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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

Effects of Frankia on field performance of Alnus clones and seedlings

Field performance of tissue cultured clones and seedlings of Alnus viridis ssp. crispa, A. glutinosa, A. incana, and A. japonica was assessed five years after outplanting in central Ontario. Half the individuals were inoculated with a mixture of four Frankia isolates prior to planting. Inoculation produced significant increases (25% to 33%) in biomass production of two clones of A. glutinosa and one of A. incana. Woody biomass increments for the first five years, averaged across all clones and seedlings, were highest in A. japonica and A. incana (4.3 and 3.7 Mg ha^–1 yr^–1, respectively). Individual tree growth improved markedly in lower slope positions, but total plot biomass did not show similar gains in downslope positions owing to higher mortality and aphid (Paraprociphilus tessellatus) infestation. Aphids occurred in 22% of Frankia-inoculated individuals, and 15% of non-inoculated individuals. The fastest growing species, A. incana and A. japonica, were most susceptible to aphid attack. Growth of the best clones of A. glutinosa and A. incana exceeded seedling growth by 51% and 76%, respectively. The high growth variation in clones of the same species with similar geographic origins and the excellent performance of tissue cultured stock suggest that rapid genetic gains in an Alnus breeding program might be obtained by clonal propagation.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Purity of Frankia preparations from root nodules of Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from the host plant. Special attention was paid to plant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5. The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0.4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be further decreased by osmotic shock. It is concluded that Frankia vesicle clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are sufficiently pure to be used for studies of Frankia metabolism.     

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.