Isolation of Frankia Strains from Alder Actinorhizal Root Nodules

A simple procedure, based on the rapid filtration and washing of Frankia vesicle clusters, was devised for the isolation of Frankia strains from alder actinorhizal root nodules. Of 46 Alnus incana subsp. rugosa nodules prepared, 42 yielded isolates. A simple medium containing mineral salts, Casamino Acids, and sodium pyruvate proved to be the most effective for isolation. In general, colonies appeared 6 to 20 days after inoculation. On the basis of hyphal morphology, two distinct types of Frankia strains were characterized. Randomly selected isolates were tested for infectivity, and all formed root nodules on A. glutinosa. Because of its simplicity and efficiency, the procedure is an improved method for the study of Frankia diversity in alder root nodules.     

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.     

Polymorphism in Alnus based Frankia of Darjeeling

DNA samples extracted from the root nodules of Alnus nepalensis, collected from 10 different locations of Darjeeling hills, were used to assess the genetic diversity of Frankia. The DNA samples from the nodules of naturally growing plants were used as templates in PCR, targeting different genomic regions of Frankia, namely distal, middle and proximal parts of 16S rRNA gene and nifH-D IGS region with locus specific primers. The PCR products were digested with a number of frequent (4-base) cutter restriction endonucleases. Bands were scored as present (1) or absent (0) and the clustering was done using NTSYSpc. Distinct polymorphism was found among the nodules collected from different parts of the region and those of same geographic area. These results demonstrate that genetic diversity is indeed present among the naturally occurring Frankia of Darjeeling, India.     

Diversity of Frankia Strains in Root Nodules of Plants from the Families Elaeagnaceae and Rhamnaceae

Partial 16S ribosomal DNAs (rDNAs) were PCR amplified and sequenced from Frankia strains living in root nodules of plants belonging to the families Elaeagnaceae and Rhamnaceae, including Colletia hystrix, Elaeagnus angustifolia, an unidentified Elaeagnus sp., Talguenea quinquenervia, and Trevoa trinervis. Nearly full-length 16S rDNAs were sequenced from strains of Frankia living in nodules of Ceanothus americanus, C. hystrix, Coriaria arborea, and Trevoa trinervis. Partial sequences also were obtained from Frankia strains isolated and cultured from the nodules of C. hystrix, Discaria serratifolia, D. trinervis, Retanilla ephedra, T. quinquenervia, and T. trinervis (Rhamnaceae). Comparison of these sequences and other published sequences of Frankia 16S rDNA reveals that the microsymbionts and isolated strains from the two plant families form a distinct phylogenetic clade, except for those from C. americanus. All sequences in the clade have a common 2-base deletion compared with other Frankia strains. Sequences from C. americanus nodules lack the deletion and cluster with Frankia strains infecting plants of the family Rosaceae. Published plant phylogenies (based on chloroplast rbcL sequences) group the members of the families Elaeagnaceae and Rhamnaceae together in the same clade. Thus, with the exception of C. americanus, actinorhizal plants of these families and their Frankia microsymbionts share a common symbiotic origin.     

Observations on the Composition and Metabolism of the Nitrogen-Fixing Root Nodules of Alnus

Nodules of Alnus glutinosa (Alder) were exposed to excess ¹⁵Neither before or after detachment from the plant. The solublenitrogen compounds were extracted from the nodules and the extractsfractionated by chromatography on an ion exchange resin. Theamino-acid composition of the extracts was thus determined,and it was confirmed that citrulline is the predominant amino-acidpresent, being accompanied by smaller amounts of aspartic, glutaniic,and -aminobutyric acids, arginine, and other constituents. Thehighest atom per cent, excess ¹⁵N always found in glutarnicacid and the next highest in citrulline or aspartic acid. Ammoniacontained a smaller proportion of ¹⁵N than these compounds andarginine showed only very small enrichment. When the citrullinewas degraded to ammonia and ornithine it was found that theammonia liberated was richer in ¹⁵N than even glutamic acid.The significance of these findings in relation to the fixationand further metabolism of nitrogen by the alder nodule is discussed.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Partial Characterisation of Cytokinins from Root Nodules of Alnus glutinosa (L.) Gaertn

The nature of the substances responsible for the major cytokininactivity in extracts of Alnus glutinosa (L.) Gaertn. root noduleswas investigated by means of chromatographic, chemical, andenzymic methods. Five cytokinins were demonstrated and a furthertwo compounds were probably present in trace amounts. The propertiesof the cytokinins were consistent with their being identicalor closely similar to trans-zeatin, trans-zeatin riboside, zeatin-O-ß-D-glucoside,and a ß-D -glucoside of zeatin riboside together withcertain of the corresponding dihydrozeatin compounds. The greatestpart of the cytokinin activity was represented by the glucosides.     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Cytokinin Transport from the Root Nodules of Alnus glutinosa (L.) Gaertn.

The movement and metabolism of [8-¹⁴C]zeatin applied to theroot nodules of Alnus glutinosa (L.) Gaertn, was investigated.Twenty-four hours after the start of uptake, zeatin and a numberof its metabolites were detected in all parts of the plant.The major radioactive compounds present in a cationic fractionof different plant parts at this time co-chromatographed onSephadex LH20 with zeatin (in nodules, stems, and leaves) andwith zeatin riboside (in roots, stems, and buds). In the roots,in addition to the peak co-chromatographing with zeatin riboside,there was also a prominent unidentified polar peak. The presence of zeatin and zeatin riboside in the stems andleaves was indicated also by chromatographic behaviour in othersystems, effects of permanganate oxidation, and cocrystallisationwith the authentic unlabelled compounds. Biological activitywas exhibited by both peaks in the soybean callus bioassay.Other metabolites in the shoot, possibly active as cytokinins,had the characteristics of dihydrozeatin, zeatin or dihydrozeatin-5'-nucleotide(s),and zeatin or dihydrozeatin glucosides. The gradual disappearancewith time of zeatin and its riboside from the shoot was accompaniedby an increase in the proportion of more polar metabolites. These results are discussed in relation to the possible exportof endogenous cytokinins by the nodules.     

Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia

Nodulation (mean number of nodules per seedling) was 5 times greater for Elaeagnus angustifolia than for Alnus glutinosa overall when seedlings were grown in pots containing either an upland or an alluvial soil from central Illinois, USA. However, the upland Alfisol had 1.3 times greater nodulation capacity for A. glutinosa than for E. angustifolia. The presence of A. glutinosa trees on either soil was associated with a two-fold increase in nodulation capacity for E. angustifolia. Nodulation increases for soils under A. glutinosa were obtained for A. glutinosa seedlings in the Alfisol, but decreased nodulation for A. glutinosa seedlings occurred in the Mollisol. Greatest nodulation of E. angustifolia seedlings occurred near pH 6.6 for soil pH values ranging from 4.9 to 7.1, while greatest nodulation of A. glutinosa occurred at pH 4.9 over the same pH range. Nodulation was not affected by total soil nitrogen concentrations ranging from 0.09 to 0.20%. Mollisol pH was significantly lower under A. glutinosa trees than under E. angustifolia trees. For 4- to 8-year-old field-grown trees, A. glutinosa nodule weights were negatively correlated with soil pH, while for similar aged E. angustifolia trees nodulation in the acidic Alfisol was not detected.     

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.     

Identification and Localization of Frankia Strains in Alnus Nodules by In Situ Hybridization of nif H mRNA with Strain-Specific Oligonucleotide Probes

In situ hybridization of Frankia mRNA with specific probes wasused to localize the strains Arl3 and AcoN24d in Alnus nodulesobtained after inoculation with one or both strains. The probesconsisted of 18-mer oligonucleotides, complementary to strain-specificsequences located within the nif H gene. Sections of nodulesinoculated with only one strain revealed a specific hybridizationbetween the probe and the corresponding Frankia strain mRNA.In sections of dually-inoculated nodules the presence of thestrain AcoN24d in the nodule was clearly shown whereas thoseof the strain Arl3 could not be detected. This suggests thatthe strain Arl3 is less infective than the strain AcoN24d andis not present within the nodule. Key words: Nitrogen fixation, actinorhizae, autoradiography, histochemistry     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Metabolism of [8-14C]Zeatin in Root Nodules of Alnus glutinosa (L.) Gaertn

The metabolism of [8-¹⁴C]zeatin, supplied via micropipettesover a 24 h period to root nodules of Alnus gliutinosa (L.)Gaertn., was investigated. The major metabolites were tentativelyidentified by means of chromatographic, chemical, and enzymictreatments as adenine, adenosine, trans-zeatin riboside, dihydrozeatin,trans-zeatin-O-ß-D-glucoside, and the O-ß-D-glucosideof dihydrozeatin. In addition, a prominent water-soluble peakof radioactivity was present. This did not appear to be a ribosidebut was biologically active in the soybean callus test. The number and nature of the metabolites formed in the noduleswas similar in both dormant and non-dormant plants.     

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.     

The Auxin Content of Root Nodules and Roots of Alnus glutinosa (L.) Vill.

In the acid ether-soluble fraction of methanol extracts of rootnodules and roots of Alnus glutinosa (L.) Vill., indol.3yl .aceticacid (IAA) and indol-3yl-carboxylic acid (ICA) were demonstratedspectroflurometrically and the amounts determined quantitatively.Substantially more IAA was detected in nodule tissue than inroots. No seasonal variation in the IAA content, either forthe roots, could be found. ICA was present in measurable amountsonly in the root extracts. Biochromatographic investigations of the extracts revealed IAAto be the main auxin in the nodule tissues. These findings arediscussed with special attention to results of comparable investigationsof auxins in leguminous root nodules and roots.     

Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.     

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.     

Observations on the Formation and Function of the Root Nodules of Alnus glutinosa (L.) Gaertn

Nodulated alder plants grow vigorously in water culture withoutcombined nitrogen. Evidence is advanced to show that the fixationof atmospheric nitrogen thus implied occurs actually withinthe nodulated plant and probably in the nodule. Nodule formation occurred most freely over the pH range 5•4to 7•0, while subsequent to nodulation the best growthof plants was in the pH range 4•2 to 5•4. The capacityof the host plant to tolerate relatively low pH levels considerablyexceeds that of the nodule organism. The oxygen requirementof the nodules appears to be relatively high. The fixation ofnitrogen per unit dry weight of nodule tissue exceeds that oflegumes grown under comparable conditions.     

Capacity for hexose respiration in symbiotic Frankia from Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules containing a local source of Frankia by an improved homogenization-filtration procedure. The capacity of the vesicle clusters to metabolize hexoses was investigated by respirometric and enzymological studies. The vesicle clusters could utilize glucose, glucose-6-phosphate and 6-phosphogluconate provided that appropriate cofactors were added to the preparations. The enzymes hexokinase (EC 2.7.1.1), NADP⁺: glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and NAD⁺;6-phosphogluconate dehydrogenase (EC 1.1.1.44) were found in cell-free extracts of the vesicle clusters and kinetic constants for the enzymes were determined. Hexokinase had a lower K_m for glucose than for fructose. Extracts from both symbiotic and propionate grown Frankia AvcII also showed activity of these hexose-degrading enzymes, indicating that their presence is not necessarily dependent on sugars as carbon source. The NAD⁺- dependent 6-phosphogluconate dehydrogenase was only present in Frankia cells and not in alder root cells, which makes this enzyme a useful Frankia -specific marker in these symbiotic systems.     

Respiration of Malate and Glutamate in Symbiotic Frankia Prepared from Alnus incana

Frankia vesicle clusters were prepared from root nodules ofAlnus incana (L.) Moench inoculated either with a local sourceof Frankia or with Frankia Cpll. The capacity of vesicle clustersto respire was investigated by respirometric and enzymologicalstudies. Simultaneous addition of malate, glutamate, and NAD⁺supported respiration in both types of Frankia, though at asmaller rate compared to the substrates NADH or 6-phosphogluconate.The saturating concentrations of malate and glutamate were alsomuch higher than with the other substrates. No respiration wassupported by succinate. Activity of the enzymes malate dehydrogenase(EC 1.1.1.37 [EC] ) and glutamate oxaloacetate transaminase (EC 2.6.1.1 [EC] )was demonstrated in crude extracts from both types of symbioticFrankia. Their maximum rates were high enough to account forthe respiration of malate and glutamate. This respiration wasinhibited by mersalylic acid, an inhibitor of the dicarboxylateshuttle in mitochondria, but it was shown that inhibition ofrespiration could be due to a direct effect on the enzymes.We conclude that respiration of malate and glutamate is mostlikely mediated by malate dehydrogenase and glutamate oxaloacetatetransaminase, but no explicit evidence for or against the presenceof a dicarboxylate carrier was found. The utilization of respiratorysubstrates was largely similar in the two types of Frankia,except for some differences in maximum rates and cofactor dependency. Key words: Actinorhizal symbioses, Alnus, dicarboxylate shuttle, Frankia, reducing power, respiration     

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.