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.     

Distinct patterns of symbiosis-related gene expression in actinorhizal nodules from different plant families

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).     

Characterization of a Casuarina glauca nodule-specific subtilisin-like protease gene, a homolog of Alnus glutinosa ag12

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.     

Regulation of nodulation in the absence of N2 is different in actinorhizal plants with different infection pathways

Root nodulation in actinorhizal plants, like Discaria trinervis and Alnus incana, is subject to feedback regulatory mechanisms that control infection by Frankia and nodule development. Nodule pattern in the root system is controlled by an autoregulatory process that is induced soon after inoculation with Frankia. The final number of nodules, as well as nodule biomass in relation to plant biomass, are both modulated by a second mechanism which seems to be related to the N status of the plant. Mature nodules are, in part, involved in the latter process, since nodule excision from the root system releases the inhibition of infection and nodule development. To study the effect of N(2) fixation in this process, nodulated D. trinervis and A. incana plants were incubated under a N(2)-free atmosphere. Discaria trinervis is an intercellularly infected species while A. incana is infected intracellularly, via root hairs. Both symbioses responded with an increment in nodule biomass, but with different strategies. Discaria trinervis increased the biomass of existing nodules without significant development of new nodules, while in A. incana nodule biomass increased due to the development of nodules from new infections, but also from the release of arrested infections. It appears that in D. trinervis nodules there is an additional source for inhibition of new infections and nodule development that is independent of N(2) fixation and nitrogen assimilation. It is proposed here that the intercellular Frankia filaments commonly present in the D. trinervis nodule apex, is the origin for the autoregulatory signals that sustain the blockage of initiated nodule primordia and prevent new roots from infections. When turning to A. incana plants, it seems likely that this signal is related to the early autoregulation of nodulation in A. incana seedlings and is no longer present in mature nodules. Thus, actinorhizal symbioses belonging to relatively distant phylogenetic groups and displaying different infection pathways, show different feedback regulatory processes that control root nodulation by Frankia.     

Carbon metabolism of Frankia spp. in root nodules of Alnus glutinosa and Hippophaë rhamnoides

The occurrence and localization of enzymes involved in glycolysis, tricarboxylic acid cycle and glyoxylate cycle in root nodules of Alnus glutinosa (L.) Vill. and Hippophaë rhamnoides L. ssp. rhamnoides were studied. The following enzymes, catalyzing reversible steps in the glycolysis, were found in both the endophyte Frankia spp. and the plant cytosol of Alnus nodules: fructose-1,6-diphosphate aldolase, glyceralde-hyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase. The enzymes catalyzing irreversible steps in glycolysis, viz. hexokinase and pyruvate kinase, were detectable only in the plant cytosol. Similar results were obtained with nodule homogenates of Hippophaë. This indicates the absence of a complete glycolysis in the endophyte. Vesicle clusters of the nodule endophyte of Alnus contained various dehydrogenases of the tricarboxylic acid cycle and showed activity of glutamate oxaloacetate transaminase. Respiration studies showed that vesicle clusters take up oxygen when supplied with NAD, glutamate and malate together. No oxygen uptake was found when any of these compounds was omitted. Vesicle clusters from both Alnus and Hippophaë nodules showed no detectable activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. Since these enzymes are known to be present in Frankia Avcll, when grown in a medium with Tween 80 as carbon source, it is suggested that the glyoxylate cycle enzymes are repressed in the root-nodule symbioses.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

Water-retentive polymers increase nodulation of actinorhizal plants inoculated with Frankia

Actinorhizal plants form a nodular, nitrogen-fixing root symbiosis with the actinomycete Frankia and are economically and ecologically important due to their ability to improve the nitrogen fertility of disturbed and infertile substrates. In this study, water-retentive polymer inoculum carriers were applied as a root dip. This treatment significantly increased nodulation and in some cases early growth of Alnus glutinosa (L.) Gaertn. and Casuarina equisetifolia var. equisetifolia Forst. & Forst. in a controlled environment and also of A. glutinosa under field conditions. Nodule number and nodule dry weight per plant were at least two to three times greater after 56 to 140 days for plants inoculated with Frankia carried in a water-retentive polymer base compared with plants inoculated with Frankia in water. Nodules on the roots of the plants that were inoculated with Frankia in a polymer slurry were distributed throughout the entire root system, rather than concentrated near the root collar. When amended with water-retentive polymers, actinorhizal plants inoculated with 5- to 10-fold lower titers of Frankia exhibited early growth and nodule numbers equal to or greater than those plants inoculated with standard titers without polymers. The water-retentive, superabsorbent polymers clearly increased the nodulation of two actinorhizal plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa . Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell-free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae . No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.     

The acetylene reduction assay inactivates root nodule uptake hydrogenase in some actinorhizal plants

Actinorhizal nodules do not usually evolve H₂ due to the action of an uptake hydrogenase. We have found that nodules of several Frankia symbioses evolved large amounts of H₂ gas when returned to air following exposure to 10 kPa C₂HT₂ during an acetylene reduction assay. Increased H₂ evolution in air persisted for several days when intact root systems of Alnus incana (L.) Moench (inoculated with Frankia UGL 011101) were treated with 10 kPa C.H₂ for 1 h. Full recovery of uptake hydrogenase activity required 4 to 8 days. Studies with crude homogenates of nodules of the same plants showed that hydrogenase (measured amperometrically with phenazine metho-sulfate as electron acceptor) was directly affected, since activity in treated nodules was only 10% of that in untreated nodules. A survey of actinorhizal symbioses revealed variation in the effect of an acetylene reduction assay on hydrogen metabolism. Nodules of three species, including Alnus rubra Bong, inoculated with Frankia HFPArD. showed complete inactivation of hydrogenase. H₂ evolution in air was 25% of the C₂H₂ reduction rate and H, evolution in Ar/O₂ was equal to the QH₂ reduction rate. Two symbioses, Ceanothus americanus L. (soil inoculant) and Batista glomerata Baill. (soil inoculant) showed no change following an acetylene reduction assay. A third group of symbioses showed an intermediate response.     

Reactive oxygen species in legume and actinorhizal nitrogen-fixing symbioses: the microsymbiont's responses to an unfriendly reception

The plant responses to infection by pathogenic bacteria have been extensively reviewed in recent years, including the spatial and temporal production of reactive oxygen species (ROS). The immediate and localized release of ROS upon infection, known as the oxidative burst, was shown not only to be part of the hypersensitive response but also likely responsible for mediating, directly or via signal transduction pathways, other plant defence strategies. This paradigm inspired studies in nitrogen-fixing root nodule symbioses, and a parallelism is unavoidable. In rhizobia–legume symbioses, histochemical data revealed the presence of ROS in the host infection threads and in the root nodules primordia. On the other hand, in actinorhizal infections, it has been shown that Alnus glutinosa root exudates induce several oxidative stress response-related proteins in compatible Frankia . These data suggest that the nitrogen-fixing microsymbionts must have had to evolve adaptations to overcome and possibly regulate an unfriendly environment. In this review, particular emphasis will be given to the bacteria antioxidant mechanisms at different developmental stages of the nitrogen-fixing root nodule symbioses.     

Features of the intrageneric Alnus-Frankia specificity

Host specificity between local Frankia strains and native alders [Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn.] was evaluated in inoculation experiments. Pure cultures of Frankia , whether originating from A. incana or A. glutinosa , were infective and effective on both host species. These pure cultures were isolated from spore-negative (Sp⁻) nodules. From spore-positive (Sp⁺) nodules no Frankia isolates were obtained. This strain type resisted all our isolation attempts and therefore crushed nodules had to be used for Sp⁺ type inoculations.
The Sp⁺ type Frankia populations differed in their host specificity. Sp⁺ nodules from A. glutinosa were effective on both alder species, but Sp⁺ nodules from A. incana induced effective nodules only on the original host; on A. glutinosa only small (1-3mm) prenodule-like structures were found. Such A. glutinosa plants died on N-free medium, thus showing that these nodules were ineffective. In the effective nodules the middle cortex was dominated by infected cells filled with vesicle clusters. In the ineffective nodules only a few cortical cells were infected and sporangia predominated in these cells. Surprisingly enough they also contained vesicle-like structures as demonstrated in electron micrographs.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

Relationships among pure cultured strains of Frankia based on host specificity

Fifty strains of Frankia were tested for their ability to nodulate six species of actinorhizal plants. Pure cultured strains were used to inoculate seedlings of Alnus glutinosa (L.) Gaertn., Alnus rubra Bong., Casuarina equisetifolia L., Elaeagnus angustifolia L., Hippophaë rhamnoides L. and Myrica cerifera L. in nutrient solution culture. From the results of this study, host inoculation groups among the actinorhizal plants were defined. Although overlap between host inoculation groups appears to be common, the results from this study did not support the view that Frankia strains are promiscuous. All Frankia strains tested in this study could easily be classified into four major host-specificity groups.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

欧洲赤杨根瘤及其内生菌的形态学研究

对在云南生长的欧洲赤杨自然发生的根瘤及其内生菌──Ｆｒａｎｋｉａ进行了光学显微镜和扫描电镜的观察,结果表明,这种根瘤与原产地生长的欧洲赤杨所结根瘤十分相似;与原产地在云南的蒙自桤木的根瘤结构也很相近。从这种根瘤中分离到的内生菌的纯培养具有典型的Ｆｒａｎｋｉａ的特征。     

The diversity of actinorhizal symbiosis

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.     

Rhizobium strain effects on pea: The relation between nitrogen accumulation, phosphoenolpyruvate carboxylase activity in nodules and asparagine in root bleeding sap

Pisum sativum L. cv. Bodil was infected with various strains of Rhizobium leguminosarum (R501, 128c53, B155, 18a or 1044). The Rhizobium genotype influenced the activity of the plant enzyme phosphoenoipyruvate (PEP) carboxylase (EC 4.1.1.31), and the assimilation of fixed N in the root nodules. The specific activity of nodule PEP carboxylase was lowest in the symbioses, which accumulated the least total N (R501 and 128c53). The root bleeding sap of the less effective symbioses contained a lower proportion of asparagine and a higher proportion of glutamine than the more effective symbioses (B155,18a and 1044). The N yield of the symbioses was related neither to the net respiratory CO₂ evolution of the root system nor to the nitrogenase linked nodule respiration. The lower yielding symbioses accumulated a larger proportion of the fixed N in the nodules due to a higher proportion of total dry weight contained in the nodule tissue. However, the concentration of soluble protein in the nodules of the lower-yielding symbioses was lower than that recorded for the higher yileding symbioses. The effect of the Rhizobium strains on N yield was maintained at maturity, and reflected in seed yields.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Identification and subgrouping of Frankia strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Polypeptide patterns of soluble proteins from 35 Frankia strains from different plants of various geographical origins, belonging to Alnus and Elaeagnus host-specificity groups were determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide pattern was qualitatively the same for each strain whatever the number of subcultures or the age. Two gel electrophoresis groups A and E were observed which matched with the Alnus and Elaeagnus host-specificity groups, but with some exceptions. The polypeptide patterns of the 35 Frankia strains tested were separated into 13 gel electrophoresis subgroups. Five Frankia strains were inoculated separately or in 3 mixed combinations of 2 strains on Alnus glutinosa (L.) Gaertn. plants. The polypeptide patterns of the re-isolates obtained from 5-month-old nodules were identical to the corresponding strains used initially in the inoculum. Dual infection was observed on single plantlets.