Symbiotic N2 Fixation Research Articles

Maize/faba bean intercropping with rhizobial inoculation in a reclaimed desert soil enhances productivity and symbiotic N2 fixation and reduces apparent N losses

Although intensive modern agriculture, featured with high inputs of chemicals, contributed to global food production, simultaneously caused a series of serious issues, such as loss of biodiversity, soil degradation, and environment risk. The objective of our study is to integrate various inherit biological potentials of cropping system to gain higher productivity with lower external inputs. Field experiments were conducted over a two-year period (2008-2009) with five fertilizer-N application rates (0, 75, 150, 225 and 300 kg N ha−1) to study the influence of fertilizer N application on the productivity and N utilization of a Rhizobium-inoculated maize (Zea mays L.)/faba bean (Vicia faba L.) intercropping system on a reclaimed desert soil (Sierozems). Average grain yields of faba bean and maize with inoculation increased by 50.0 and 19.6 %, respectively, and reached a maximum land equivalent ratio of 1.78 with zero fertilizer N application in 2009 compared with sole crops. The nodulation of intercropped faba bean was significantly higher with Rhizobium inoculation, especially with an optimum N application rate of < 150 kg N ha−1. Average %Ndfa and Ndfa increased by 42.5 and 86.6 %, respectively, based on interspecific interaction and inoculation. The average apparent N recovery of the intercropping system with inoculation increased by 146.5 % at 75 kg N ha−1 relative to the weighted mean values of sole crops. Intercropping saved fertilizer N compared to sole crops suggested by a fertilizer N equivalent ratio (NFER) > 1. Furthermore, intercropping with rhizobium inoculation saved more fertilizer N than that without rhizobium inoculation. After the faba bean harvest, intercropped maize made better use of the nitrogen taken up to grow faster than sole maize. At the maize harvest the intercropping system showed higher grain yield and N acquisition in the aboveground parts and lower Nmin accumulation in the soil profile, an effect that declined with fertilizer N application. The results therefore indicate that Rhizobium-inoculated maize/faba bean intercropping on the reclaimed desert soil with a moderate fertilizer N application rate enhancede productivity, biological N2 fixation and apparent N recovery with lower environmental risk.

Rhizobium Inoculation and Chemical Fertilisation Improve Faba Bean Yield and Yield Components in Northwestern Ethiopia

The productivity of the faba bean has declined in Ethiopia, owing to poor management practices, such as blanket fertilisation. In 2018, a field experiment was conducted in a Nitisol soil during the main cropping season in Northwestern Ethiopia, to determine the amount of chemical fertiliser and Rhizobium inoculant to be used for the optimum yield within economic feasibility. The experiment consisted of a factorial combination of five rates of blended NPSZnB fertiliser (0, 60, 121, 180 and 240 kg ha−1) and three rates of inoculant (0, 500 and 750 g ha−1). Sole chemical fertilisation, as well as inoculation, individually produced a seed yield of 2.3–2.5 t ha−1, about 1.0–1.2 t ha−1 more than the control. However, the maximum seed yield (3.3 t ha−1) was recorded from the combined application of both the chemical fertiliser and the inoculant. The seed yield correlated closely with the number of active nodules (R2 = 0.78 **), suggesting a substantial contribution of symbiotic N2 fixation. Inoculation increased the N content of the seed yield by at least 30 kg ha−1. Chemical fertilisation, containing at least 44 kg ha−1 of mineral N does not appear to have an adverse effect on N2 fixation. The combined use of 180 kg ha−1 blended fertiliser with 750 g ha−1 inoculant, producing a maximum net profit of 72,918 birr ha−1 (EUR 2232), is recommended for the study area. This study emphasises that (1) inoculation alone can produce as much seed as the maximum rate of chemical fertilisation, but (2) the maximum yield was produced with a combined use of inoculant and chemical fertiliser, by promoting the vigour of the nodules and N2 fixation.

Twenty years of paradigm-breaking studies of taxonomy and symbiotic nitrogen fixation by beta-rhizobia, and indication of Brazil as a hotspot of Paraburkholderia diversity.

Twenty years ago, the first members of the genus Burkholderia capable of nodulating and fixing N2 during symbiosis with leguminous plants were reported. The discovery that β-proteobacteria could nodulate legumes represented a breakthrough event because, for over 100years, it was thought that all rhizobia belonged exclusively to the α-Proteobacteria class. Over the past 20years, efforts toward robust characterization of these bacteria with large-scale phylogenomic and taxonomic studies have led to the separation of clinically important and phytopathogenic members of Burkholderia from environmental ones, and the symbiotic nodulating species are now included in the genera Paraburkholderia and Trinickia. Paraburkholderia encompasses the vast majority of β-rhizobia and has been mostly found in South America and South Africa, presenting greater symbiotic affinity with native members of the families Mimosoideae and Papilionoideae, respectively. Being the main center of Mimosa spp. diversity, Brazil is also known as the center of symbiotic Paraburkholderia diversity. Of the 21 symbiotic Paraburkholderia species described to date, 11 have been isolated in Brazil, and others first isolated in different countries have also been found in this country. Additionally, besides the symbiotic N2-fixation capacity of some of its members, Paraburkholderia is considered rich in other beneficial interactions with plants and can promote growth through several direct and indirect mechanisms. Therefore, these bacteria can be considered biological resources employed as environmentally friendly alternatives that could reduce the agricultural dependence on agrochemical inputs.

Supply of acetyl-CoA to N&lt;sub&gt;2&lt;/sub&gt;-fixing bacteroids: insights from the mutational and proteomic analyses of &lt;em&gt;Sinorhizobium meliloti&lt;/em&gt;

Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.

The relative contribution of indigenous and introduced arbuscular mycorrhizal fungi and rhizobia to plant nutrient acquisition in soybean/maize intercropping in unsterilized soils

It has been demonstrated that intercropped crops may benefit from inoculation with rhizobia or arbuscular mycorrhizal (AM) fungi in soybean/maize intercropping systems under sterilized conditions. Little is known about the relative contribution of indigenous and introduced AM fungi and rhizobia to nutrient acquisition of intercropped soybean and maize in unsterilized soils. In this study, we evaluated the effectiveness of indigenous and introduced AM fungi and rhizobia in a soybean/maize intercropping system under unsterilized soil conditions. The results show that introduced rhizobial inoculation promoted soybean nodulation and growth. Single AM fungal inoculation promoted indigenous rhizobial nodulation and increased maize AM root colonization, but had little effect on maize growth. Co-inoculation strongly increased plant dry weight, and nitrogen (N) and phosphorus (P) content of the intercropped soybean compared to those with only indigenous rhizobial and AM fungal communities, but had no effect on those of maize in the same intercropped system. These results indicate that AM fungal inoculation promoted nodulation by indigenous rhizobia. Effective rhizobial inoculation combined with introduced AM fungi may not only promote symbiotic N2 fixation, but also enhance rhizosphere processes, which has the potential to improve nutrient acquisition in the field. Soybean is a weak competitor compared with maize in intercropping systems. Inoculation with introduced AM and rhizobia ameliorated the growth suppression of intercropped soybean, but inhibited growth of intercropped maize, indicating that inoculation of introduced AM fungi and/or rhizobia partly shifted the competitive advantage from maize to soybean. In conclusion, introduced AM fungi and rhizobia contributed more than indigenous populations to promoting growth and nutrient acquisition of the intercropped soybean in unsterilized soils.

Why Should Nodule Cysteine-Rich (NCR) Peptides Be Absent From Nodules of Some Groups of Legumes but Essential for Symbiotic N-Fixation in Others?

In nitrogen-fixing nodules of legumes such as pea (Pisum) and Medicago spp. the plant induces terminal differentiation in the rhizobial endosymbionts by targeting nodule-specific cysteine-rich defensin-like peptides into the bacteria. However, in nodules of other legumes such as soybean and Lotus spp. terminal bacterial differentiation does not occur; these legumes lack genes encoding equivalent peptides controlling rhizobial development. Here, we review the effects of some of these peptides on rhizobia and address the question as to how and why such peptides may have evolved to enslave rhizobia and become essential for nitrogen fixation in some clades of legumes but not in others.

The symbiotic nitrogen fixation by legumes in a legume‐companion and a legume‐dominant alpine steppe on the central Tibetan Plateau

AbstractSymbiotic N2‐fixing plant species occur in many stressful environments, including the central Tibetan Plateau, where the legume species Oxytropis microphylla, Oxytropis glacialis, Astragalus arnoldii occur in the alpine steppe at elevations as high as 4700 m above sea level, either as companion or as dominant species, accounting for 13.91–60.25% of the total aboveground steppe biomass. However, the degree to which they are nutritionally reliant upon symbiotic N2 fixation is still unclear. We measured the symbiotic nitrogen fixation rate in a legume‐companion (LC) and a legume‐dominant (LD) alpine steppe community using the 15N isotope dilution technique. The N derived from the atmosphere (%Ndfa) in the three legume species ranged from 17% to 61% in aboveground tissues and from 70% to 88% in belowground tissues. The N2 fixation rate was estimated to be 0.25 ± 0.04 and 1.39 ± 0.20 g N m−2 year−1 for LC and LD based on the aboveground biomass. It was 0.86 ± 0.04 and 2.77 ± 0.22 gNm−2 year−1 for LC and LD, respectively, when both above and belowground tissues were considered. The total biological nitrogen fixation (BNF) of the alpine steppe on the Tibetan Plateau (ca. 0.7 million km2) can then be conservatively estimated at approximately 0.6 T g N year−1 using the BNF estimate from the LC community. Our findings indicate that estimates based solely on aboveground biomass and LC communities might drastically underestimate the N inputs arising from BNF by legumes to the alpine ecosystem N budget.

Influence of nitrogen supply on gas exchange, chlorophyll fluorescence and grain yield of breeding lines of common bean evaluated in the Amazon region of Colombia

The common bean (Phaseolus vulgaris L.) is a food crop that provides fiber, vitamins and carbohydrates and it is produced by smallholders in developing countries under nitrogen (N)-depleted and low-fertility soil conditions. Soil acidity and the associated aluminum toxic and phosphorus deficient conditions are known to reduce symbiotic N-fixing ability of common bean. Breeding lines of common bean have been developed to adapt to acid-soil and high-temperature conditions in the Amazon region but the response of these lines to N application was not determined. The present study was conducted with an objective to identify genotypic differences among ten common bean breeding lines (4 Andean, 5 Mesoamerican, 1 interspecific) in their physiological characteristics (gas exchange, chlorophyll fluorescence and photosynthetic efficiency) and their contribution to superior agronomic performance under three different levels of N (0, 60 and 120 kg N ha−1; N0, N60 and N120) supply. We tested the hypothesis that common bean genotypes that combine greater capacity to assimilate carbon with improved ability to mobilize photosynthates to developing pods and seeds could enhance their agronomic performance at different levels of N supply under high-temperature and acid soil-stress conditions in the field. A randomized complete block design with factorial arrangement (3 levels of N × 10 genotypes) in three replications was used for the study. Measurements included photosynthetic gas exchange characteristics (light and CO2-saturated photosynthesis), chlorophyll fluorescence, viability of pollen, leaf N concentration, photosynthetic N use efficiency and grain yield. The increase in supply of N had a significant effect on grain yield and on different photosynthetic characteristics. Results showed that lines with indeterminate growth habit responded better to increase in N supply through their physiological response in improving grain yield. Among the genotypes evaluated, two lines (BFS 10 and SEF 10) were outstanding in their agronomic performance due to greater photosynthetic capacity combined with greater ability to mobilize photosynthates to grain with increase in N supply. These two lines can serve as parents for further improvement in resource use efficiency and multiple-stress resistance of common bean in the tropics.

Genome-wide analysis and expression profiling of H-type Trx family in Phaseolus vulgaris revealed distinctive isoforms associated with symbiotic N2-fixing performance and abiotic stress response

Thioredoxins (Trxs) are implicated in plant development and stress tolerance through redox regulation of target proteins. Trxs of Type h (Trxhs) constitute the largest and the most complicated cluster in the Trx family because of their unknown individual functions. Here, we identified and characterized the Phaseolus vulgaris Trxh family during development, mutualistic interactions and in response to abiotic stress. P. vulgaris (common bean) Trxh gene family (PvTrxh) encompasses 12 isoforms (PvTrxh1-h12), subdivided into 3 groups according to their amino acid sequence features. In silico RNA-seq -based expression analysis showed a differential expression of PvTrxh genes during development. RT-qPCR analysis of PvTrxh genes during nodule organogenesis revealed their highest expression in the nodule primordium (NP). Interestingly, in response to symbiosis, specific PvTrxh isoforms (PvTrxh3 and h5) were found to be highly upregulated compared to mock-inoculated plants. In addition, their expression patterns in the NP positively correlated with the symbiotic N2-fixing efficiency of the Rhizobium strain, as revealed by a number of symbiotic efficiency parameters (ARA, leghemoglobin content, biomass, and total soluble proteins), concomitantly with increased amounts of hydrogen peroxide (H2O2). On the other hand, distinctive PvTrxh isoforms were found to be upregulated in plant leaves, where H2O2 amounts were elevated, in response to both salt and drought constraints. When exogenously applied, H2O2 upregulated specific PvTrxh isoforms in plant leaves and roots. These findings point to a specific, rather than redundant, function for Trxh proteins in common bean beside the association of distinctive Trxh isoforms with symbiosis and abiotic stress response.

Linking rhizospheric diazotrophs to the stimulation of soybean N2 fixation in a Mollisol amended with maize straw

Crop residue amendment is likely to stimulate symbiotic N2 fixation, and clarifying its effect on N2-fixing bacteria, i.e., diazotrophs in the rhizosphere of legume crops, is important for sustainable N management in legume-cereal cropping systems. Therefore, this study aimed to reveal the diazotrophic community composition in the rhizosphere of soybean in response to maize residue amendment. Being designed with treatments of maize residue, chemical fertilizer, and non-fertilizer applications, this study deployed the 15N-labeling technology combined with high-throughput sequencing of the nifH gene as a molecular marker for diazotrophs to quantify the symbiotically-fixed N2 in soybean plants and link symbiotically fixed N2 to the diazotrophic community diversity in the rhizosphere. Residue amendment increased the abundance of diazotrophs and fundamentally altered the composition of its community in the rhizosphere. It increased the relative abundances of Bradyrhizobium and Azohydromonas compared to the chemical fertilizer treatment. The copy number of nifH in the rhizosphere was associated with dissolved organic carbon and N2 fixation. Residue-induced increase in dissolved organic carbon may provide sufficient carbon sources for diazotroph enrichment and thus enhance nodulation. The maize residue amendment may enrich N2 fixers to facilitate nodulation and subsequent N2 fixation of soybean, highlighting the eco-functional importance of diazotrophs fixing extra N into the rotation system.

Symbiotic nitrogen fixation contributes to divergent patterns of foliar and soil nitrogen isotopic composition in an alpine legume shrub species

Legumes that possess the capacity to fix atmospheric nitrogen (N) via symbiotic N2 fixation are widely considered to have the potential to regulate ecosystem N cycling. However, the N dynamics of those N2-fixing species in response to changes of climatic, edaphic and biotic factors are not well-understood, especially for alpine plants that are particularly susceptible to climate change. In this work, foliar and soil N isotopic composition (δ15N) of Caragana jubata, a legume shrub species mainly growing in alpine regions, was measured from 35 sites across a 2666 km east–west transect. Results showed that there were significant differences between foliar δ15N (ranging from − 0.67 to 0.60‰) and soil δ15N (ranging from 0.60 to 5.71‰), but no correlation was observed between them. Foliar δ15N increased with growing season temperature, foliar N concentration, and decreased with altitude, foliar carbon (C) concentration, foliar carbon to nitrogen ratio (C/N), and soil pH, whereas soil δ15N increased with altitude and decreased with soil C, soil N, soil C/N, and soil electrical conductivity. A principal component analysis indicated that the significant influential factors on foliar δ15N were foliar N, foliar C/N, growing season precipitation, growing season temperature and soil pH, whereas those on soil δ15N were soil electrical conductivity, soil C, soil N, soil C/N, foliar C, growing season precipitation and growing season temperature. Structural equation modeling indicated that the temperature and precipitation during the growing season affected foliar δ15N mainly via their effects on soil pH and foliar N, while affected soil δ15N mainly by their effects on soil electrical conductivity and soil C/N. This study provides strong evidence that C. jubata in the alpine regions obtains N mainly by symbiotic N2 fixation, which has resulted in the decoupling of N dynamics between plant and soil, and in different patterns and controls of foliar and soil δ15N across climatic, edaphic and biotic gradients.

Nitrogen Fixing Azotobacter Species as Potential Soil Biological Enhancers for Crop Nutrition and Yield Stability.

Biological nitrogen fixation (BNF) refers to a microbial mediated process based upon an enzymatic “Nitrogenase” conversion of atmospheric nitrogen (N2) into ammonium readily absorbable by roots. N2-fixing microorganisms collectively termed as “diazotrophs” are able to fix biologically N2 in association with plant roots. Specifically, the symbiotic rhizobacteria induce structural and physiological modifications of bacterial cells and plant roots into specialized structures called nodules. Other N2-fixing bacteria are free-living fixers that are highly diverse and globally widespread in cropland. They represent key natural source of nitrogen (N) in natural and agricultural ecosystems lacking symbiotic N fixation (SNF). In this review, the importance of Azotobacter species was highlighted as both important free-living N2-fixing bacteria and potential bacterial biofertilizer with proven efficacy for plant nutrition and biological soil fertility. In addition, we described Azotobacter beneficial plant promoting traits (e.g., nutrient use efficiency, protection against phytopathogens, phytohormone biosynthesis, etc.). We shed light also on the agronomic features of Azotobacter that are likely an effective component of integrated plant nutrition strategy, which contributes positively to sustainable agricultural production. We pointed out Azotobacter based-biofertilizers, which possess unique characteristics such as cyst formation conferring resistance to environmental stresses. Such beneficial traits can be explored profoundly for the utmost aim to research and develop specific formulations based on inoculant Azotobacter cysts. Furthermore, Azotobacter species still need to be wisely exploited in order to address specific agricultural challenges (e.g., nutrient deficiencies, biotic and abiotic constraints) taking into consideration several variables including their biological functions, synergies and multi-trophic interactions, and biogeography and abundance distribution.

A Stringent-Response-Defective Bradyrhizobium diazoefficiens Strain Does Not Activate the Type 3 Secretion System, Elicits an Early Plant Defense Response, and Circumvents NH4NO3-Induced Inhibition of Nodulation.

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.

Evolution of diverse effective N2-fixing microsymbionts of Cicer arietinum following horizontal transfer of the Mesorhizobium ciceri CC1192 symbiosis integrative and conjugative element.

Rhizobia are soil bacteria capable of forming N2-fixing symbioses with legumes, with highly effective strains often selected in agriculture as inoculants to maximize symbiotic N2 fixation. When rhizobia in the genus Mesorhizobium have been introduced with exotic legumes into farming systems, horizontal transfer of symbiosis Integrative and Conjugative Elements (ICEs) from the inoculant strain to soil bacteria has resulted in the evolution of ineffective N2-fixing rhizobia that are competitive for nodulation with the target legume. In Australia, Cicer arietinum (chickpea) has been inoculated since the 1970's with Mesorhizobium ciceri sv. ciceri CC1192, a highly effective strain from Israel. Although the full genome sequence of this organism is available, little is known about the mobility of its symbiosis genes and the diversity of cultivated C. arietinum-nodulating organisms. Here, we show the CC1192 genome harbors a 419-kb symbiosis ICE (ICEMcSym1192) and a 648-kb repABC-type plasmid pMC1192 carrying putative fix genes. We sequenced the genomes of 11 C. arietinum nodule isolates from a field site exclusively inoculated with CC1192 and showed they were diverse unrelated Mesorhizobium carrying ICEMcSym1192, indicating they had acquired the ICE by environmental transfer. No exconjugants harboured pMc1192 and the plasmid was not essential for N2 fixation in CC1192. Laboratory conjugation experiments confirmed ICEMcSym1192 is mobile, integrating site-specifically within the 3' end of one of the four ser-tRNA genes in the R7ANS recipient genome. Strikingly, all ICEMcSym1192 exconjugants were as efficient at fixing N2 with C. arietinum as CC1192, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.Importance Symbiotic N2 fixation is a key component of sustainable agriculture and in many parts of the world legumes are inoculated with highly efficient strains of rhizobia to maximise fixed N2 inputs into farming systems. Symbiosis genes for Mesorhizobium spp. are often encoded chromosomally within mobile gene clusters called Integrative and Conjugative Elements or ICEs. In Australia, where all agricultural legumes and their rhizobia are exotic, horizontal transfer of ICEs from inoculant Mesorhizobium strains to native rhizobia has led to the evolution of inefficient strains that outcompete the original inoculant, with the potential to render it ineffective. However, the commercial inoculant strain for Cicer arietinum (chickpea), M. ciceri CC1192, has a mobile symbiosis ICE (ICEMcSym1192) which can support high rates of N2 fixation following either environmental or laboratory transfer into diverse Mesorhizobium backgrounds, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.

Topographical Controls on Hillslope‐Scale Hydrology Drive Shrub Distributions on the Seward Peninsula, Alaska

AbstractObservations indicate shrubs are expanding across the Arctic tundra, mainly on hillslopes and primarily in response to climate warming. However, the impact topography exerts on hydrology, nutrient dynamics, and plant growth can make untangling the mechanisms behind shrub expansion difficult. We examined the role topography plays in determining shrub expansion by applying a coupled transect version of a mechanistic ecosystem model (ecosys) in a tundra hillslope site in the Seward Peninsula, Alaska. Modeled biomass of the dominant plant functional types agreed well with field measurements (R2 = 0.89) and accurately represented shrub expansion over the past 30 years inferred from satellite observations. In the well‐drained crest position, canopy water potential and plant nitrogen (N) uptake was modeled to be low from plant and microbial water stress. Intermediate soil water content in the mid‐slope position enhanced mineralization and plant N uptake, increasing shrub biomass. The deciduous shrub growth in the mid‐slope position was further enhanced by symbiotic N2 fixation primed by increased root carbon allocation. The gentle slope in the poorly drained lower‐slope position resulted in saturated soil conditions that reduced soil O2 concentrations, leading to lower root O2 uptake and lower nutrient uptake and plant biomass. A simulation that removed topographical interconnectivity between grid cells resulted in (1) a 28% underestimate of mean shrub biomass and (2) over or underestimated shrub productivity at the various hillslope positions. Our results indicate that land models need to account for hillslope‐scale coupled surface and subsurface hydrology to accurately predict current plant distributions and future trajectories in Arctic ecosystems.

Energetic supply regulates heterotrophic nitrogen fixation along a glacial chronosequence

Ice-free areas are expanding on the Tibetan Plateau and in its surroundings, and these recently exposed lands are typically deficient in nitrogen (N). Free-living N fixation (FLNF) represents a vital source of N input, especially when symbiotic N-fixing plants are absent. However, we know little about how FLNF changes with ecosystem succession after glacier retreat. In 2018, we measured the potential rates of FLNF in soil profiles at four sites (exposed since 2012, 1980, 1970 and 1930, respectively) along the Hailuogou glacier chronosequence and at a reference site with well-developed Podosols. We also tested the role of nifH gene abundance and resource availability in regulating FLNF rates. The results showed that photosynthetic cyanobacteria likely contributed the majority of FLNF in the biological soil crusts (exposed since 2012), while heterotrophic FLNF may prevail at later successional stages with closed canopies. FLNF rates were the highest in the litter layer of the soil profiles and decreased with soil depth. In the litter layer, FLNF rates were lower at the site dominated by N-fixing shrubs (exposed since 1980) than at the older sites dominated by non-N-fixing trees (exposed since 1970 and 1930, and the reference site). Carbon (C) availability was the major factor explaining variation in heterotrophic FLNF rates measured in the lab without light, while the effect of nifH gene abundance was nonsignificant. Overall, our results highlighted the energetic control on heterotrophic FLNF rates in relatively young ecosystems, which likely resulted in the poor relationship between nifH gene abundance and heterotrophic FLNF rates. The findings will help in understanding and modeling the coupled C and N cycles in glacier forefield ecosystems which are being exposed at accelerated rates.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.

MiR169c-NFYA-C-ENOD40 modulates nitrogen inhibitory effects in soybean nodulation.

Legume crops contribute a great portion of clean nitrogen (N) to agro-ecosystems through symbiotic N2 fixation in the nodule; however, the nodulation is always inhibited by high N availability which is known as the N inhibitory effect through largely unknown mechanisms. We functionally investigated miR169c-GmNFYA-C-GmENOD40 under multiple N conditions in soybean (Glycine max) (ENOD, Early Nodulin; NFYA, Nuclear Factor-Y Subunit A). We elucidated their regulatory roles in soybean nodulation through analyzing expression patterns, micro-messenger RNA (miRNA-mRNA) interactions, phenotypes of transgenic soybean plants and genetic interactions. We found that miR169c expression was induced by high N, whereas its target GmNFYA-C was preferentially expressed in nodules and induced by rhizobium inoculation. Overexpression of miR169c inhibited nodulation through targeting 3'-UTR of GmNFYA-C, whereas knockout miR169c through CRISPR-cas9 promoted nodulation. However, overexpression of GmNFYA-C promoted soybean nodulation through facilitating rhizobium infection and increasing the expression of symbiotic signaling gene GmENOD40. Besides, GmNFYA-C directly induced the expression of GmENOD40. In addition, overexpression of GmNFYA-C without the target site of miR169c partially attenuated the inhibitory effect of high N on soybean nodulation. We discovered a new regulatory pathway involving the miR169c-NFYA-C-ENOD40 module that regulates soybean nodulation in response to N availability. This pathway provides substantial new insights into the mechanisms underlying the N inhibitory effect on nodulation.

Urochloa Grasses Swap Nitrogen Source When Grown in Association with Legumes in Tropical Pastures

The degradation of tropical pastures sown with introduced grasses (e.g., Urochloa spp.) has dramatic environmental and economic consequences in Latin America. Nitrogen (N) limitation to plant growth contributes to pasture degradation. The introduction of legumes in association with grasses has been proposed as a strategy to improve N supply via symbiotic N2 fixation, but the fixed N input and N benefits for associated grasses have hardly been determined in farmers’ pastures. We have carried out on-farm research in ten paired plots of grass-alone (GA) vs. grass-legume (GL) pastures. Measurements included soil properties, pasture productivity, and sources of plant N uptake using 15N isotope natural abundance methods. The integration of legumes increased pasture biomass production by about 74%, while N uptake was improved by two-fold. The legumes derived about 80% of their N via symbiotic N2 fixation. The isotopic signature of N of grasses in GA vs. GL pastures suggested that sources of grass N are affected by sward composition. Low values of δ15N found in some grasses in GA pastures indicate that they depend, to some extent, on N from non-symbiotic N2 fixation, while δ15N signatures of grasses in GL pastures pointed to N transfer to grass from the associated legume. The role of different soil–plant processes such as biological nitrification inhibition (BNI), non-symbiotic N2 fixation by GA pastures and legume–N transfer to grasses in GL pastures need to be further studied to provide a more comprehensive understanding of N sources supporting the growth of grasses in tropical pastures.

Potential of Native Arbuscular Mycorrhizal Fungi, Rhizobia, and/or Green Compost as Alfalfa (Medicago sativa) Enhancers under Salinity.

Salinity is one of the devastating abiotic stresses that cause reductions in agricultural production. The increased salinization affects alfalfa growth, metabolism, and rhizobium capacity for symbiotic N2 fixation negatively. This study was undertaken to investigate the efficiency of green compost (C; made from green waste), arbuscular mycorrhizal fungi (M; field-sourced native consortium), and/or rhizobium (R; a salt-tolerant rhizobium strain) individually or in combination as an effective strategy to improve alfalfa productivity under non-saline and high-saline (120 mM NaCl) conditions. In addition, we aimed to understand the agro-physiological and metabolic basis as well as glomalin content in the soil of biofertilizers-induced salt tolerance in alfalfa. Here, we show that mycorrhizal infection was enhanced after MR inoculation, while C application decreased it significantly. Salinity reduced growth, physiological functioning, and protein concentration, but the antioxidant system has been activated. Application of the selected biofertilizers, especially C alone or combined with M and/or R improved alfalfa tolerance. The tri-combination CMR mitigated the negative effects of high salinity by stimulating plant growth, roots and nodules dry matters, mineral uptake (P, N, and K), antioxidant system, synthesis of compatible solutes, and soil glomalin content, sustaining photosynthesis-related performance and decreasing Na+ and Cl− accumulation, lipid peroxidation, H2O2 content, and electrolyte leakage.