Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-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 [(15)N]NO(3) 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 N(2) 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 N(2) 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 N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-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 N(2)-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.     

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.     

An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

An ineffective strain type of Frankia of unknown strain composition, coded AgI-WD1 was discovered in the soil of wet dune slacks where A. glutinosa was the dominant tree species. Strain type AgI-WD1 was recognized by the development of slow growing root nodules on A. glutinosa testplants inoculated with soil suspensions. Microscopical examination of these nodules showed extremely reduced development of vesicles, normal development of intracellular clusters of hyphae and absence of sporangia. The stability of characteristics of this strain type such as the expression of root nodule symbiosis and ineffectivity of symbiontic N-fixation was demonstrated through ‘subculture’ of ineffective root nodules in successive hydrocultures of A. glutinosa. The nodulation process also differed from normal effective root nodules by the occurrence of resistance to strain type AgI-WD1 among part of the half-siblings of A. glutinosa used in the nodulation tests. Strain type AgI-WD1 was detected in the soil of different dune slacks which are inundated for a large part of the year and in a nearby peatbog covered with alder. The contribution of this strain type to soil populations of Frankia was demonstrated by nodulation potentials that were up to 500 times higher than that of the concurrent effective strain type AgSp^-. The distribution of strain type AgI-WD1 appeared to be restricted to sites with water-logged soil conditions. Nodulation experiments pointed to potentials for competitive interactions between effective and ineffective strain thpes, especially to a density dependent reduction of nodule type AgI-WD1 by strain type AgSp^-. The impact of competitive interactions is also affected by host trees that are resistant to AgI-WD1. The occurrence of resistance in the study areas was suggested by resistance among seedlings of a local seedbatch (±70% of the half-siblings) and by the absence of ineffective root nodules at site VD7-1, despite a high nodulation potential of the soil population of strain type AgI-WD1.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

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.     

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.     

Arthrobacter globiformis and Its Bacteriophage in Soil

Bacteriophages in soil for Arthrobacter globiformis were rarely detected unless the soil was nutritionally amended and incubated. In amended soil, phage were continuously produced for at least 48 h, and this did not require the addition of host cells. Rod and spheroid stage host cells added to the amended soil encountered indigenous bacteriophage, but added phage did not encounter sensitive indigenous host cells for some time, if at all. The indigenous phage in nonincubated soil seemed to be present in a masked state which was not merely a loose physical adsorption to soil materials but required growth conditions other than lysogeny for them to increase their titers. The possibility is discussed that the indigenous host cells in nonamended soil are present in a nonsensitive spheroid state, with the cells becoming sensitive to the phage in a rate-limiting fashion as nonsynchronous outgrowth occurs for a portion of the spheroid cells.     

Effect of juglone on growthin vitro ofFrankia isolates and nodulation ofAlnus glutinosa in soil

Summary In vitro growth (total protein content) of 5Frankia isolates was significantly inhibited at 10^–4 M juglone (5-hydroxy-1, 4-napthoquinone) concentration, but the degree of inhibition varied with theFrankia isolate. Isolates fromAlnus crispa [Alnus viridis ssp.crispa (Ait.) Turril] were most tolerant of 10^–4 M juglone relative to controls, while an isolate fromPurshia tridentata (Pursh.) D.C. was most inhibited, displaying a dramatic decrease in growth and greatly altered morphology.Nodulation of black alder [Alnus glutinosa L. (Gaertn.)] in an amended prairie soil inoculated with aFrankia isolate from red alder (Alnus rubra Bong.) was significantly decreased by the addition of aqueous suspensions of 10^–3 M and 10^–4 M juglone. This decrease was partially independent of decreased plant growth. The addition of an equal volume of sand to the soil mixture further decreased nodulation of black alder.Frankia inoculation of the soil mixtures significantly increased the total number of nodules formed per seedling, and the degree of differences in seedling nodulation owing to juglone and soil treatments.     

Ineffective Frankia and host resistance in natural populations of Alnus glutinosa (L.) gaertn

Alnus glutinosa (black alder) populations are known to exhibit a variable degree of incompatibility to root nodule formation by ineffective Frankia. The relationship between the occurrence of ineffective Frankia in wet stands of black alder and the degree of resistance to nodulation by ineffective Frankia of seed-lots and clones of alder trees from these particular locations was studied through soil inoculation experiments. The average percentage of resistant plants (R-frequency) among the seed-lots from locations with an ineffective Frankia soil population was equal to, or higher than, the R-frequencies of locations without ineffective Frankia. The mean R-frequency was highest for the seed-lots from the location from which the soil inoculant was taken. These results strongly suggest that ineffective Frankia are not strictly dependent on susceptible A. glutinosa for the maintenance of their population size. The fungus Penicillium nodositatum also nodulated A. glutinosa seedlings. Whereas a negative interaction with the ineffective Frankia nodulation was found, this did not have a significant effect on the R-frequencies of the seed-lots that were tested, suggesting that the ineffective Frankia nodulation adversely affected the myco-nodulation, and not vice versa.     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.     

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.      

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.     

Host range differentiation of spore-positive and spore-negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore-positive (Sp⁺)and spore-negative (Sp^?) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp ⁺ types of Frankia (coded AiSp⁺ and AiSp⁺ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp ⁺ and AiSp ⁺ H caused small, ineffective root nodules with sporangia (coded Ineff ^?), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff ^?) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore-negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp^? type root nodules (coded AgSp ^?) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp ⁺ strain type of Frankia. coded AgSp⁺ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp ⁺ by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp ⁺ nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp⁺ and AiSp⁺ H were found in the soil environment of dominant AiSp ⁺ nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp ^? nodule populations on A. glutinosa and in the former field at Hämeen-linna near the Sp⁺ nodule area in Hämeenlinna forest. Strain type AgSp^? had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp⁺ and AiSp ^? H and A. incana and between strain type AgSp^? and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp ⁺ and Sp ^? to strains with some variation in spore development was stressed by the low potentials of strain type AiSp ⁺ H to develop spores in symbioses with hydrocultures of A. incnna.     

Diversity and Specificity of Frankia Strains in Nodules of Sympatric Myrica gale, Alnus incana, and Shepherdia canadensis Determined by rrs Gene Polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia

Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N₂-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.     

Oligonucleotide Probes That Hybridize with rRNA as a Tool To Study Frankia Strains in Root Nodules

Oligonucleotide probes that hybridize with specific sequences in variable regions of the 16S rRNA of the nitrogen-fixing actinomycete Frankia were used for the identification of Frankia strains in nodules. Frankia cells were released from plant tissue by grinding glutaraldehyde-fixed root nodules in guanidine hydrochloride solution. rRNA was obtained after sonication, precipitation with ethanol, and purification by phenolchloroform extraction. Degradation of rRNA, evident in Northern blots, did not affect hybridization with the oligonucleotides. Nodules of about 1 mg (fresh weight) provided sufficient rRNA for reliable detection of the Frankia strain. The utility of this rRNA extraction method was tested in a competition experiment between two effective Frankia strains on cloned Alnus glutinosa plants.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

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.     

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.     

Symbiotic Deficiencies Associated with a coxWXYZ Mutant of Bradyrhizobium japonicum

The terminal oxidase complexes encoded by coxMNOP and coxWXYZ were studied by analysis of mutations in each of the two oxidases. Carbon monoxide difference spectra obtained from membranes of coxMNOP mutant bacteroids were like those obtained for the wild type, whereas bacteroid membranes of a coxWXYZ mutant were deficient in CO-reactive cytochrome b. Experiments involving cyanide inhibition of oxidase activity were consistent with the conclusion that the coxX mutant is deficient in a membrane-associated O₂-binding component. The viable cell number (bacteria that could be recultured from crushed nodules) was 20 to 29% lower for the coxX mutant than for the wild-type or the CoxN⁻ strain. In three separate greenhouse studies, nodules of a coxX mutant had significantly lower (28 to 34% less) acetylene reduction rates than the wild-type nodules did, and plants inoculated with a double mutant (coxMNOP coxWZYZ) had rates 30% lower than those of wild-type-inoculated plants.