Comparing the benthic nitrogenase activity and diazotrophic community assembly of three large river-connected freshwater lakes in eastern China.

Biological nitrogen fixation (BNF) is a crucial process that provides bioavailable nitrogen and supports primary production in freshwater lake ecosystems. However, the characteristics of diazotrophic community and nitrogenase activity in freshwater lake sediments remain poorly understood. Here, we investigated the diazotrophic communities and nitrogenase activities in the sediments of three large river-connected freshwater lakes in eastern China using 15N-isotope tracing and nifH sequencing. The sediments in these lakes contained diverse nitrogenase genes that were phylogenetically grouped into Clusters I and III. The diazotrophic communities in the sediments were dominated by stochastic processes in Hongze Lake and Taihu Lake, which had heterogeneous habitats and shallower water depths, while in Poyang Lake, which had deeper water and a shorter hydraulic retention time, the assembly of the diazotrophic community in the sediments was dominated by homogeneous selection processes. Temperature and water depth were also found the key environmental factors affecting the sediment diazotrophic communities. Sediment nitrogenase activities varied in the three lakes and within distinct regions of an individual lake, ranging from 0 to 14.58 nmol/(kg·hr). Nitrogenase activity was significantly correlated with ferric iron, total phosphorus, and organic matter contents. Our results suggested that freshwater lake sediment contain high diversity of nitrogen-fixing microorganisms with potential metabolic diversity, and the community assembly patterns and nitrogenase activities varied with the lake habitat.

Strategy of constructing D-A structure and accurate active sites over graphitic carbon nitride nanowires for high efficient photocatalytic nitrogen fixation.

Constructing photocatalysts for the stable and efficient production of NH3 is of excellent research significance and challenging. In this paper, the electron acceptor 5-amino-1,10-phenanthroline (AP) is introduced into the electron-donor graphitic carbon nitride (CN) framework by a simple heated copolymerization method to construct a donor-acceptor (D-A) structure. Subsequently, the phenanthroline unit is coordinated with transition metal Fe3+ ions to obtain the photocatalyst Fe(III)-0.5-AP-CN with better nitrogen fixation performance, and the average NH3 yield can reach 825.3 µmol g-1 h-1. Comprehensive experimental results and theoretical calculations show that the presence of the D-A structure can induce intramolecular charge transfer, effectively separating photogenerated electrons and holes. The Fe active sites can improve the chemisorption energy for N2, enhance the N-Fe bonding, and better activate the N2 molecule. Therefore, the synergistic effect between the construction of the D-A structure and the stably dispersed Fe active sites can enable CN to achieve high-performance N2 reduction to produce NH3.

SiO2 nanoparticles can enhance nitrogen retention and reduce copper resistance genes during aerobic composting of swine manure.

SiO2 nanoparticles (SiO2 NPs) are low-cost, environmentally friendly materials with significant potential to remove pollutants from complex environments. In this study, SiO2 NPs were used for the first time as an additive in aerobic composting to enhance nitrogen retention and reduce the expression of copper resistance genes. The addition of 0.5 g kg-1 SiO2 NPs effectively reduced nitrogen loss by 72.33 % by decreasing denitrification genes (nosZ, nirK, and napA) and increasing nitrogen fixation gene (nifH). The dominant factors affecting nitrification and denitrification genes were Firmicutes and C/N ratio. Additionally, SiO2 NPs decreased copper resistance genes by 28.96 % - 37.52 % in compost products. Copper resistance genes decreased most in the treatment with 0.5 g kg-1 SiO2 NPs. In summary, 0.5 g kg-1 SiO2 NPs have the potential to reduce copper resistance genes and enhance nitrogen retention during aerobic composting, which may be used to improve compost quality.

Symbiotic nitrogen fixation reduces belowground biomass carbon costs of nitrogen acquisition under low, but not high, nitrogen availability.

Many plant species form symbiotic associations with nitrogen-fixing bacteria. Through this symbiosis, plants allocate photosynthate belowground to the bacteria in exchange for nitrogen fixed from the atmosphere. This symbiosis forms an important link between carbon and nitrogen cycles in many ecosystems. However, the economics of this relationship under soil nitrogen availability gradients is not well understood, as plant investment toward symbiotic nitrogen fixation tends to decrease with increasing soil nitrogen availability. Here, we used a manipulation experiment to examine how costs of nitrogen acquisition vary under a factorial combination of soil nitrogen availability and inoculation with Bradyrhizobium japonicum in Glycine max L. (Merr.). We found that inoculation decreased belowground biomass carbon costs to acquire nitrogen and increased total leaf area and total biomass, but these patterns were only observed under low fertilization and were the result of increased plant nitrogen uptake and no change in belowground carbon allocation. These results suggest that symbioses with nitrogen-fixing bacteria reduce carbon costs of nitrogen acquisition by increasing plant nitrogen uptake, but only when soil nitrogen is low, allowing individuals to increase nitrogen allocation to structures that support aboveground growth. This pattern may help explain the prevalence of plants capable of forming these associations in less fertile soils and provides useful insight into understanding the role of nutrient acquisition strategy on plant nitrogen uptake across nitrogen availability gradients.

Marine N2-fixer Crocosphaera waterburyi.

Marine N2-fixing cyanobacteria, including the unicellular genus Crocosphaera, are considered keystone species in marine food webs. Crocosphaera are globally distributed and provide new sources of nitrogen and carbon, which fuel oligotrophic microbial communities and upper trophic levels. Despite their ecosystem importance, only one pelagic, oligotrophic, phycoerythrin-rich species, Crocosphaera watsonii, has ever been identified and characterized as widespread. Herein, we present a new species, named Crocosphaera waterburyi, enriched from the North Pacific Ocean. C. waterburyi was found to be phenotypically and genotypically distinct from C. watsonii, active in situ, distributed globally, and preferred warmer temperatures in culture and the ocean. Additionally, C. waterburyi was detectable in 150- and 4000-meter sediment export traps, had a relatively larger biovolume than C. watsonii, and appeared to aggregate in the environment and laboratory culture. Therefore, it represents an additional, previously unknown link between atmospheric CO2 and N2 gas and deep ocean carbon and nitrogen export and sequestration.

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase.

The Azotobacter vinelandii molybdenum nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires an existing [Fe-S] cluster to form a [Mo-Fe3-S4] group, which acts as specific molybdenum donor during nitrogenase FeMo-co biosynthesis. Here, we show biochemical evidence supporting the role of NifU as the [Fe-S] cluster donor. Protein-protein interaction studies involving apo-NifQ and as-isolated NifU demonstrated their interaction, which was only effective when NifQ lacked its [Fe-S] cluster. Incubation of apo-NifQ with [Fe4-S4]-loaded NifU increased the iron content of the former, contingent to both proteins being able to interact with one another. As a result of this interaction, a [Fe4-S4] cluster was transferred from NifU to NifQ. In A. vinelandii , NifQ was preferentially metalated by NifU rather than by the [Fe-S] cluster scaffold protein IscU. These results indicate the necessity of co-expressing NifU and NifQ to efficiently provide molybdenum for FeMo-co biosynthesis when engineering nitrogenase in plants.

Nitrogen-Fixing Gamma Proteobacteria Azotobacter vinelandii-A Blueprint for Nitrogen-Fixing Plants?

The availability of fixed nitrogen limits overall agricultural crop production worldwide. The so-called modern "green revolution" catalyzed by the widespread application of nitrogenous fertilizer has propelled global population growth. It has led to imbalances in global biogeochemical nitrogen cycling, resulting in a "nitrogen problem" that is growing at a similar trajectory to the "carbon problem". As a result of the increasing imbalances in nitrogen cycling and additional environmental problems such as soil acidification, there is renewed and increasing interest in increasing the contributions of biological nitrogen fixation to reduce the inputs of nitrogenous fertilizers in agriculture. Interestingly, biological nitrogen fixation, or life's ability to convert atmospheric dinitrogen to ammonia, is restricted to microbial life and not associated with any known eukaryotes. It is not clear why plants never evolved the ability to fix nitrogen and rather form associations with nitrogen-fixing microorganisms. Perhaps it is because of the large energy demand of the process, the oxygen sensitivity of the enzymatic apparatus, or simply failure to encounter the appropriate selective pressure. Whatever the reason, it is clear that this ability of crop plants, especially cereals, would transform modern agriculture once again. Successfully engineering plants will require creating an oxygen-free niche that can supply ample energy in a tightly regulated manner to minimize energy waste and ensure the ammonia produced is assimilated. Nitrogen-fixing aerobic bacteria can perhaps provide a blueprint for engineering nitrogen-fixing plants. This short review discusses the key features of robust nitrogen fixation in the model nitrogen-fixing aerobe, gamma proteobacteria Azotobacter vinelandii, in the context of the basic requirements for engineering nitrogen-fixing plants.

The Application of a Foliar Spray Containing Methylobacterium symbioticum Had a Limited Effect on Crop Yield and Nitrogen Recovery in Field and Pot-Grown Maize.

In this study, the effectiveness of an inoculant containing a nitrogen (N)-fixing microorganism (Methylobacterium symbioticum) was evaluated on maize (Zea mays L.) grown both in the field (silage maize) and in pots over two years (2021 and 2022). The field trial included the following two treatments: with (Yes) and without (No) the inoculant. The pot experiment was designed as a factorial arrangement with two factors: the application of the inoculant (Yes and No) and N applied to the soil (0, 0.4, 0.8, and 1.6 g pot-1). In the field, total dry matter yield (DMY) did not differ significantly between treatments, although the average DMY was higher in the inoculant treatment. In pots, the total DMY varied significantly across all N rates but was only significantly affected by the inoculant application in 2022. N fixation estimates in the field were 58.8 and 14.5 kg ha-1 for 2021 and 2022, respectively, representing 23.7% and 9.1% of the N recovered in the aboveground plant parts. In pots, the estimated fixed N values were -49.2 and 199.2 mg pot-1 in 2021 and 2022, respectively, which corresponded to -5.2% and 18.5% of the N found in the aboveground plant parts. Considering the average values obtained across the four cultivation conditions, there was a positive outcome for the treated plants. However, these values cannot be considered significant when compared to nitrogen removal in maize crops. A commercial product should provide an unequivocal and quantitatively relevant contribution to plant nutrition, which did not appear to be the case. Thus, for this inoculant to provide reliable guarantees of positive outcomes for farmers and become a useful tool in promoting more sustainable agriculture, further studies appear necessary. These studies should aim to determine in which crops and under what cultivation conditions the application of the inoculant is truly effective in enhancing N fixation and improving crop productivity.

Two-dimensional Boridene Nanosheets for Efficient Electrochemical Nitrogen Fixation under Ambient Conditions.

The carbon-free electrocatalytic nitrogen reduction reaction (NRR) is an alternative technology to the current Haber-Bosch method, that can be conducted under ambient conditions, and directly converting water and nitrogen (N2) into ammonia (NH3). However, the limited activity and selectivity of NH3 electrosynthesis hinder the practical applications of NRR. In this study, we present a novel type of electrocatalyst called boridene nanosheets enriched with metal vacancies that are specifically designed for efficient electrocatalytic NRR under ambient conditions. Electrochemical testing in a 0.1 M phosphate-buffered saline (PBS) electrolyte demonstrates that boridene exhibits a high Faradaic efficiency of 66.7% for NH3 production at -0.2 V vs. RHE, with a maximum NH3 yield rate of 23.6 µg h-1 mg cat-1 at -0.4 V vs. RHE. Durability tests show that boridene maintains significant stability throughout multiple cycles of NRR. Mechanistic insights are obtained through in situ FTIR spectroscopy, revealing that boridene exhibits a preference for the distal pathway during the process of NRR. These findings highlight the potential of boridene as an efficient and stable catalyst for sustainable NH3 synthesis.

Reactivity of Polynuclear Niobium Oxynitride Cluster Anions Nb4N5-xOx- (x = 0-5) toward N2.

The activation of N2 under mild conditions remains a significant challenge in chemistry. Understanding how the composition of ligands modulates the reactivity of metal centers is pivotal for the rational design of efficient catalysts for nitrogen fixation. Herein, the reactions between polynuclear niobium oxynitride anions Nb4N5-xOx- (x = 0-5) and N2 were investigated by employing mass spectrometry, photoelectron imaging spectroscopy, and theoretical calculations. The rate constants of Nb4N5-xOx-/N2 gradually decrease for x = 0 to x = 4, and then increase again for x = 5. The sharp increase of the rate constants of Nb4O5-/N2 corresponds to a decrease in the electron detachment energy of the Nb4O5- cluster in the photoelectron spectroscopic experiment. Theoretical calculations suggest that the low-coordinated Nb-Nb site in Nb4N5-xOx- (x = 0-5) behaves as the active center to bind N2 in the side-on/end-on manner. Mechanistic analysis reveals that raising the O/N ratio leads to higher electron densities on the active Nb-Nb center and decreased positive charge on the metal atoms, which hinders the approach of N2 to the clusters. This finding discloses fundamental insights into the impact of N/O ratios in fine-tuning the reactivity of metal centers towards N2 adsorption in related catalytic processes.

Synergistic Tuning of Heterovalent States and Oxygen-Vacancy Defect Engineering in Hydrophilic W-Doped Sb2OS2 for Enhanced Nitrogen Photoreduction to Ammonia.

Nitrogen fixation reaction via photocatalysis offers a green and promising strategy for renewable NH3 synthesis, and catalysts with high-efficiency photocatalytic properties are essential to the process. Herein, we demonstrate a W-doped Sb2OS2 bimetal oxysulfide catalyst (labeled as SbWOS) with abundant oxygen vacancies, heterovalent metal states, and hydrophilic surfaces for nitrogen photoreduction to ammonia. The SbWOS-3 with suitable W-doping exhibited excellent nitrogen fixation activity of 408.08 µmol·g-1·h-1 and an apparent quantum efficiency (AQE) of 1.88% at 420 nm and a solar-to-ammonia (STA) conversion efficiency of 0.082% in pure water under AM1.5G light irradiation. The W-doping not only transforms hydrophobic Sb2OS2 into a hydrophilic catalyst, making it easier for H2O molecules adsorbed on the SbWOS surface and catalyzed into protons, but also endows the SbWOS catalyst with rich oxygen vacancies, acting as the active sites for trapping and activating the N2 molecule, and for trapping and activating H2O to produce the protons for the N2 photocatalytic reduction reaction. The hydrazine drives the SbWOS catalyst with the heterovalent metal states, which acts as the photogenerate electrons quickly hopping between W5+ and W6+ to transfer for the N2 reduction reaction. This study provides a feasible scheme for applying oxygen vacancy defects, heterovalent metal states, and surface hydrophobic-to-hydrophilic wetting engineering in bimetal oxysulfide for N2 photoreduction to ammonia.

Assessing the combined impacts of microplastics and nickel oxide nanomaterials on soybean growth and nitrogen fixation potential.

The excessive presence of polystyrene microplastic (PS-MPx) and nickel oxide nanomaterials (NiO-NPs) in agriculture ecosystem have gained serious attention about their effect on the legume root-nodule symbiosis and biological nitrogen fixation (BNF). However, the impact of these contaminants on the root-nodule symbiosis and biological N2-fixation have been largely overlooked. The current findings highlighted that NiO-NMs at 50 mg kg-1 improved nodule formation and N2-fixation potential, leading to enhanced N2 uptake by both roots and shoots, resulting in increased plant growth and development. While single exposure of PS-MPx (500 mg kg-1) significantly reduced the photosynthetic pigment (8-14 %), phytohormones (9-25 %), nodules biomass (24 %), N2-related enzymes (12-17 %) that ultimately affected the N2-fixation potential. Besides, co-exposure of MPx and NiO at 100 mg kg-1 altered the nodule morphology. Additionally, single and co-exposure of MPx and NiO-NMs at 100 mg kg-1 reduced the relative abundance of Proteobacteria, Gemmatimonadota, Actinobacteria, Firmicutes, and Bacteroidetes is associated with N2-cycling and N2-fixation potential. The findings of this study will contribute to understanding the potential risks posed by MPx and NiO-NMs to leguminous crops in the soil environment and provide scientific insights into the soybean N2-fixation potential.

Glucose addition in natural forest soils has higher biological nitrogen fixation capacity than other types of soils.

Land use changes soil microbial and chemical properties, but the mechanism of biological nitrogen fixation under different land use patterns is rarely reported, so we used four types of soil: Natural forest soil (NS), healthy banana soil (HS), diseased banana soil (DS) and paddy soil (PS). Treatments included the control (CK), addition of glucose (G), addition of glucose and ammonium nitrate (GN), addition of banana straw (BS), addition of banana straw and ammonium nitrate (BSN), addition of banana root (BR), and addition of banana root and ammonium nitrate (BRN). The study found that the change of soil utilization types, glucose addition increased carbon dioxide emissions (Compared with the control, increased by 963.11%, 508.39%, 794.77% and 511.34%, respectively) and enhanced the ability of soil microbial nitrogen fixation. Importantly, natural forest soil microorganisms have a higher biological nitrogen fixation capacity compared to other types of soils. Glucose addition caused the accumulation of ammonium nitrogen (Compared with the control, increased by 426.08%, 934.21%, 420% and 1065.95%, respectively), indicating that microorganisms had higher utilization efficiency of soluble carbon and enhanced the biological nitrogen fixation capacity, and nitrogen addition caused the accumulation of ammonium nitrogen, thereby weakening the biological nitrogen fixation capacity. At the same time, glucose significantly increased the Fimicutes phylum (83.73%, 66.38%, 67.18% and 70.36%) and lowered the level of other bacterial phylums, thereby reducing the bacterial network structure, and the stability of the soil environment has decreased. Forest analysis showed that CO2 was an important factor in predicting the bacterial community structure of different soil types, an increase in CO2 content can predict drastic changes in the bacterial community. Bacteria at the Fimicutes phylum level preferred glucose, which may also have a negative effect on bacteria at the level of other phylums.

Development of two quantitative PCR assays for the detection of emerging opportunistic human pathogens belonging to the genus Phytobacter in routine diagnostics.

The genus Phytobacter (fam. Enterobacteriaceae) includes species like Phytobacter diazotrophicus and Phytobacter ursingii, which have emerged as opportunistic human pathogens, particularly affecting vulnerable populations such as pre-term infants and immunocompromised patients. Traditional biochemical and molecular methods have struggled to accurately identify Phytobacter species in clinical diagnostics. This study addresses the issue by developing and validating two quantitative PCR (qPCR) assays using SYBR® Green I and TaqMan® technologies, targeting the nitrogen fixation regulatory gene (nifL) of Phytobacter spp. The SYBR® Green I assay showed a detection limit of a single cell per reaction, while the TaqMan® assay was easier to interpret due to the absence of background noise. These assays, validated with clinical isolates from Brazil, identified multiple new Phytobacter isolates, including a potentially novel species, providing improved diagnostic tools for detecting Phytobacter spp. and aiding in better clinical management.

Increased dependence on nitrogen-fixation of a native legume in competition with an invasive plant.

Suppression of roots and/or their symbiotic microorganisms, such as mycorrhizal fungi and rhizobia, is an effective way for alien plants to outcompete native plants. However, little is known about how invasive and native plants interact with the quantity and activity of nutrient-acquisition agents. Here a pot experiment was conducted with monoculture and mixed plantings of an invasive plant, Xanthium strumarium, and a common native legume, Glycine max. We measured traits related to root and nodule quantity and activity and mycorrhizal colonization. Compared to the monoculture, fine root quantity (biomass, surface area) and activity (root nitrogen (N) concentration, acid phosphatase activity) of G. max decreased in mixed plantings; nodule quantity (biomass) decreased by 45%, while nodule activity in N-fixing via rhizobium increased by 106%; mycorrhizal colonization was unaffected. Contribution of N fixation to leaf N content in G. max increased in the mixed plantings, and this increase was attributed to a decrease in the rhizosphere soil N of G. max in the mixed plantings. Increased root quantity and activity, along with a higher mycorrhizal association was observed in X. strumarium in the mixed compared to monoculture. Together, the invasive plant did not directly scavenge N from nodule-fixed N, but rather depleted the rhizosphere soil N of the legume, thereby stimulating the activity of N-fixation and increasing the dependence of the native legume on this N source. The quantity-activity framework holds promise for future studies on how native legumes respond to alien plant invasions.

Interactive Effects of Temperature, Aridity, and Plant Stoichiometry on Insect Herbivory: Past and Present.

AbstractThe influence of climate on deep-time plant-insect interactions is becoming increasingly well known, with temperature, CO2 increases (and associated stoichiometric changes in plants), and aridity likely playing a critical role. In our modern climate, all three factors are shifting at an unprecedented rate, with uncertain consequences for biodiversity. To investigate effects of temperature, stoichiometry (specifically that of nitrogen), and aridity on insect herbivory, we explored insect herbivory in three modern floral assemblages and in 39 fossil floras, especially focusing on eight floras around a past hyperthermal event (the Paleocene-Eocene Thermal Maximum) from Bighorn Basin (BB). We find that higher temperatures were associated with increased herbivory in the past, especially among BB sites. In these BB sites, non-N2-fixing plants experienced a lower richness but higher frequency of herbivory damage than N2-fixing plants. Herbivory frequency but not richness was greater in BB sites compared with contemporaneous, nearby, but less arid sites from Hanna Basin. Compared with deep-time environments, herbivory frequency and richness are higher in modern sites, suggesting that current accelerated warming uniquely impacts plant-insect interactions. Overall, our work addresses multiple aspects of climate change using fossil data while also contextualizing the impact of modern anthropogenic change on Earth's most diverse interactions.

Development of Modular Expression Across Phylogenetically Distinct Diazotrophs.

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

Assembly strategies for microbe-material hybrid systems in solar energy conversion.

Microbe-material hybrid systems which facilitate the solar-driven synthesis of high-value chemicals, harness the unique capabilities of microbes, maintaining the high-selectivity catalytic abilities, while concurrently incorporating exogenous materials to confer novel functionalities. The effective assembly of both components is essential for the overall functionality of microbe-material hybrid systems. Herein, we conducted a critical review of microbe-material hybrid systems for solar energy conversion focusing on the perspective of interface assembly strategies between microbes and materials, which are categorized into five types: cell uptake, intracellular synthesis, extracellular mineralization, electrostatic adsorption, and cell encapsulation. Moreover, this review elucidates the mechanisms by which microbe-material hybrid systems convert elementary substrates, such as carbon dioxide, nitrogen, and water, into high-value chemicals or materials for energy generation.

Insect frass fertilizer as a regenerative input for improved biological nitrogen fixation and sustainable bush bean production.

Bush bean (Phaseolus vulgaris L.) production is undermined by soil degradation and low biological nitrogen fixation (BNF) capacity. This study evaluated the effect of black soldier fly frass fertilizer (BSFFF) on bush bean growth, yield, nutrient uptake, BNF, and profitability, in comparison with commercial organic fertilizer (Phymyx, Phytomedia International Ltd., Kiambu, Kenya), synthetic fertilizer (NPK), and rhizobia inoculant (Biofix, MEA Fertilizers, Nairobi, Kenya). The organic fertilizers were applied at rates of 0, 15, 30, and 45 kg N ha-1 while the NPK was applied at 40 kg N ha-1, 46 kg P ha-1, and 60 kg K ha-1. The fertilizers were applied singly and in combination with rhizobia inoculant to determine the interactive effects on bush bean production. Results showed that beans grown using BSFFF were the tallest, with the broadest leaves, and the highest chlorophyll content. Plots treated with 45 kg N ha-1 BSFFF produced beans with more flowers (7 - 8%), pods (4 - 9%), and seeds (9 - 11%) compared to Phymyx and NPK treatments. The same treatment also produced beans with 6, 8, and 18% higher 100-seed weight, compared to NPK, Phymyx, and control treatments, respectively. Beans grown in soil amended with 30 kg N ha-1 of BSFFF had 3-14-fold higher effective root nodules, fixed 48%, 31%, and 91% more N compared to Phymyx, NPK, and rhizobia, respectively, and boosted N uptake (19 - 39%) compared to Phymyx and NPK treatments. Application of 45 kg N ha-1 of BSFFF increased bean seed yield by 43%, 72%, and 67% compared to the control, NPK and equivalent rate of Phymyx, respectively. The net income and gross margin achieved using BSFFF treatments were 73 - 239% and 118 - 184% higher than the values obtained under Phymyx treatments. Our findings demonstrate the high efficacy of BSFFF as a novel soil input and sustainable alternative for boosting BNF and improving bush bean productivity.

Challenges to rhizobial adaptability in a changing climate: Genetic engineering solutions for stress tolerance.

Rhizobia interact with leguminous plants in the soil to form nitrogen fixing nodules in which rhizobia and plant cells coexist. Although there are emerging studies on rhizobium-associated nitrogen fixation in cereals, the legume-rhizobium interaction is more well-studied and usually serves as the model to study rhizobium-mediated nitrogen fixation in plants. Rhizobia play a crucial role in the nitrogen cycle in many ecosystems. However, rhizobia are highly sensitive to variations in soil conditions and physicochemical properties (i.e. moisture, temperature, salinity, pH, and oxygen availability). Such variations directly caused by global climate change are challenging the adaptive capabilities of rhizobia in both natural and agricultural environments. Although a few studies have identified rhizobial genes that confer adaptation to different environmental conditions, the genetic basis of rhizobial stress tolerance remains poorly understood. In this review, we highlight the importance of improving the survival of rhizobia in soil to enhance their symbiosis with plants, which can increase crop yields and facilitate the establishment of sustainable agricultural systems. To achieve this goal, we summarize the key challenges imposed by global climate change on rhizobium-plant symbiosis and collate current knowledge of stress tolerance-related genes and pathways in rhizobia. And finally, we present the latest genetic engineering approaches, such as synthetic biology, implemented to improve the adaptability of rhizobia to changing environmental conditions.