Equilibrium between the "genuine mutualists" and "symbiotic cheaters" in the bacterial population co-evolving with plants in a facultative symbiosis

The mathematical model for evolution of the plant-microbe facultative mutualistic interactions based on the partners’ symbiotic feedbacks is constructed. Using the example of rhizobia-legume symbiosis, we addressed these feedbacks in terms of the metabolic exchange resulting in the parallel improvements of the partners’ fitness (positive feedbacks). These improvements are correlated to the symbiotic efficiency dependent on the ratio of N₂-fixing bacterial strains (“genuine mutualists”) to the non- N₂-fixing strains (“symbiotic cheaters”) in the root nodules. The computer experiments demonstrated that an interplay between the frequency-dependent selection (FDS) and the Darwinian (frequency-independent) selection pressures implemented in the partners’ populations ensures an anchoring or even domination for the newly generated host-specific mutualists (which form N₂-fixing nodules only with one of two available plant genotypes) more successfully than for the non-host-specific mutualists (which form N₂-fixing nodules with both plant genotypes). The created model allows us to consider the mutualistic symbiosis as a finely balanced polymorphic system wherein the equilibrium in bacterial population may be shifted in favor of “genuine mutualists” due to the partner-stipulated selection for an improved symbiotic efficiency implemented in the plant population.     

Evolution of legume-rhizobium symbiosis for an improved ecological efficiency and genotypic specificity of partner interactions

Mathematical simulation of the evolution of polymorphic legume-rhizobium symbiosis showed that co-evolution of the partners for an improved ecological efficiency of symbiosis is greatly stimulated when low-active N₂-fixing and non-N₂-fixing strains of nodule bacteria are prohibited from colonizing nodules. The results of analysis of the model were collated with the comparative morphology of the infection process in various legumes, and its was assumed that mechanisms controlling bacterial reproduction in nodules arose in early evolution of symbiosis in primitive legumes owing to a transition from mixed to clonal infection. The development of such mechanisms was associated with adaptively valuable macroevolutionary transformations of symbiosis and directed its microevolution towards a parallel increase in the specificity and efficiency of mutualism. The increase was due to a reorganization of selective processes in endosymbiotic bacterial populations, which was based on changes in their genetic and spatial structures and optimized metabolic feedbacks between the partners (preferential allocation of photosynthesis products to the most active N₂-fixing strains).     

Construction of highly-effective symbiotic bacteria: Evolutionary models and genetic approaches

Using the example of N₂-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve: (i) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; (ii) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes encoding for nitrogenase synthesis or for the energy supply of N₂ fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars and the production of polysaccharides and storage compounds, as well as for oxidative-reductive processes).     

Nitrogen fixation in lichens is important for improved rock weathering

It is known that cyanobacteria in cyanolichens fix nitrogen for their nutrition. However, specific uses of the fixed nitrogen have not been examined. The present study shows experimentally that a mutualistic interaction between a heterotrophic N₂ fixer and lichen fungi in the presence of a carbon source can contribute to enhanced release of organic acids, leading to improved solubilization of the mineral substrate. Three lichen fungi were isolated fromXanthoparmelia mexicana, a foliose lichen, and they were cultured separately or with a heterotrophic N₂ fixer in nutrient broth media in the presence of a mineral substrate. Cells of the N₂-fixing bacteria attached to the mycelial mats of all fungi, forming biofilms. All biofilms showed higher solubilizations of the substrate than cultures of their fungi alone. This finding has bearing on the significance of the origin and existence of N₂-fixing activity in the evolution of lichen symbiosis. Further, our results may explain why there are N₂-fixing photobionts even in the presence of non-fixing photobionts (green algae) in some remarkable lichens such asPlacopsis gelida. Our study sheds doubt on the idea that the establishment of terrestrial eukaryotes was possible only through the association between a fungus and a phototroph.     

Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N₂-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N₂-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N₂ fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C₄ metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

Simulation of bacteria-plant coevolution in the mutualistic symbiosis

We present the mathematical model for coevolution of root nodule bacteria (rhizobia) and leguminous plants which is based on the partners’ positive feedbacks resulted from their metabolic integration. The model parameters were introduced which determine: (1) coordinated changes in plant and bacterial population structures; (2) increase of fitness (reproductive potentials) in both partners as dependent on the symbiotic efficiency determined by proportion of N₂-fixing rhizobia strain in root nodules. Computer experiments demonstrated that microevolution of the simulated system may follow either oscillatory or quasi-monotonous regime as dependent of frequency-dependent selection (FDS) in plant population. Negative FDS occurring in the bacterial population during competition for nodulation in combination with the positive partners’ feedback may lead to anchoring the bacterial mutations which lead either to acquisition of mutualistic traits or to changes in specificity of their expression. Anchoring of the mutualistic strains occurs most successfully in the quasi-monotonous regime and results in the improvement of genetic stability in symbiotic system.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Nitrogen-Fixing (Acetylene Redution) Activity and Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland Rice

Nitrogen-fixing activity associated with different wetland rice varieties was measured at various growth stages by an in situ acetylene reduction method after the activities of blue-green algae (cyanobacteria) in the flood water and on the lower portion of the rice stem were eliminated. Nitrogen-fixing activities associated with rice varieties differed with plant growth stages. The activities increased with plant age, and the maximum was about at heading stage. The nitrogen fixed during the whole cropping period was estimated at 5.9 kg of N per ha for variety IR26 (7 days) and 4.8 kg of N per ha for variety IR36 (95 days). The population of aerobic heterotrophic N₂-fixing bacteria associated with rice roots and stems was determined by the most-probable-number method, using semisolid glucose-yeast extract and semisolid malate-yeast extract media. The addition of yeast extract to the glucose medium increased the number and activity of aerobic heterotrophic N₂-fixing bacteria. The glucose-yeast extract medium gave higher counts of aerobic N₂-fixing bacteria associated with rice roots than did the malate-yeast extract medium, on which Spirillum-like bacteria were usually observed. The lower portion of the rice stem was also inhabited by N₂-fixing bacteria and was an active site of N₂ fixation.     

Cyanobacterial biofertilizers in rice agriculture

Floodwater and the surface of soil provide the sites for aerobic phototrophic nitrogen (N) fixation by free-living cyanobacteria and theAzolla-Anabaena symbiotic N₂-fixing complex. Free-living cyanobacteria, the majority of which are heterocystous and nitrogen fixing, contribute an average of 20–30 kg N ha^-1, whereas the value is up to 600 kg ha^-1 for theAzollaAnabaena system (the most beneficial cyanobacterial symbiosis from an agronomic point of view). Synthesis and excretion of organic/growth-promoting substances by the cyanobacteria are also on record. During the last two or three decades a large number of studies have been published on the various important fundamental and applied aspects of both kinds of cyanobacterial biofertilizers (the free-living cyanobacteria and the cyanobacteriumAnabaena azollae in symbiotic association with the water fernAzolla), which include strain identification, isolation, purification, and culture; laboratory analyses of their N₂-fixing activity and related physiology, biochemistry, and energetics; and identification of the structure and regulation of nitrogenfixing (nif) genes and nitrogenase enzyme. The symbiotic biology of theAzolla-Anabaena mutualistic N₂-fixing complex has been clarified. In free-living cyanobacterial strains, improvement through mutagenesis with respect to constitutive N₂ fixation and resistance to the noncongenial agronomic factors has been achieved. By preliminary meristem mutagenesis inAzolla, reduced phosphate dependence was achieved, as were temperature tolerance and significant sporulation/spore germination under controlled conditions. Mass-production biofertilizer technology of free-living and symbiotic (Azolla-Anabaena) cyanobacteria was studied, as were the interacting and agronomic effects of both kinds of cyanobacterial biofertilizer with rice, improving the economics of rice cultivation with the cyanobacterial biofertilizers. Recent results indicate a strong potential for cyanobacterial biofertilizer technology in rice-growing countries, which opens up a vast area of more concerted basic, applied, and extension work in the future to make these self-renewable natural nitrogen resources even more promising at the field level in order to help reduce the requirement for inorganic N to the bare minimum, if not to zero.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

Root-based N2-fixing Symbioses: Legumes, Actinorhizal Plants, Parasponia sp. and Cycads

In the mutualistic symbioses between legumes and rhizobia, actinorhizal plants and Frankia, Parasponia sp. and rhizobia, and cycads and cyanobacteria, the N₂-fixing microsymbionts exist in specialized structures (nodules or cyanobacterial zones) within the roots of their host plants. Despite the phylogenetic diversity among both the hosts and the microsymbionts of these symbioses, certain developmental and physiological imperatives must be met for successful mutualisms. In this review, phylogenetic and ecological aspects of the four symbioses are first addressed, and then the symbioses are contrasted and compared in regard to infection and symbio-organ development, supply of carbon to the microsymbionts, regulation of O₂ flux to the microsymbionts, and transfer of fixed-N to the hosts. Although similarities exist in the genetics, development, and functioning of the symbioses, it is evident that there is great diversity in many aspects of these root-based N₂-fixing symbioses. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere.     

Root-Associated N2 Fixation (Acetylene Reduction) by Enterobacteriaceae and Azospirillum Strains in Cold-Climate Spodosols

N₂ fixation by bacteria in associative symbiosis with washed roots of 13 Poaceae and 8 other noncultivated plant species in Finland was demonstrated by the acetylene reduction method. The roots most active in C₂H₂ reduction were those of Agrostis stolonifera, Calamagrostis lanceolata, Elytrigia repens, and Phalaris arundinacea, which produced 538 to 1,510 nmol of C₂H₄·g⁻¹ (dry weight)· h⁻¹ when incubated at pO₂ 0.04 with sucrose (pH 6.5), and 70 to 269 nmol of C₂H₄· g⁻¹ (dry weight)·h⁻¹ without an added energy source and unbuffered. Azospirillum lipferum, Enterobacter agglomerans, Klebsiella pneumoniae, and a Pseudomonas sp. were the acetylene-reducing organisms isolated. The results demonstrate the presence of N₂-fixing organisms in associative symbiosis with plant roots found in a northern climatic region in acidic soils ranging down to pH 4.0.     

Enumeration and Localization of N2-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N₂-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N₂-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N₂-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N₂-fixing bacteria recoverable from the rhizoplane, although high numbers of N₂-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N₂ fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules

Legumes form a symbiotic interaction with Rhizobiaceae bacteria, which differentiate into nitrogen‐fixing bacteroids within nodules. Here, we investigated in vivo the pH of the peribacteroid space (PBS) surrounding the bacteroid and pH variation throughout symbiosis. In vivo confocal microscopy investigations, using acidotropic probes, demonstrated the acidic state of the PBS. In planta analysis of nodule senescence induced by distinct biological processes drastically increased PBS pH in the N₂‐fixing zone (zone III). Therefore, the PBS acidification observed in mature bacteroids can be considered as a marker of bacteroid N₂ fixation. Using a pH‐sensitive ratiometric probe, PBS pH was measured in vivo during the whole symbiotic process. We showed a progressive acidification of the PBS from the bacteroid release up to the onset of N₂ fixation. Genetic and pharmacological approaches were conducted and led to disruption of the PBS acidification. Altogether, our findings shed light on the role of PBS pH of mature bacteroids in nodule functioning, providing new tools to monitor in vivo bacteroid physiology.     

Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw

Much of the crop residues, including cereal straw, that are produced worldwide are lost by burning. Plant residues, and in particular straw, contain large amounts of carbon (cellulose and hemicellulose) which can serve as substrates for the production of microbial biomass and for biological N₂ fixation by a range of free-living, diazotrophic bacteria. Microorganisms with the dual ability to utilise cellulose and fix N₂ are rate, but some strains that utilize hemicellulose and fix N₂ have been found. Generally, cellulolysis and diazotrophy are carried out by a mixed microbial community in which N₂-fixing bacteria utilise cellobiose and glucose produced from straw by cellulolytic microorganisms. N₂-fixing bacteria include heterotrophic and phototrophic organisms and the latter are apparently more prominent in flooded soils such as rice paddies than in dryland soils. The relative contributions of N₂ fixed by heterotrophic diazotrophic bacteria compared with cyanobacteria and other phototrophic bacteria depend on the availability of substrates from straw decomposition and on environmental pressures. Measurements of asymbiotic N₂ fixation are limited and variable but, in rice paddy systems, rates of 25 kg N ha^-1 over 30 days have been found, whereas in dryland systems with wheat straw, in situ measurements have indicated up to 12 kg N ha^-1 over 22 days. Straw-associated N₂ fixation is directly affected by environmental factors such as temperature, moisture, oxygen concentration, soil pH and clay content as well as farm management practices. Modification of managements and use of inoculants offer ways of improving asymbiotic N₂ fixation.In laboratory culture systems, inoculation of straws with cellulolytic and diazotrophic microorganisms has resulted in significant increases in N₂ fixation in comparison to uninoculated controls and gains of N of up to 72 mg N fixed g^-1 straw consumed have been obtained, indicating the potential of inoculation to improve N gains in composts that can then be used as biofertilisers. Soils, on the other hand, contain established, indigenous microbial populations which tend to exclude inoculant microorganisms by competition. As a consequence, improvements in straw-associated N₂ fixation in soils have been achieved mostly by specific straw-management practices which encourage microbial activity by straw-decomposing and N₂-fixing microorganisms.Further research is needed to quantify more accurately the contribution of asymbiotic N₂ fixation to cropping systems. New strains of inoculants, including those capable of both cellulolytic and N₂-fixing activity, are needed to improve the N content of biofertilisers produced from composts. Developments of management practices in farming systems may result in further improvements in N₂ fixation in the field.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.     

Biological nitrogen fixation: A key to world protein

Summary 1. Characteristics and methodology of the C₂H₂-C₂H₄ assay forin situ measurement of N₂ fixation are outlined. 2. Electron micrographic analysis of the developmental morphology of the natural soybean symbiosis and C₂H₂-C₂H₄ analysis indicate that increasing N₂-fixing activity from 12–35 days of age is accompanied by an increase in bacteroid number per cell, bacteroid number per vesicle and inclusions per bacteroid. The mole ratio of leghemoglobin to nitrogenase also increases from 50 to a relatively constant plateau of 500 to 1500 during this period. The quantitative validity of the C₂H₂-C₂H₄ assay as a measure of N₂ fixation during a complete growth cycle of soybeans on nitrogen-free medium is demonstrated by Σ (C₂H₂→C₂H₄)×28/3 values which are 75–95% of the values determined for N₂ fixed by Kjeldahl analyses. 3. A technique for the establishment of the first callus N₂-fixing symbiosis in mixed cultures ofRhizobium legume provides a defined experimental system for exploration of legume symbiosis. N₂-fixing activity is about 1% of the natural system and is influenced by exogenous auxins and cytokinins. Morphology, including infection threads and vesicle enclosed bacteroids, is similar to the nodule system. 4. N₂-fixing activity of field-grown soybeans, including varieties which differed in flowering characteristics and maturity dates, and of peanuts was determined biweekly with the C₂H₂-C₂H₄ assay. Activity extended from nodule initiation to senescence and correlated with the nitrogen demands of the plant and in most cases >90% of the N₂ fixed during the 60–70 day period of fruit formation and maturation. A logarithmic relationship between N₂-fixing activity and age, and N₂ fixed and age was demonstrated as a fundamental characteristic of these annual symbionts,i.e. log N₂ fixed =k(t−t ₀), wheret ₀ is age at activity initiation. The resultant parameters: 1) age at activity initiation, 2) calculated rate of daily increase (7–9% for soybeans and 7–10% for peanuts), 3) age at end of logarithmic phase (about 80 days for soybeans), and 4) total N₂ fixed (about 250 mg per soybean plant) are useful bases for evaluation of environmental, bacterial and host effects on N₂ fixation. Various N fertilizers applied at planting and flowering inhibited N₂ fixation of soybeans by decreasing the rate of daily increase. 5. Physical and chemical characteristics of nitrogenase, including those of crystalline Mo-Fe protein, reactions of nitrogenase, and model studies are consistent with a proposed mechanism. 6. Potential utilities of N₂ fixation research include increased food protein production via initially enhanced N₂ fixation of legumes such as soybeans and eventually extension of N₂-fixing symbioses to non-legumes and new chemistry of N₂, including the direct incorporation of aerial N₂ into important organic compounds. Contribution No. 1748.     

Co-evolution of partners and the integrity of symbiotic systems

Symbioses are very suitable models for studying the integrity of biosystems which characterizes their structural/functional organization enabling the partners to respond adequately to the environmental changes. Analysis of different forms of plant-microbe and animal-microbe symbiosis suggests that a qualitative increase of its integrity occurs under the facultative and ecologically obligatory interactions and is culminated under the genetically obligatory interactions. By use of mathematical models, we demonstrate that the functional integrity of N2-fixing legume-rhizobia symbiosis (concordance of changes in partners' genotypic frequencies induced by environmental fluctuations) correlates to its ecological efficiency which increases under force of natural selection. It results in the tight partners' regulatory feedbacks leading to their genetic integration manifested in the establishment of "symbiogenome". The genetic integrity of symbiosis determines its high evolutionary potential based on: a) epigenetic inheritance of symbiotic traits by hosts which may occur in the form of vertical transmission of either microsymbionts themselves or genes obtained from them; b) interspecies altruism interactions related to the positive partners' feedbacks which determine the ecological efficiency of mutualistic interactions. Realization of this potential results in the deep genetic integration of initially independent partners including their fusions into the novel integral organisms.     

The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.