Symbiotic Nitrogen Fixation Efficiency Research Articles

What determines symbiotic nitrogen fixation efficiency in rhizobium: recent insights into Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.

Nitrogen Distribution and Soil Microbial Community Characteristics in a Legume–Cereal Intercropping System: A Review

Intercropping systems can flexibly use resources such as sunlight, heat, water, and nutrients in time and space, improve crop yield and land utilization rates, effectively reduce continuous cropping obstacles and the occurrence of diseases and insect pests, and control the growth of weeds. Thus, intercropping is a safe and efficient ecological planting mode. The legume–cereal intercropping system is the most common planting combination. Legume crops fix nitrogen from the atmosphere through their symbiotic nitrogen fixation abilities, and the fixed nitrogen can be transferred to and utilized by cereal crops in various ways. The symbiotic nitrogen fixation efficiency of legume crops was improved by reducing the inhibition of soil nitrogen on nitrogenase activity through competitive absorption of soil nitrogen. However, the effects of nitrogen transformation and distribution in intercropping systems and microbial community structure characteristics on nitrogen transfer need to be further explored. In this review, (ⅰ) we present the transformation and distribution of nitrogen in the legume–cereal intercropping system; (ⅱ) we describe the soil microbial community characteristics in intercropping systems; and (ⅲ) we discuss the advantages of using modern biological molecular techniques to study soil microorganisms. We conclude that intercropping can increase the diversity of soil microorganisms, and the interaction between different plants has an important impact on the diversity and composition of the bacterial and fungal communities. The extensive application of modern biological molecular techniques in soil microbial research and the great contribution of intercropping systems to sustainable agriculture are particularly emphasized in this review.

Isolation and Characterization of Chickpea (Cicer arietinum L.) Nodulating Rhizobia Collected from South Wollo Zone, Ethiopia

This study aimed at screening rhizobial isolates of chickpea and evaluating their symbiotic nitrogen fixation efficiency and tolerance to abiotic stresses. A total of 107 (100%) isolates were collected of which 52 (48.6%) were confirmed as chickpea rhizobia using preliminary tests. Among 52 (48.6%) isolates, 46 (88.5%) have induced nodulation on chickpea under greenhouse and were screened under in vitro conditions and 6 (11.5%) of them were discarded due to fail to nodulate. The greenhouse data showed the highest nodule number (68.67 plant−1), nodule dry weight (0.17 g plant−1), and shoot dry weight (0.81 g plant−1) were scored by plants inoculated with isolates WUCR 17, WUCR 1, and WUCR 66, respectively. Among authenticated isolates, 73.9%, 21.7%, and 4.3% were highly effective, effective, and lowly effective, respectively. The physiological test showed 15% of isolates tolerated 13% salt concentration and 10.9% of isolates grew at pH 4–10 range. All isolates grew at 20–35°C and 13% grew at maximum temperature (50°C). The isolates showed better resistance to the tested antibiotics at low concentration (2.5 μg/ml) but the majorities were sensitive at high concentration (10 μg/ml). Among the isolates, 13% tolerated all tested heavy metals but 48% were sensitive to mercury. Regarding the carbohydrate utilization test, 52.2% catabolized all the tested 11 carbon sources but 6.5% of them utilized only 63.6% carbon sources. Amino acid utilization showed isolates (85%) utilized D-alanine except WUCR (14, 25, 31, 34, 39, 59, and 76) and 76% of the isolates utilized arginine and phenylalanine and 74% utilized leucine as a nitrogen source. Of the isolates, 43.5% of them utilized both D-alanine and arginine as a nitrogen source. In almost all tests conducted, isolates WUCR 1 and 5 performed top and were recommended as potential candidates for microbial inoculants.

Phenotypic, stress tolerance, and plant growth promoting characteristics of rhizobial isolates of grass pea.

Grass pea (Lathyrus sativus L.) is widely cultivated for food and feed in some developing countries including Ethiopia. However, due to its overexaggerated neuro-lathyrism alkaloid causing paralysis of limbs, it failed to attract attention of the research community and is one of the most neglected orphan crops in the world. But, the crop is considered an insurance crop by resource-poor farmers due to its strong abiotic stress tolerance and ability to produce high yields when all other crops fail due to unfavorable environmental conditions. This study was aimed at screening rhizobial isolates of grass pea and evaluating their symbiotic nitrogen fixation efficiency and tolerance to abiotic stresses. Fifty rhizobial isolates collected from grass pea nodules were isolated, screened, and characterized based on standard microbiological methods. The rhizobial isolates showed diversity in nodulation, symbiotic nitrogen fixation, and nutrient utilization. The 16S rRNA gene sequencing of 14 rhizobial isolates showed that two of them were identified as Rhizobium leguminosarum and the remaining twelve as Rhizobium species. Based on their overall performance, strains AAUGR-9, AAUGR-11, and AAUGR-14 that performed top and identified as Rhizobium species were recommended for field trials. This study screened and identified effective and competitive rhizobial isolates enriched with high nitrogen-fixing and abiotic stress tolerant traits, which contributes much to the application of microbial inoculants as alternative to chemical fertilizers.

Research status of soybean symbiosis nitrogen fixation

Soybean (Glycine max) is one of the most important economic legume crops with largest planting area, and is also an important oil crop, as well as food and feed material. Soybean-rhizobia symbiosis plays important roles in plant cultivation and fertilizer application. However, there are many problems in agricultural application of soybean symbiotic nitrogen fixation. In this review, we summarized three restriction factors (host specificity, low nitrogen fixation efficiency and abiotic stress) and discussed research progresses of these factors. Clarification of host specific mechanism will help to select and apply rhizobia inoculants. Both maintaining high nitrogenase activity and delaying nodule senescence can improve the efficiency of symbiotic nitrogen fixation. Abiotic stress-tolerant rhizobia can improve the abiotic stress tolerance of soybean. Breeding stress tolerant genotypes of soybean and rhizobia, obtaining correlated genes are the common strategies to improve soybean symbiotic nitrogen fixation under extreme conditions. Regulatory mechanisms of these restriction factors are still poorly understood and needs further clarification.

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

Fenotipska i genotipska raznolikost sojeva Sinorhizobium meliloti izoliranih iz tala Zadarske županije

Soil bacteria, Sinorhizobium meliloti is of great agricultural importance because of its ability to fix atmospheric nitrogen in symbiosis with alfalfa very valuable forage crop. The main aim of this study was to evaluate tolerance of indigenous S. meliloti strains to stress environmental conditions. Twenty rhizobial strains, isolated from different regions in Croatia, were genotipically characterized to assess diversity amongst natural population. Stress tolerance assays were performed in order to select indigenous rhizobia with tolerance to unfavorable soil conditions. The growth of the strains was studied at different pH values, temperatures, NaCl and heavy metals concentrations. The results of 16S rDNA genotyping using PCR/RFLP analysis showed that 17 isolates could be assigned to S. meliloti while RAPD and ERIC-PCR fingerprints revealed significant genetic diversity among indigenous rhizobial strains. In comparison to the reference strain, higher resistance to adverse growth conditions of indigenous S. meliloti strains was determined. Most of the strains grew at temperatures higher than optimal and tolerated both acidic and alkaline environment. It was found that indigenous strains tolerate extremely high NaCl concentrations. Better understanding of rhizobial response to adverse environmental conditions is of potential value for improving rhizobial inoculants and efficiency of symbiotic nitrogen fixation.

Water-Soluble Humic Materials Regulate Quorum Sensing in Sinorhizobium meliloti Through a Novel Repressor of expR.

Quorum sensing (QS) plays an important role in the growth, nodulation, and nitrogen fixation of rhizobia. In this study, we show that water-soluble humic materials (WSHM) repress the expression of the QS related genes sinI, sinR, and expR in Sinorhizobium meliloti. This decreased the production of N-acetyl homoserine lactones (AHL) and exopolysaccharides (EPS), and ultimately increased S. meliloti cell density. We also identified a novel regulator, SMc03890 (renamed QsrR), which binds directly to the expR promoter. Deletion of qsrR increased expR expression. WSHM repressed the expression of expR by augmenting the interaction between QsrR and the expR promoter; this was determined by a bacterial-one-hybrid assay. These effects of WSHM on the QS system in S. meliloti may be the underlying mechanism by which WSHM increase the symbiotic nitrogen fixation of Medicago sativa inoculated with S. meliloti. This study provides the first evidence that humic acids regulate the QS of rhizobia and suggests that WSHM could be used as fertilizers to improve the efficiency of symbiotic nitrogen fixation.

Quantification of leghaemoglobin content in pea nodules based on near infrared hyperspectral imaging spectroscopy and chemometrics

Leghaemoglobin content in nodules is closely related to the amount of nitrogen fixed by the legume–rhizobium symbiosis. It is, therefore, commonly measured in order to assess the effect of growth-promoting parameters such as fertilisation on the symbiotic nitrogen fixation efficiency of legumes. The cyanmethaemoglobin method is a reference method in leghaemoglobin content quantification, but this method is time-consuming, requires accurate and careful technical operations and uses cyanide, a toxic reagent. As a quicker, simpler and non-destructive alternative, a method based on near infrared (NIR) hyperspectral imaging was tested to quantify leghaemoglobin in dried nodules. Two approaches were evaluated: (i) the partial least squares (PLS) approach was applied to the full spectrum acquired with the hyperspectral device and (ii) the potential of multispectral imaging was also tested through the preselection of the most relevant wavelengths and the building of a multiple linear regression model. The PLS approach was tested on mean spectra acquired from samples containing several nodules and acquired separately from individual nodules. Peas (Pisum sativum L.) were cultivated in a greenhouse. The nodules were harvested on four different dates in order to obtain variations in leghaemoglobin content. The leghaemoglobin content measured with the cyanmethaemoglobin method in fresh nodules ranged between 1.4 and 4.2 mg leghaemoglobin g–1 fresh nodule. A PLS regression model was calibrated on leghaemoglobin content measured with the reference method and mean NIR spectra of dried nodules acquired with a hyperspectral imaging device. On a validation dataset, the PLS model predicted the leghaemoglobin content in nodule samples well (R2 = 0.90, root mean square error of prediction = 0.26). The multispectral approach showed similar performance. Applied to individual nodules, the PLS model highlighted a wide variability of leghaemoglobin content in nodules harvested from the same plant. These results show that NIR hyperspectral imaging could be used as a rapid and safe method to quantify leghaemoglobin in pea nodules.

Zn-use efficiency for optimization of symbiotic nitrogenfixation in chickpea (Cicer arietinum L.)

Zn deficiency is widespread in traditional areas of chickpea cultivation worldwide. It limits chickpea productivity and causes significant losses to the economies of the world?s largest chickpea exporters. This review may be of interest to researchers who would like to contribute to the improvement of chickpea cultivation on Zn-depleted soils in an environmentally sustainable manner, namely via identification of genotypes with superior symbiotic performance under Zn-limited conditions. The primary aim of the current work is to familiarize the readers with the biology and symbiotic characteristics of chickpea, and also to provide the necessary background on Zn as an essential nutrient for symbiotic nitrogen fixation (SNF). Special attention has been paid to the choice of rhizobial strains compatible with chickpea. Strains that can serve as an inoculum for simultaneous analysis of many genetically diverse chickpea lines have been suggested. The genotypes listed in this work can be good starting material for identification of chickpea lineages useful for unraveling the molecular basis of Zn-use efficiency, SNF efficiency, or both.

RNA-Seq Analysis of Differential Gene Expression Responding to Different Rhizobium Strains in Soybean (Glycine max) Roots.

The root nodule symbiosis (RNS) between legume plants and rhizobia is the most efficient and productive source of nitrogen fixation, and has critical importance in agriculture and mesology. Soybean (Glycine max), one of the most important legume crops in the world, establishes a nitrogen-fixing symbiosis with different types of rhizobia, and the efficiency of symbiotic nitrogen fixation in soybean greatly depends on the symbiotic host-specificity. Although, it has been reported that rhizobia use surface polysaccharides, secretion proteins of the type-three secretion systems and nod factors to modulate host range, the host control of nodulation specificity remains poorly understood. In this report, the soybean roots of two symbiotic systems (Bradyrhizobium japonicum strain 113-2-soybean and Sinorhizobium fredii USDA205-soybean)with notable different nodulation phenotypes and the control were studied at five different post-inoculation time points (0.5, 7–24 h, 5, 16, and 21 day) by RNA-seq (Quantification). The results of qPCR analysis of 11 randomly-selected genes agreed with transcriptional profile data for 136 out of 165 (82.42%) data points and quality assessment showed that the sequencing library is of quality and reliable. Three comparisons (control vs. 113-2, control vs. USDA205 and USDA205 vs. 113-2) were made and the differentially expressed genes (DEGs) between them were analyzed. The number of DEGs at 16 days post-inoculation (dpi) was the highest in the three comparisons, and most of the DEGs in USDA205 vs. 113-2 were found at 16 dpi and 21 dpi. 44 go function terms in USDA205 vs. 113-2 were analyzed to evaluate the potential functions of the DEGs, and 10 important KEGG pathway enrichment terms were analyzed in the three comparisons. Some important genes induced in response to different strains (113-2 and USDA205) were identified and analyzed, and these genes primarily encoded soybean resistance proteins, NF-related proteins, nodulins and immunity defense proteins, as well as proteins involving flavonoids/flavone/flavonol biosynthesis and plant-pathogen interaction. Besides, 189 candidate genes are largely expressed in roots and\\or nodules. The DEGs uncovered in this study provides molecular candidates for better understanding the mechanisms of symbiotic host-specificity and explaining the different symbiotic effects between soybean roots inoculated with different strains (113-2 and USDA205).

Improvement of symbiotic nitrogen fixation in plants: molecular-genetic approaches and evolutionary models

Efficiency of symbiotic nitrogen fixation in legumes depends on bringing together the processes of N2 fixation, assimilation of its products, supply of nitrogenase with energy, and development of nodule tissue and cellular structures. Coordination of these processes could arise from the evolutionary old functions of the nodules associated with deposition of the products of photosynthesis governed by systemic signals traveling between the above-ground organs and the roots. Further increase in symbiotic efficiency was associated with a pronounced ability to fix N2 by intracellular bacteroids that lost capability to propagate (as observed in galegoid legumes from the tribes Viciae, Trifolieae, and Galegae producing indeterminate nodules). However, efficiency of these symbioses is restricted by a slow removal from the nodules of the products of N2 fixation, which are assimilated along the same amide pathway as nitrogen compounds arriving from the soil. In legumes from the tribe Phaseoleae, such a restriction was overcome owing to a particular way of nitrogen assimilation via its incorporation into ureides (in determinate nodules). Development of symbioses where specialization of bacteroids in symbiotic fixation of atmospheric nitrogen is combined with its ureide assimilation will make it possible to produce new forms of plants highly efficient in symbiotic nitrogen fixation.

The nodule conductance to O2 diffusion increases with high phosphorus content in the Phaseolus vulgaris-rhizobia symbiosis

Although recent studies have addressed the effects of phosphorus (P) deficiency on nodule O2 permeability, little attention has been given to the relationship between nodule P status and nodule permeability. To study these traits, four recombinant inbred lines, namely RILs 34, 83, 115, 147 and one local variety (Concesa) of common bean (Phaseolus vulgaris) were inoculated with RhM11 (a native rhizobial strain from Haouz area of Marrakesh), and grown in hydroaeroponic culture under P-sufficiency (250 μmol P plant−1 week−1) versus P-deficiency (75 μmol P plant−1 week−1) conditions. At the flowering stage, the biomass of plants and nodules and their P contents was determined after measuring O2 uptake by nodulated roots (Conr) and nodule conductance to O2 diffusion (gn). The results showed that P-deficiency significantly decreased plant growth and nodulation, though there were differences between bean genotypes. P-deficiency also induced a decrease in nodule P content (31%) in both sensitive (83, 147) and tolerant lines (34, 115), a 42 and 27% reduction in shoots of sensitive and tolerant lines, respectively. These decreases were associated with significant variations in nodule surface and O2 permeability among bean genotypes and P-nutrition. Under P-deficiency, gn increased more for the sensitive (39%) than for the tolerant lines (27%). This increase was linked with a rise both in the P levels in nodules and shoots, as well as in the efficiency of symbiotic nitrogen fixation as determined by nodule-dependent biomass production for the sensitive lines. Furthermore, positive correlations were found between O2 permeability, gn and P content both in nodules and shoots (r2 = 0.94** and r2 = 0.96**). We conclude that nodule variations in Conr and gn are related to nodule P content, and may contribute to the adaptation of energy metabolism in N2-fixing bean nodules to P-deficiency.

Rizóbios nativos do Rio Grande do Sul simbioticamente eficientes em Lotus glaber

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.

The importance of nodule CO2 fixation for the efficiency of symbiotic nitrogen fixation in pea at vegetative growth and during pod formation.

Nodule CO2 fixation is of pivotal importance for N2 fixation. The process provides malate for bacteroids and oxaloacetate for nitrogen assimilation. The hypothesis of the present paper was that grain legume nodules would adapt to higher plant N demand and more restricted carbon availability at pod formation through increased nodule CO2 fixation and a more efficient N2 fixation. Growth, N2 fixation, and nodule composition during vegetative growth and at pod formation were studied in pea plants (Pisum sativum L.). In parallel experiments, 15N2 and 13CO2 uptake, as well as nodule hydrogen and CO2 release, was measured. Plants at pod formation showed higher growth rates and N2 fixation per plant when compared with vegetative growth. The specific activity of active nodules was about 25% higher at pod formation. The higher nodule activity was accompanied by higher amino acid concentration in nodules and xylem sap with a higher share of asparagine. Nodule 13CO2 fixation was increased at pod formation, both per plant and per 15N2 fixed unit. However, malate concentration in nodules was only 40% of that during vegetative growth and succinate was no longer detectable. The data indicate that increased N2 fixation at pod formation is connected with strongly increased nodule CO2 fixation. While the sugar concentration in nodules at pod formation was not altered, the concentration of organic acids, namely malate and succinate, was significantly lower. It is concluded that strategies to improve the capability of nodules to fix CO2 and form organic acids might prolong intensive N2 fixation into the later stages of pod formation and pod filling in grain legumes.

Factors Affecting the Efficiency of Symbiotic Nitrogen Fixation by Rhizobium

Factors Affecting the Efficiency of Symbiotic Nitrogen Fixation by Rhizobium

Dictyostelium discoideum Fatty-acyl Amidase II Has Deacylase Activity on Rhizobium Nodulation Factors

Dictyostelium discoideum (Amoebidae) secretes cell-lysing enzymes: esterases, amidases, and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty-acyl amidases FAA I and FAA II, act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of Gram-negative bacterial lipopolysaccharide. FAA I selectively hydrolyzes the 3-hydroxymyristoyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. bis-N-acylated lipid A is not a substrate. We have synthesized a 14C-labeled substrate analog for FAA II and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease, but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicates that Rhizobium fredii Nod factors are selectively de-N-acylated by purified FAA II, suggesting that the enzyme may reduce the N2-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal.

Translocation ‐ A key factor limiting the efficiency of nitrogen fixation in legume nodules

A hypothesis is presented that the availability of water for export of nitrogenous products from legume nodules is a major factor limiting the efficiency of symbiotic nitrogen fixation. Water for export of solutes in the xylem probably depends largely on the import of water and reduced carbon in the phloeum, and one function of respiration may be to dispose of reduced carbon in order to increase the supply of water. A second hypothesis presented is that control of gas diffusion in soybean nodules is largely restricted to the cortex nearby the vascular bundles, thus making possible the linkage of solute balances in xylem and phloem with resistance to diffusion. These concepts are used in a re‐examination of literature on manipulations of nodules and nodulated plants such as lowering of light levels, water stress, defoliation, stem girdling, and alteration of oxygen supply. The concept of translocation as a major factor limiting efficiency of symbiotic fixation is consistent with the failure of superior rhizobial isolates to improve N input significantly, and this limitation could also prevent exploitation of superior bacterial symbionts in the future

Enhanced efficiency of symbiotic nitrogen fixation by a derivative of Rhizobium meliloti

Summary The derivative of Rhizobium meliloti Bp, that had just been used as inoculant strain was isolated and characterized. The symbiotic efficiency of the derivative Bp 17 was compared with the parental Bp strain in laboratory and field conditions. In both kinds of experiments the increase of the yield of plants was higher in the case of Bp 17 strain. In field experiment that had been conducted during 3 years the total green mass of plants infected with Bp 17 was about 10% higher than the total green mass of plants inoculated with the parental strain. Rhizobium meliloti Bp 17 formed nodules earlier than its parental strain and occupied more than half of nodules even in the ratio Bp/Bp 17 = 10:1.

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia)

Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif (r) showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.