found, indicating that some strains are cosmopolitan. Geographic separation, host selectivity for Frankia symbionts and soil environment may account for the diversity of Frankia strains and differences in Frankia populations found in M. rubra nodules. Several very closely related local Frankia populations in M. rubra nodules could be distinguished from one another by our approach.     

Different genetic groups of Frankia within the root nodules of Casuarina growing in Mexico

This study was carried out to examine the possible genetic diversity that may occur among Frankia strains that nodulate C. equisetifolia in Mexico growing in the site of introduction, the cost of the Golf of Mexico, as well in the highlands. DNA extracted from reference cultures of Casuarina infecting Frankia strains, from field collected nodules (two trees of each of 14 sites and 3–5 analyzed lobes of each nodule) or from young nodules obtained from plants growing at the growth chamber and inoculated with field nodules, were used as the template in PCR reaction with primers targeting two DNA regions, one of the ribosomal operon and the other in the nif operon. PCR products were analyzed by using a set of recommended restriction enzymes. Six PCR-RFLP groups were detected after digestion with combination of enzymes BstUI and CfoI of the nif region, and four PCR-RFLP groups with enzymes NciI and ScrFI in the ribosomal region. Reference strains showed similar patterns and were assigned in group 1 along with an uncultured strain. nifD-K PCR-RFLP groups allowed a better understanding of the diversity among nodular Frankia strains than those observed in the rrs PCR-RFLP groups.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Diversity of Frankia Populations in Root Nodules of Geographically Isolated Arizona Alder Trees in Central Arizona (United States)

The diversity of uncultured Frankia populations in root nodules of Alnus oblongifolia trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of nifH gene fragments. Sequences were retrieved from Frankia populations in nodules of four trees from each of three mountaintops (n = 162) and their levels of diversity compared using spatial genetic clustering methods and single-nucleotide or 1, 3, or 5% sequence divergence thresholds. With the single-nucleotide threshold level, 45 different sequences with significant differences between the mountaintops were retrieved, with the southern site partitioning in a separate population from the two other sites. Some of these sequences were identical in nodules from different mountaintops and to those of strains isolated from around the world. A high level of diversity that resulted in the assignment of 14 clusters of sequences was also found on the 1% divergence level. Single-nucleotide and 1% divergence levels thus demonstrate microdiversity of frankiae in root nodules of A. oblongifolia trees and suggest a partitioning of diversity by site. At the 3 and 5% divergence levels, however, diversity was reduced to three clusters or one cluster, respectively, with no differentiation by mountaintop. Only at the 5% threshold level do all Frankia strains previously assigned to one genomic group cluster together.Frankia spp. are nitrogen-fixing actinomycetes that form root nodules in symbiosis with more than 200 species of nonleguminous woody plants in 24 genera of angiosperms (5, 24, 43). These actinorhizal plants have an almost worldwide distribution and can live in soils with low nitrogen availability and thus exploit habitats not favorable for growth of many other plant species (12). Alnus oblongifolia Torr. (Arizona alder) is an actinorhizal plant that can be found in mountainous regions in northern Mexico and the southwestern United States. Within the southwestern United States, isolated populations of Arizona alder trees are frequently found along streams draining the southern edge of the Colorado Plateau and the scattered mountain ranges found throughout central Arizona. The alder sites are in mountains that are surrounded by deserts, grasslands, brush or woodland types, and forests and as such are home to many endemic species that have developed as a consequence of geographic isolation (47).Alnus oblongifolia grows in unique moist environments in this desert region, specifically along perennial streams of canyons, primarily at elevations between 1,400 and 2,300 m, and has been shown to form effective root nodules in nature (13). Because mountainous sites inhabited by A. oblongifolia are geographically isolated, analyses of Frankia populations in nodules of A. oblongifolia trees growing on different mountaintops may provide an opportunity to get new insights into the diversity and biogeography of these Frankia populations.Specific factors that drive Frankia diversification are presently unclear, even though there are preferences among Frankia strains for specific host plants, separating strains into host infection groups and subgroups (15, 22, 28). Frankia strains infecting Casuarina plants have been shown to have coevolved with their host plant, illustrating the importance of the host plant in shaping the diversity and evolution of these strains (44). However, for most Frankia strains, no simple pattern of coevolution is present (3). While phylogenetic analyses reveal three clades for each of the partners in this symbiosis, Frankia populations within one clade may form root nodules with plants in more than one clade (4). This lack of correlating phylogenies is likely due to Frankia populations occupying two distinct ecological niches, root nodules and soil, where symbiotic or saprophytic growth conditions may drive diversification of Frankia populations differently (3). Thus, the complex divergence patterns in Frankia phylogeny may best be explained in a geographic mosaic theory of coevolution in which multiple confounding factors, like geographic isolation, plant host preferences, and environmental factors, converge to shape the evolutionary patterns of Frankia (3, 46).One aspect of the geographic mosaic theory of coevolution is allopatric speciation, the divergent evolution of geographically isolated populations, which may be a potential driver of Frankia diversification (38, 50). Comparative analysis of Frankia populations on isolated mountainous habitats may be a unique opportunity to test if geographic divergence is indeed driving Frankia evolution. The isolation of Frankia populations in root nodules of trees growing on different mountaintops may permit differentiation, as the effects of neutral drift, population bottlenecks and adaptation to even slight environmental differences cause the accumulation of mutations which may lead to allopatry (38), as indicated for other bacteria (37, 51). However, Frankia populations are capable of forming spores that allow them to survive transport from one hospitable habitat to another (26). Additionally, Frankia strains, particularly those of the Alnus host infection group, seem to have a cosmopolitan distribution (4) because strains from the same species or genomic group have been isolated from all over the world (see references 1 and 19) and have been found in soils with no extant actinorhizal plants (9, 25, 30, 39).The aim of this study was to determine if uncultured Frankia populations in root nodules of A. oblongifolia trees isolated on mountaintops within different geologic regions of Arizona showed signs of endemism in a functional gene, nifH, and whether that unique diversity could be correlated with differences in Frankia populations from root nodules among mountaintops. Nodules were collected in June of 2008 from four trees at each of three mountaintop sampling sites, each separated from the nearest by 150 km, proceeding from north to south, within 1° longitude of each other along a 300-km latitudinal gradient in southern Arizona (Fig. ​(Fig.1).1). Site 1 (Oak Creek in the Coconino National Forest, 35°00.6′N, 111°44.3′W) was a sandy alluvial soil located near Oak Creek at an elevation of 1,703 m, site 2 (Workman Creek watershed in the Sierra Ancha Experimental Forest of Tonto National Forest, 33°49.1′N, 110°55.8′W) a streamside soil high in organic matter at an elevation of 2,073 m, and site 3 (Sabino Canyon in Coronado National Forest, 32°26.1′N, 110°45.5′W) a sandy loam soil adjacent to Sabino Creek at an elevation of 2,310 m. Each site was in a different geologic province: the Colorado Plateau Province was in the north, the Central Highlands Province in a transition zone, and the Basin and Range Province within the Madrean Archipelago in the south (8, 32). Nodules were stored in cold 95% ethanol until analyzed.Open in a separate windowFIG. 1.Locations of the mountaintops in central and southern Arizona sampled for uncultured Frankia populations from the root nodules of Alnus oblongifolia trees growing near perennial streams on these mountains. The scale bar indicates 100 km. Site 1 is Oak Creek in the Coconino National Forest (35°00.6′N, 111°44.3′W), site 2 is the Workman Creek watershed in the Sierra Ancha Wilderness of Tonto National Park (33°49.1′N, 110°55.8′W), and site 3 is the Sabino Canyon watershed in the Coronado National Forest (32°26.1′N, 110° 45.5′W).DNA was extracted from individual lobes of different root nodules, and a 606-bp fragment of nifH, the structural gene for nitrogenase reductase, was amplified using Frankia-specific primers nifHf1 and nifHr (34, 49) and sequenced as described previously (49). Initially, nifH gene sequences were obtained from 24 nodules from one tree from each mountaintop to determine the level of sampling required to capture the diversity present. A rarefaction curve was generated using DOTUR (41) with a threshold level of divergence set to 3%, which was found to group Frankia strains into appropriate genomic groups in a previous study (34). Based on rarefaction analyses, 10 nodules were sampled from the remaining three trees from each mountaintop, for a total of 54 nodules from each mountain. Sequences of amplified nifH gene fragments of uncultured Frankia populations from 54 A. oblongifolia nodules from each of three mountaintops (GenBank accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"FJ977167 to FJ977328","start_term":"FJ977167","end_term":"FJ977328","start_term_id":"262234671","end_term_id":"262234993"}}FJ977167 to FJ977328) were aligned with those of the three pure cultures of Frankia populations (GenBank accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"FJ977329 to FJ977331","start_term":"FJ977329","end_term":"FJ977331","start_term_id":"262234995","end_term_id":"262234999"}}FJ977329 to FJ977331) and sequences of 46 other strains or uncultured populations analyzed in previous studies (34, 49) or retrieved from public databases and analyzed using maximum likelihood, maximum parsimony, neighbor-joining, and Bayesian analyses as described previously (49).Phylogenetic analyses of this data set of 211 sequences produced similar topologies independent of the methodology used (data not shown) and assigned all sequences in nodules of A. oblongifolia to frankiae of the Alnus host infection group (see Fig. S1 in the supplemental material). The analysis retrieved 45 different sequences in these nodules from A. oblongifolia, differing from each other by at least one nucleotide, with most of the changes being synonymous. For presentation purposes, to show the relationship of frankiae from root nodules of A. oblongifolia to available pure cultures, the complete data set was reduced to 51 representative sequences, including 21 sequences from frankiae in root nodules of A. oblongifolia, and was reanalyzed using the above-mentioned parameters (Fig. ​(Fig.2).2). Several of the sequences obtained from root nodules were identical or nearly identical (i.e., single-nucleotide differences) to those of strains or uncultured Frankia populations from other parts of the world (Fig. ​(Fig.2).2). For example, sequence AO3-14nodF was identical to nifH gene fragment sequences from four Frankia strains isolated from around the world (CpI1 in Massachusetts [7], ArI3 in Oregon [6], AvsI4 in Washington [2], and Ai14a in Finland [48]). Identical sequence does not mean that these are identical strains but does suggest that certain genotypes may have a ubiquitous distribution (34).Open in a separate windowFIG. 2.Maximum parsimony tree of a subset of uncultured Frankia populations from root nodules of Alnus oblongifolia and other pure cultures and uncultured Frankia populations created using 522 bp of the nifH gene. Numbers outside parentheses reflect bootstrap support values, and numbers in parentheses reflect bootstrap support or posterior probabilities from neighbor-joining, maximum likelihood, and Bayesian analyses. Representatives from clusters A and B are indicated. Strains in boldface belong to Frankia genospecies 1 (see reference 20 for a summary). Strain EAN1pec was used as the outgroup.Sequences retrieved from Frankia populations in root nodules of A. oblongifolia were organized into populations by the tree that they were isolated from and by mountaintop (18) for analysis of molecular variance (AMOVA) using Arlequin version 3.01 (17). The AMOVA settings included 16,000 permutations and a more conservative proportion of differences for matrix criteria. AMOVA includes both differences in sequences at the single-nucleotide level and differences in abundance of sequences present (40) and indicated significant differences in sequence diversity within trees (P < 0.001), with most of the variation in diversity (83.6%) found within populations of Frankia from each A. oblongifolia tree. Differences in sequence diversity among trees on each mountaintop, however, were not significant (P = 0.165). Significant differences in the levels of diversity of Frankia populations among mountaintops were also recovered, accounting for 14.6% of the variation in diversity, suggesting differences by site in the diversity of Frankia populations recovered at the single-nucleotide level of differences.To explore this geographic component in more detail, spatial genetic clustering methods were used in GENELAND version 3.1.4, which utilizes a Bayesian algorithm to make population assignments and weights by using geographic coordinates (20). The analyses of 54 variant characters among the 162 root nodule sequences proceeded in two stages, similar to what was observed by Coulton et al. in 2006, except that the number of populations in the data set initially fluctuated between 1 and 24 and 1 million generations were run with a burn-in of 100,000 (10). Spatial and nonspatial settings were used, and uncertainty in coordinates was tested at 3 m and 10 m; however, all analyses yielded two populations in the data set and assigned individuals in the same way. Sequences of 108 root nodule Frankia strains were unambiguously assigned to one population, corresponding to those from sites 1 and 2, and 54 sequences of root nodule Frankia strains were unambiguously assigned to the other population, corresponding to site 3. Regions encompassing both site 1 and site 2 have some geographic connectivity by forest along the Mongollon Rim and have been placed in the Central Highlands Floristic Subdivision and thus might have been expected to be more similar (8, 31). However, site 3 is isolated from the other two sites on account of being surrounded by desert on four sides and was placed in the Southeastern Arizona Floristic Subdivision, supporting the comparative uniqueness of site 3 revealed by our spatial clustering analysis (8, 31).Three additional threshold levels, i.e., the 1, 3, and 5% divergence levels, were subsequently used to compare the levels of diversity of nodule populations among mountaintops. While the 1% level, corresponding to ∼5-nucleotide differences, was arbitrarily chosen, the 3 and 5% levels represented thresholds previously used to assign Frankia strains of the same species or genomic group (34) and uncultured root nodule frankiae of the Alnus or Elaeagnus host infection groups (34, 49) into the same cluster on the basis of comparative sequence analyses of nifH gene fragments. To formulate the assignment of clusters at these three levels of differentiation, the complete data set was reduced by removing all sequences but those representing frankiae in nodules of A. oblongifolia. This data set of 162 nifH gene fragments was executed using PAUP* 4.05b, where an uncorrected distance matrix was created and analyzed using DOTUR to assign taxa to clusters at various thresholds of diversity and then in SONS to compare memberships in these clusters by mountaintop (42). A similar DOTUR/SONS analysis was completed on the entire data set of 211 sequences to describe the groupings of uncultured nodule populations with pure cultures representing various genomic groups. Pie charts displaying the clusters of frankiae in root nodules of A. oblongifolia at 1%, 3%, and 5% differences were generated (Fig. ​(Fig.33).Open in a separate windowFIG. 3.Graphical representation of output from SONS (42) for clusters from uncultured Frankia populations from root nodules of Alnus oblongifolia trees (n = 54) isolated on three different mountaintops in central and southern Arizona (sites 1 to 3). The inner circle represents 14 clusters recovered using a 1% diversity threshold, the middle circle represents 3 clusters recovered using a 3% diversity threshold, and the outer circle represents 1 cluster recovered using a 5% diversity threshold. A and B designate the only clusters at the 1% diversity threshold, representing ∼5-nucleotide differences, for uncultured frankiae recovered from all three mountaintops.At the 1% threshold level, analyses using the SONS program demonstrated the presence of 14 clusters of sequences (Fig. ​(Fig.3).3). Half of these clusters were represented by three or fewer sequences. Seven sequence clusters were found only in nodules of trees from one mountaintop, five were present in nodules from trees of two mountaintops, and two were detected on all three mountaintops (identified as A and B in Fig. ​Fig.3).3). Cluster A was dominant overall (n = 73; 44% of all nodules recovered) and was found on all three mountaintops at various frequencies (Fig. ​(Fig.3).3). Cluster B was also found on all three mountaintops (n = 23; 14% of all nodules recovered) but was dominant on one site (site 3, Sabino Canyon) and barely detected on the other mountaintops, supporting the geographic uniqueness of this site (Fig. ​(Fig.3).3). The number of clusters decreased to three when a 3% divergence threshold was used. All three clusters were present on all three mountaintops, but in various frequencies (Fig. ​(Fig.3).3). At this threshold, however, pure cultures belonging to the same genomic group still did not cluster in the same group. Only when the threshold was set to 5% did all the pure cultures from Frankia genomic group 1 (see reference 21 for a summary) cluster together. At a threshold of 5%, all Frankia populations in nodules of A. oblongifolia were placed in one group, suggesting no differentiation by mountaintop and a limited overall level of diversity, with one cluster present, compared to the potential presence of at least six clusters of frankiae within the Alnus host infection group described in previous studies (34, 49). Low overall diversity has also been described to occur in other studies of natural Frankia diversity in root nodules of various alder species (11, 23, 27, 29).Differences in nitrogenase activity and nodulation capacity have been reported for Frankia strains of the same genomic group on the same alder species in the same soils (14, 15). These differences in effectiveness and infectivity of strains in the same species group have been suggested to be evidence of the effects of plant host shaping symbiotic Frankia diversity under different environmental conditions (3). Thus, the variations in diversity and abundance seen at the single-nucleotide or 1% diversity level may reflect preferences by A. oblongifolia for one strain over another in the different environmental conditions on each mountaintop and microscale differences among trees on the same mountaintop. Genetic differences among Arizona alder populations are unknown, as is the extent to which seed and pollen dispersals occur among these isolated populations. Nonetheless, there were no morphological differences among trees in populations sampled, and the trees were all the same species. In contrast, Frankia diversity in root nodules has been shown to be affected by different edaphic conditions, like soil type and pH (35, 45), or environmental effects, like elevation (27, 29), which are different on each mountaintop. However, these variations in diversity may also reflect random chance and small sample size (16), because rarefaction analysis at the 1% diversity level did not indicate saturation of sampling for any mountaintop (data not shown).Determination of reasons for selective nodulation by specific strains of Frankia becomes highly speculative. Some evidence suggests that active Frankia populations in the soil may be preferentially selected by the host for nodulation (33, 36). Previous research in our laboratory has confirmed the importance of the plant host in selecting Frankia strains for symbiosis when the same soil was inoculated into six different actinorhizal plant species and resulted in six different diversity profiles (34). Additionally, we have shown that the same actinorhizal plant species inoculated with soils from five different continents resulted in five different diversity profiles, demonstrating the effects of soil type and history on root nodule Frankia diversity (49).Frankia microdiversity in root nodules of A. oblongifolia recovered in this study shows a clear geographic pattern, but the reasons for these patterns are unclear. The limited Frankia diversity in A. oblongifolia root nodules is likely due to a combination of factors, including saprophytic growth capabilities, host plant preferences, and edaphic conditions acting at the microecosystem level on Frankia populations.      

Isozyme Variation among 40 Frankia Strains

Forty Frankia strains belonging to the Alnus and Elaeagnus host specificity groups and isolated from various plant species from different geographical areas were characterized by the electrophoretic separation of isozymes of eight enzymes. All the enzyme systems that were investigated showed large variation. Diaphorases and esterases gave multiple band patterns and confirmed the identification of specific Frankia strains. Less variability was observed with enzymes such as phosphoglucose isomerase, leucine aminopeptidase, and malate dehydrogenase, which allowed for the delineation of larger groups of Frankia strains. Cluster analysis, based on the pair-wise similarity coefficients calculated between strains, delineated three large, dissimilar groups of Frankia strains, although each of these groups contained a large amount of heterogeneity. However, numerous Frankia strains, mainly from the Alnus host specificity group, demonstrated a perfect homology for all the enzymes tested.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.