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UNLABELLED: 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. ONE-SENTENCE SUMMARY: In this study, broad-host plasmids and synthetic genetic parts were leveraged to enable expression tools in a library of diazotrophic bacteria.

Three strains of Gram‐negative bacterium, Rhizobium, were developed by gamma (γ)‐irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants—chickpea, field‐pea, and lentil—in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control‐uninoculated crops and crops grown with indigenous or commercial crop‐specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field‐pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro‐ and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility. [ABSTRACT FROM AUTHOR]

Marine biological nitrogen fixation (BNF) is crucial for introducing "new nitrogen" into the oceans. Over the past 30 years, numerous laboratory and on-board culture experiments have been conducted studying the effects of nutrients such as total dissolved nitrogen (TDN), total dissolved phosphorus (TDP), and dissolved iron (DFe) on marine diazotrophs such as Braarudosphaera bigelowii (B. bigelowii), Trichodesmium, Crocosphaera and noncyanobacterial diazotrophs (NCDs). Most studies concluded that elevated dissolved inorganic nitrogen levels inhibit nitrogen fixation in Trichodesmium, promote its growth, and have minimal effect on B. bigelowii. The impact on NCDs is unclear. Moreover, elevated dissolved inorganic phosphorus (DIP) levels can promote individual growth, population growth, and nitrogen fixation in most diazotrophs in P-limited marine environments. Dissolved organic phosphorus is a potential phosphorous source for diazotrophs in low-DIP environments. Elevated DFe can promote population growth and nitrogen fixation in diazotrophs in Fe-limited marine environments. At present, most diazotrophs have yet to achieve pure culture. Moreover, the effect of nutrients on diazotrophs is mainly limited to the study of a single nutrient, which cannot accurately reflect the actual Marine environment where diazotrophs live. As a result, our understanding of the effect of nutrients on diazotrophs is still insufficient. Future research focusing on the issues above and the development of innovative technologies and methodologies to investigate the impact of marine BNF is highly recommended, which will allow for a more precise assessment of the impact of marine BNF on global primary productivity while providing a scientific foundation for rational evaluation of ocean CO2 uptake and emissions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biological N2 fixation (BNF) is traced to the Archean. The nitrogen isotopic fractionation composition (δ15N) of sedimentary rocks is commonly used to reconstruct the presence of ancient diazotrophic ecosystems. While δ15N has been validated mostly using organisms grown under present-day conditions; it has not under the pre-Cambrian conditions, when atmospheric pO2 was lower and pCO2 was higher. Here, we explore δ15N signatures under three atmospheres with (i) elevated CO2 and no O2 (Archean), (ii) present-day CO2, and O2 and (iii) future elevated CO2, in marine and freshwater, heterocytous cyanobacteria. Additionally, we augment our data set from literature for more generalized dependencies of δ15N and the associated fractionation factor epsilon (ε = δ15Nbiomass – δ15NN2) during BNF in Archaea and Bacteria, including cyanobacteria, and habitats. The ε ranges between 3.70‰ and −4.96‰ with a mean ε value of −1.38 ± 0.95‰, for all bacteria, including cyanobacteria, across all tested conditions. The expanded data set revealed correlations of isotopic fractionation of BNF with CO2 concentrations, toxin production, and light, although within 1‰. Moreover, correlation showed significant dependency of ε to species type, C/N ratios and toxin production in cyanobacteria, albeit it within a small range (−1.44 ± 0.89‰). We therefore conclude that δ15N is likely robust when applied to the pre-Cambrian-like atmosphere, stressing the strong cyanobacterial bias. Interestingly, the increased fractionation (lower ε) observed in the toxin-producing Nodularia and Nostoc spp. suggests a heretofore unknown role of toxins in modulating nitrogen isotopic signals that warrants further investigation. IMPORTANCE Nitrogen is an essential element of life on Earth; however, despite its abundance, it is not biologically accessible. Biological nitrogen fixation is an essential process whereby microbes fix N2 into biologically usable NH3. During this process, the enzyme nitrogenase preferentially uses light 14N, resulting in 15N depleted biomass. This signature can be traced back in time in sediments on Earth, and possibly other planets. In this paper, we explore the influence of pO2 and pCO2 on this fractionation signal. We find the signal is stable, especially for the primary producers, cyanobacteria, with correlations to CO2, light, and toxin-producing status, within a small range. Unexpectedly, we identified higher fractionation signals in toxin-producing Nodularia and Nostoc species that offer insight into why some organisms produce these N-rich toxic secondary metabolites. [ABSTRACT FROM AUTHOR]

The interaction between bean (Phaseolus vulgaris L.) genotypes and N-fixing microorganisms has the potential to be utilized in sustainable agricultural production. Thus, the aim of this study was to verify the effects of the interaction for growth, production and nodulation characteristics between the bacteria Rhizobium tropici and Azospirillum brasilense among different bean genotypes. The experiments were conducted in the Selvíria, Mato Grosso do Sul, Brazil, in 2018 and 2019, in randomized block design with three replicates in a 20 × 3 factorial scheme; 20 common bean genotypes were tested under three forms of inoculation (no inoculation, inoculation with R. tropici, co-inoculation of R. tropici + A. brasilense). The number of grains per pod and nodule mass had the most significant direct positive effect on grain yield. For nodulation, a positive correlation of strong magnitude was observed between mass of the aerial part and number of pods per plant (0.68), stem diameter and mass of nodules (0.92) and first pod insertion height and number of nodules (0.64). In the path analysis, the number of grains per pod had the most significant direct effect (0.57) on the grain productivity variable. Regarding nodulation characteristics, selection for nodule mass was preferred due to its direct effect on grain yield as opposed to the number of nodules, which has a negative effect. This suggests an advantage of larger nodules with greater individual mass than a large number of small nodules for both improved yield and N fixing by common bean plants. [ABSTRACT FROM AUTHOR]

Biochar is a nature-based solution for sustainable agriculture but its potential adoption in some parts of sub-Saharan Africa is still minimal. In this study, we evaluated the agronomic potential of maize stover biochar in cowpea-maize sequential cropping in Uganda under field conditions. The treatments included; the common farmer practice of no inorganic fertilizer and no biochar (CTR), inorganic fertilizer (F), 10 t ha−1 biochar (B10), 40 t ha−1 biochar (B40), 10 t ha−1 biochar + inorganic Fertilizer (FB10), and 40 t ha−1 biochar + inorganic Fertilizer (FB40), arranged in a randomized complete block design (RCBD) with three replications. The results showed that cowpea seed yield was not significantly affected by biochar and fertilizer application but the haulm yield was significantly improved only in FB40 treatment. Maize grain and stover yield was significantly improved only in the FB40 treatment but biochar showed a high potential to also improve yield even without inorganic fertilizer. The potential for biochar to improve maize yield either in the presence or absence of fertilizers could be attributed to the residual soil fertility from cowpeas. In both seasons, biochar significantly improved soil pH, EC, SOC, total N, available P, exchangeable K and Ca, irrespective of fertilizer application. However, exchangeable Mg did not significantly vary among the treatments. This study further revealed that in cowpea-maize rotation, optimum yield could also be possible with sole biochar application. Therefore, instead of burning the maize stovers after harvest, farmers should convert the residues into biochar and return it to the soil so as to achieve sustainable food systems. [ABSTRACT FROM AUTHOR]

Millions of small scale legume farmers lack access to rhizobia bacterial inoculants that improve crop protein and yield, and minimize fossil-fuel based nitrogen fertilizers, through biological nitrogen fixation (BNF). BNF converts atmospheric nitrogen gas into ammonia, required to synthesize chlorophyll and amino acids. BNF is catalyzed by rhizobia that inhabit nodule organs in legume roots (e.g., soybean, cowpea, chickpeas, lentil, fava, peas, beans). Rhizobia inoculant access in remote communities is limited by centralized facilities to grow bacteria, inadequate transportation networks and refrigeration. Recently, we proposed that rhizobia inoculants can be diffused by farmers themselves, simply, by crushing nodules onto seeds, and demonstrated its efficacy under field conditions. A concern was whether nodules remain viable between growing seasons. Here we provide preliminary evidence that bean nodules, dried and stored at room temperature after 6 months, retain nodulation potential. We discuss: (1) the feasibility, constraints and risks of nodule crushing; (2) scaling up strategies; (3) entrepreneurship that benefits women farmers (e.g., noduleselling microenterprises); and (4) empowering farmers to directly select, evolve and indigenize rhizobia for the first time. Finally, we prioritize research questions and encourage the formation of a global participatory research network, with the goal of decentralizing and democratizing rhizobia inoculants. [ABSTRACT FROM AUTHOR]

The use of cover crops (CCs) based on tropical legumes, including Crotalaria ochroleuca, Crotalaria juncea, Crotalaria spectabilis, and Cajanus cajan, represents a pivotal aspect of agricultural rotations. These crops facilitate the incorporation of nitrogen through biological nitrogen fixation (BNF), thereby reducing the necessity for synthetic nitrogen fertilizers. Nevertheless, the capacity for the BNF of these species in Uruguay is relatively modest. To address this limitation, an approach is proposed that involves the immobilization of nitrogen in the soil using a highly energetic material, such as sucrose. The objective of this study was to examine the impact of incorporating sucrose into typical Uruguayan soil on aboveground dry matter production, nitrogen accumulation, and nitrogen fixation by legumes utilized as CCs. The experiments involved the planting of C. ochroleuca, C. juncea, C. spectabilis, and C. cajan in pots containing either soil alone or soil mixed with sucrose and the subsequent maintenance of these in a plant growth chamber for a period of 90 days. The addition of sucrose had a positive impact, with nearly double the aboveground dry matter production and nitrogen content observed. The percentage of nitrogen derived from the atmosphere (%Ndfa) increased significantly in all species, rising from an average of 83% to 96% in the sucrose-amended soil compared to the control. In the case of C. juncea, there was a notable threefold increase in aboveground dry matter and nitrogen accumulation across different treatments, accompanied by a 26% rise in %Ndfa and a fourfold increase in nitrogen fixation amounts. These findings indicate that C. juncea has the potential to significantly enhance performance and ecosystem services in typical Uruguayan soil. [ABSTRACT FROM AUTHOR]

In maize-soybean intercropping systems (ICS), the morphophysiological growth traits and grain yield of component crops (CC), especially of soybean suffer greatly, owing to the dominance of maize in acquiring growth resources and shading effect. Thus, a field trial was conducted entailing maize hybrids (H1= YH-1898 and H2= YH-5427) and defoliation treatments (R1= removal of top two leaves, R2= removal of top four leaves, R3= removal of top six leaves, and R4= no removal of leaves) for boosting soybean (cv. AARI-soybean) growth and yield in maize-soybean ICS. The response variables included plant height (PH), 1000 grains weight, grain yield (GY), and biological yield (BY) along with the physiological growth traits (leaf area index LAI, leaf area duration LAD, crop growth rate CGR, and net assimilation rate NAR) of CC. The H1 hybrid of maize outperformed the other hybrid by recording 8% and 9% higher PH and BY, respectively, while the H2 hybrid depicted 18% and 6% greater 1000 grain weight and GY, respectively, along with NAR. Additionally, R1H2 exhibited the maximum LAI, LAD, and CGR at 30, 60, 90, and 110 days after sowing (DAS). Contrastingly, soybean recorded 11% higher PH in intercropping with the H1 hybrid under R4 defoliation treatment along with significantly higher 1000 grains weight (13%), GY (57%), BY (10%), and NAR (157%). Moreover, soybean exhibited the maximum physiological growth in response to the R4H1 treatment combination. On the basis of the recorded findings, the H2R1 treatment combination could be recommended for boosting maize yield, whereas H1R4 or H1R3 could be adopted to bolster the growth and productivity of soybean intercrop, but at the cost of a significant reduction in maize yield. [ABSTRACT FROM AUTHOR]

The use of cover crops (CCs) based on tropical legumes, including Crotalaria ochroleuca, Crotalaria juncea, Crotalaria spectabilis, and Cajanus cajan, represents a pivotal aspect of agricultural rotations. These crops facilitate the incorporation of nitrogen through biological nitrogen fixation (BNF), thereby reducing the necessity for synthetic nitrogen fertilizers. Nevertheless, the capacity for the BNF of these species in Uruguay is relatively modest. To address this limitation, an approach is proposed that involves the immobilization of nitrogen in the soil using a highly energetic material, such as sucrose. The objective of this study was to examine the impact of incorporating sucrose into typical Uruguayan soil on aboveground dry matter production, nitrogen accumulation, and nitrogen fixation by legumes utilized as CCs. The experiments involved the planting of C. ochroleuca, C. juncea, C. spectabilis, and C. cajan in pots containing either soil alone or soil mixed with sucrose and the subsequent maintenance of these in a plant growth chamber for a period of 90 days. The addition of sucrose had a positive impact, with nearly double the aboveground dry matter production and nitrogen content observed. The percentage of nitrogen derived from the atmosphere (%Ndfa) increased significantly in all species, rising from an average of 83% to 96% in the sucrose-amended soil compared to the control. In the case of C. juncea, there was a notable threefold increase in aboveground dry matter and nitrogen accumulation across different treatments, accompanied by a 26% rise in %Ndfa and a fourfold increase in nitrogen fixation amounts. These findings indicate that C. juncea has the potential to significantly enhance performance and ecosystem services in typical Uruguayan soil.

Abstract Biochar is a nature-based solution for sustainable agriculture but its potential adoption in some parts of sub-Saharan Africa is still minimal. In this study, we evaluated the agronomic potential of maize stover biochar in cowpea-maize sequential cropping in Uganda under field conditions. The treatments included; the common farmer practice of no inorganic fertilizer and no biochar (CTR), inorganic fertilizer (F), 10 t ha−1 biochar (B10), 40 t ha−1 biochar (B40), 10 t ha−1 biochar + inorganic Fertilizer (FB10), and 40 t ha−1 biochar + inorganic Fertilizer (FB40), arranged in a randomized complete block design (RCBD) with three replications. The results showed that cowpea seed yield was not significantly affected by biochar and fertilizer application but the haulm yield was significantly improved only in FB40 treatment. Maize grain and stover yield was significantly improved only in the FB40 treatment but biochar showed a high potential to also improve yield even without inorganic fertilizer. The potential for biochar to improve maize yield either in the presence or absence of fertilizers could be attributed to the residual soil fertility from cowpeas. In both seasons, biochar significantly improved soil pH, EC, SOC, total N, available P, exchangeable K and Ca, irrespective of fertilizer application. However, exchangeable Mg did not significantly vary among the treatments. This study further revealed that in cowpea-maize rotation, optimum yield could also be possible with sole biochar application. Therefore, instead of burning the maize stovers after harvest, farmers should convert the residues into biochar and return it to the soil so as to achieve sustainable food systems. Graphical Abstract

The fixation and transfer of biological nitrogen from peanuts to maize in maize–peanut intercropping systems play a pivotal role in maintaining the soil nutrient balance. However, the mechanisms through which root interactions regulate biological nitrogen fixation and transfer remain unclear. This study employed a 15N isotope labelling method to quantify nitrogen fixation and transfer from peanuts to maize, concurrently elucidating key microorganisms and genera in the nitrogen cycle through metagenomic sequencing. The results revealed that biological nitrogen fixation in peanut was 50 mg and transfer to maize was 230 mg when the roots interacted. Moreover, root interactions significantly increased nitrogen content and the activities of protease, dehydrogenase (DHO) and nitrate reductase in the rhizosphere soil. Metagenomic analyses and structural equation modelling indicated that nrfC and nirA genes played important roles in regulating nitrogen fixation and transfer. Bradyrhizobium was affected by soil nitrogen content and DHO, indirectly influencing the efficiency of nitrogen fixation and transfer. Overall, our study identified key bacterial genera and genes associated with nitrogen fixation and transfer, thus advancing our understanding of interspecific interactions and highlighting the pivotal role of soil microorganisms and functional genes in maintaining soil ecosystem stability from a molecular ecological perspective. Summary Statement: The metagenomic analyses and structural equation modelling indicated that Bradyrhizobium significantly affected the nitrogen fixation, whereas the nrfC and nirA influenced the nitrogen transfer. Overall, our study identified key bacterial genus and genes associated with nitrogen fixation and transfer, thus advancing our understanding of interspecific interactions and highlighting the pivotal role of soil microorganisms and functional genes in maintaining soil ecosystem stability from a molecular ecological perspective. [ABSTRACT FROM AUTHOR]

Abstract Background The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. Results The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. Conclusions This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Background: A soil suitability evaluation of Kanamadi South sub-watershed in the Northern Dry Zone of India was carried out during April 2019 to define the soil fitness for the production of pigeonpea, chickpea, greengram and cowpea the four major pulses that are widely grown in the area. Methods: A detailed soil survey of Kanamadi South sub-watershed was carried out using IRS P6 LISS-IV image and Vijayapura district toposheet. The soils of Kanamadi south sub watershed were grouped into 19 mapping units based on studied 50 profiles. The studied physical and chemical properties were evaluated using the FAO (1976) framework for land evaluation was followed in the evaluation of soil-site suitability for major crops grown in Kanamadi South sub-watershed. This classification recognized two orders of land suitability, order 'S' (suitable) and order 'N' (not suitable) which are further subdivided into land suitability classes. Result: The studied 19 mapping units were moderately (S2) to marginally suitable (S3) for pigeonpea and greengram having marginal to severe limitations of rainfall and soil physico-chemical properties and none to slight limitation of land form characteristics. The mapping units were moderately suitable (S2) to marginally suitable (S3) for chickpea and cowpea having moderate to severe limitation of temperature, rainfall, depth and pH and none to slight limitation of land form characteristics. The suitability of the study area for all the pulses were said to be moderately suitable (S2) for growing pulse crop for improving productivity and improving biological nitrogen fixation. [ABSTRACT FROM AUTHOR]

Plant community biodiversity can be maintained, at least partially, by shifts in species interactions between facilitation and competition for resources as environmental conditions change. These interactions also drive ecosystem functioning, including productivity, and can promote over‐yielding‐ an ecosystem service prioritized in agro‐ecosystems, such as pastures, that occurs when multiple species together are more productive than the component species alone. Importantly, species interactions that can result in over‐yielding may shift in response to rising CO2 concentrations and changes in resource availability, and the consequences these shifts have on production is uncertain especially in the context of tropical mixed‐species grasslands.We examined the relative performance of two species pairs of tropical pasture grasses and legumes growing in monoculture and mixtures in a glasshouse experiment manipulating CO2. We investigated how over‐yielding can arise from nitrogen (N) niche partitioning and biotic facilitation using stable isotopes to differentiate soil N from biological N fixation (BNF) within N acquisition into above‐ground biomass for these two‐species mixtures.We found that N niche partitioning in species‐level use of soil N versus BNF drove species interactions in mixtures. Importantly partitioning and overyielding were generally reduced under elevated CO2. However, this finding was mixture‐dependent based on biomass of dominant species in mixtures and the strength of selection effects for the dominant species.This study demonstrates that rising atmospheric CO2 may alter niche partitioning between co‐occurring species, with negative implications for the over‐yielding benefits predicted for legume‐grass mixtures in working landscapes with tropical species. Furthermore, these changes in inter‐species interactions may have consequences for grassland composition that are not yet considered in larger‐scale projections for impacts of climate change and species distributions. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

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Background: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16, Sinorhizobium T21, Bacillus J1, Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. Results: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. Conclusions: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus. [ABSTRACT FROM AUTHOR]

Aims: Inoculation with climate-adapted rhizobia is able to increase legume productivity in drought-prone regions of Sub-Saharan Africa. Enhanced nodulation might additionally affect plant-soil interactions and control rhizosphere carbon (C) and nitrogen (N) pools. Methods: We investigated inoculation effects on nodulation and biological N2 fixation (BNF) of Vigna unguiculata and consequent effects on C and N pools in two Namibian soils. Three treatments (Bradyrhizobium sp.1–7 inoculant, non-inoculated, N-fertilised with 50 kg N ha−1) were applied in rhizoboxes at 45% and 20% maximum water holding capacity. Nodule development was photo-documented, and rhizobia-DNA sequences were identified. BNF was assessed by δ15N enrichment, and organic C and N pools were analysed in bulk and root adherent soil. Results: Plant growth initially enhanced mineral N losses from the rhizosphere at flowering stage (6 weeks growth), but led to a re-increase of N, and organic C contents after ripening (10 weeks). Inoculation had no effect on nodulation and soil C and N pools, indicating that both soils contained sufficient indigenous rhizobia to allow effective nodulation. However, the inoculant strain was more competitive in establishing itself in the root nodules, depending on the local conditions, showing a need for regional adjustment of inoculation strategies. Conclusion: Water stress was the main limitation for nodulation and, in combination with soil type, substantially affected rhizosphere and bulk soil C and N contents. The temporally enhanced rhizodeposition after ripening could be able to maintain soil C and N pools after legume cultivation. [ABSTRACT FROM AUTHOR]

Besides replacing the mineral fertilization, the biological nitrogen fixation promoted by bacteria from genus Bradyrhizobium provides enough nitrogen to enable high yields in the soybean crop. The objective of this study was to compare the standard inoculation of a product containing Bradyrhizobium spp., either applied alone or associated to Azospirillum brasilense, to the preinoculation by seed treatment in the plant nodulation and productivity of soybean crop grains. The trial was implanted in november, 2020 in four locations (Mandaguaçu/PR, Marialva/PR, Paranavaí/PR, and Itaquiraí/MS) with different edaphoclimatic features. The design with randomized blocks was used, with seven treatments and five repetitions. The evaluated variables were number and dry mass of nodules in the beginning of flowering, dry biomass of the aerial portion, N content and total N in the aerial portion and grains, and grain yield. The results obtained in the four tests enabled to conclude that the inoculation with Bradyrhizobium spp. + Azospirillum brasilense by seed treatment of leaf spraying with Azospirillum showed to be technically feasible in the soybean crop. [ABSTRACT FROM AUTHOR]

Cold plasma enhances various biological processes in plants. This study assessed the impact of cold plasma seed treatments on nodulation, root, and shoot growth in pea and lentil under controlled environmental conditions. Seeds were treated with cold plasma generated by a dielectric barrier discharge (DBD) and a pin electrode reactor (PER), with three different exposure durations (3, 6, and 12 min). At 4 weeks, notable enhancements were observed in nodule number and dry weight, root dry weight, length, volume, surface area, and shoot dry weight. The 3‐ and 6‐min exposure using the DBD and the 3‐min exposure using the PER system demonstrated the most significant increases or upward trends in these traits, highlighting the intricate nature of seed–plasma interactions. [ABSTRACT FROM AUTHOR]

To prevent environmental pollution, promote ecological restoration and impart production sustainability in biomass crops, optimization of mineral fertilization regimes is strategically required under changing climatic scenarios. There exist research gaps regarding optimal use of nitrogen (N), phosphorous (P) and potassium (K) fertilizers for the fertilizer-responsive cultivars of forage legumes like cowpea under decreasing soil fertility in semi-arid regions. Therefore, a multi-year field experiment was executed to study yield attributes, green and dry matter yields along with nutritional quality attributes of forage cowpea. The treatments were comprised of different N-P-K levels viz. F0 = (0-0-0), F1 = (150-0-0 kg·ha-1), F2 = (150-100-0 kg·ha-1) and F3 = (150-100-100 kg·ha-1). The findings revealed that F3 fertilization regime surpassed rest of treatments by recording the maximum plant population, plant height, leaf area index, plants fresh and dry weights, which led to the highest green forage yield (73% and 5.8% higher than control and following treatment of F2, respectively). For dry matter yield, all fertilization regimes performed better than control, however those were statistically at par to each other. Moreover, F3 treatment exhibited 4.4% and 1.6% higher crude protein and ether extractable fat respectively, compared to the following treatment of F2 treatment that remained at par with F3 for total ash content. Contrastingly, the control treatment remained superior by giving the minimum crude fiber content which could be attributed to dwarf plants produced in the absence of fertilizers because stem length tends to contribute the major portion of fiber content in cowpea. Thus, 150-100-100 kg·ha-1 N-P-K might be recommended to cowpea growers for boosting biomass productivity and nutritional quality, however further field investigations need to assess the impact of these fertilization regimes on biological N fixation process and solar radiation capture by cowpea plants under irrigated and dry semi-arid conditions. [ABSTRACT FROM AUTHOR]

Introduction: Biological nitrogen fixation (BNF), an unparalleled metabolic novelty among living microorganisms on earth, globally contributes ~88-101 Tg N year-1 to natural ecosystems, ~56% sourced from symbiotic BNF while ~22-45% derived from free-living nitrogen fixers (FLNF). The success of symbiotic BNF is largely dependent on its interaction with host-plant, however ubiquitous environmental heterotrophic FLNFs face many limitations in their immediate ecological niches to sustain unhindered BNF. The autotrophic FLNFs like cyanobacteria and oceanic heterotrophic diazotrophs have been well studied about their contrivances acclimated/adapted by these organisms to outwit the environmental constraints for functional diazotrophy. However, FLNF heterotrophs face more adversity in executing BNF under stressful estuarine/marine/aquatic habitats. Methods: In this study a large-scale cultivation-dependent investigation was accomplished with 190 NCBI accessioned and 45 non-accessioned heterotrophic FLNF cultivable bacterial isolates (total 235) from halophilic estuarine intertidal mangrove niches of Indian Sundarbans, a Ramsar site and UNESCO proclaimed World Heritage Site. Assuming ~1% culturability of the microbial community, the respective niches were also studied for representing actual bacterial diversity via cultivation-independent next-generation sequencing of V3-V4 rRNA regions. Results: Both the studies revealed a higher abundance of culturable Gammaproteobacteria followed by Firmicutes, the majority of 235 FLNFs studied belonging to these two classes. The FLNFs displayed comparable selection potential in media for free nitrogen fixers and iron-oxidizing bacteria, linking diazotrophy with iron oxidation, siderophore production, phosphorus solubilization, phosphorus uptake and accumulation as well as denitrification. Discussion: This observation validated the hypothesis that under extreme estuarine mangrove niches, diazotrophs are naturally selected as a specialized multidimensional entity, to expedite BNF and survive. Earlier metagenome data from mangrove niches demonstrated a microbial metabolic coupling among C, N, P, S, and Fe cycling in mangrove sediments, as an adaptive trait, evident with the co-abundant respective functional genes, which corroborates our findings in cultivation mode for multiple interrelated metabolic potential facilitating BNF in a challenging intertidal mangrove environment. [ABSTRACT FROM AUTHOR]

Perennial tall grasses show promise as bioenergy crops due to high productivity and efficient nutrient use. Ongoing research on bioenergy grasses seeks to reduce their reliance on synthetic nitrogen (N) fertilizer, the manufacture of which relies on fossil fuel combustion. Excessive use of fertilizers also causes adverse environmental consequences and leads to the evolutionary loss of plant traits beneficial to sustainable N cycle. Notably, perennial tall grasses have exhibited the potential to maintain high biomass yield without the need for N fertilizer or causing soil N depletion. Perennial grasses can be adept at interacting with their microbial partners to facilitate N acquisition and retention via mechanisms such as biological N fixation and nitrification inhibition. These inherent N management traits should be preserved and optimized at the this early stage of bioenergy grass breeding programs. This review examines the impact of external N on bioenergy grass production and explores the potential of leveraging advantageous N‐cycling attributes of perennial tall grasses, laying groundwork for future management and research efforts. With minimized dependency on external N input, the cultivation of perennial energy grasses will pave the way toward more resilient agricultural systems and play a significant role in addressing key global energy and environmental challenges. [ABSTRACT FROM AUTHOR]

The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF. [ABSTRACT FROM AUTHOR]

Given the importance of biological nitrogen (N) fixation in agroecosystems, using inoculants with phyllosphere N fixers effective across various crops would revolutionize agriculture. In this study, the application of an inoculant prepared from Methylobacterium symbioticum was tested on young olive trees. The pot experiment was arranged in a factorial design, with inoculant (Yes and No) and mineral N applied to the soil [0 (N0), 25 (N25), 50 (N50), and 100 (N100) kg ha−1], and four replicates. The inoculant application did not increase plant dry matter yield (DMY), whereas the application of mineral N had a significant and pronounced effect. The inoculant also did not significantly increase N concentration in tissues, unlike the strong increase observed with N applied to the soil. The inoculant significantly increased plant N recovery, a cumulative effect resulting from small increases in DMY and N concentration in tissues. This increase represented 5.2% more N in plants receiving the inoculant compared to untreated ones. However, only treatments receiving mineral N recorded positive values of fixed N, with the highest value observed in the N50 treatment (12.4%), whereas a negative value (−7.7%) was observed in the N0 treatment. Overall, these low values of fixed N question the economic rationale of using this inoculant by farmers and especially render it unsuitable for organic farming systems, where plants tend to have lower N levels in tissues. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is a limiting ecological factor for plant growth in most agroecosystems. Biological N fixation, especially from nodulated legumes, has been promoted in recent decades as an alternative or complement to industrially synthesized N fertilizers. The possibility of utilizing N-fixing organisms from the phyllosphere that demonstrate effectiveness across a wide range of crops is particularly exciting. In this study, we examined the N-fixing capacity and the impact on lettuce growth of an inoculant recently introduced to the market, which contains the microorganism Methylobacterium symbioticum and is recommended for various cultivated species. A pot experiment was conducted using a factorial design, which included the inoculant (No and Yes) and four N rates (0 (N0), 25 (N25), 50 (N50), and 100 (N100) kg ha−1 of N), with four replicates, over four lettuce growing cycles. The inoculant had a significant effect on dry matter yield (DMY) only during the second of the four growing cycles. The mean values of the four growing cycles ranged from 9.9 to 13.7 g pot−1 and 9.9 to 12.6 g kg−1 in pots that received and did not receive the inoculant, respectively. On the other hand, plants exhibited a robust response to N applied to the soil, showing significant increases in both DMY and tissue N concentration across all growing cycles. Mean values of DMY in the treatments N0 and N100 ranged from 5.6 to 8.9 g pot−1 and 12.5 to 16.1 g pot−1, respectively. N concentration in tissues varied inversely with DMY, indicating a concentration/dilution effect. The difference in N concentration between treated and untreated plants, used as an estimate of fixed N, was very low for each of the soils' applied N rates, assuming average values for the four growing cycles of −1.5, −0.9, 2.4, and 6.3 kg ha−1 for N0, N25, N50, and N100, respectively. This study emphasized the low amount of N supplied to lettuce by the inoculant and its limited effect on DMY. Generally, in biological systems with N-fixing microorganisms, achieving high fixation rates requires a high level of specificity between the microorganism and host plant, a condition that seems not to have been met with lettuce. Considering the importance of the subject, is imperative that further studies be conducted to determine more precisely in which crops and under what growing conditions the inoculant proves to be a valuable input for farmers and an effective method for reducing N mineral fertilization. [ABSTRACT FROM AUTHOR]

ABSTRACT Biological nitrogen (N) fixation, an important pathway of N, inputs from the atmosphere to Earth’s ecosystems, is well demonstrated to decline under N input. However, it remains unclear why N fixers sustain N fixation in many forests under high atmospheric N deposition. To address this knowledge gap, we analyzed the response of the diazotroph community to low N loads (short-term and low N addition; 3-year N addition at the rates of 25–50 kg N ha−1 year−1) vs high loads (chronic and high N addition; 9-year N addition at the rate of 150 kg N ha−1 year−1) in forest soils using high-throughput sequencing. Rates of N fixation decreased under low and high N loads (by 13%–27% and 10%–12%, respectively). Richness and alpha diversity (ACE and Chao1) of the soil diazotroph community decreased under low but not high N loads. Approximately 67.1%–74.4% of the nifH gene sequences at the OTU level overlapped between the control and low N loads, but only 52.0%–53.6% of those overlapped between the control and high N loads, indicating a larger shift of diazotroph community composition under high N loads. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ concentrations under high N loads did not have negative impacts on the structure and function of the diazotroph community. These findings indicate that diazotrophs sustain N fixation under high N deposition via adjustment of their community composition in forest soils.IMPORTANCEThis study examined the changes in soil diazotroph community under different loads of simulated N deposition and analyzed its relationship with N fixation rates in in five forests using high-throughput sequencing. The magnitudes of N fixation rates reduced by low N loads were higher than those by high N loads. Low N loads decreased richness and diversity of diazotroph community, whereas diazotroph community structure remained stable under high N loads. Compared with low N loads, high N loads resulted in a less similarity and overlap of nifH gene sequences among the treatments and a larger adjustment of diazotroph community. Low N loads increased soil NH4+ concentrations, which decreased diazotroph community richness, diversity, and N fixation rates, whereas the increased soil NH4+ under high N loads did not have negative impacts on diazotroph community structure and N fixation. Based on these findings, it is urgently needed to incorporate the loads of N deposition and the composition of diazotroph community into terrestrial N-cycling models for accurate understanding of N inputs in forest ecosystems.

Cowpea (Vigna unguiculata L. Walp.) is a promiscuous crop, nodulating with a wide range of rhizobia strains native to the soil, what often leads to low inoculation responses with selected rhizobia, reducing the efficiency of introduced inoculants. This study aimed to assess the nodule occupancyof the Bradyrhizobium pachyrhizi BR 3262 elite strain in two cowpea genotypes (BRS Pujante and BRS Acauã), in soil with established rhizobial population (higher than 103 g-1 of soil), in a greenhouse. For the BR 3262 nodule occupancy, a strain-specific PCR approach was applied, assessing all the 1,237 nodules obtained from the root crown and the entire root (secondary roots) regions of the inoculated plants. The BR 3262 inoculation increased the root dry mass and nodulation, but not the shoot dry mass. Both genotypes showed a nodular occupancy above 80 % in the crown and secondary roots, indicating a high competitiveness and persistence in the soil.

Inadequate management of nitrogen fertilization, due to high costs, compromises the nutrition and productivity of winter forage plants, highlighting the need to adopt sustainable alternatives, such as using nitrogen-fixing bacteria. This research evaluated the growth promotion of black oat (Avena strigosa) and ryegrass (Lolium multiflorum) plants inoculated with Azospirillum brasilense rhizobacteria. The experiments were carried out over two consecutive harvests in Santa Maria, Brazil. The sowing density was 300 viable seeds per square meter for black oats and 100 viable seeds per square meter for ryegrass. A dose of 5 ml per kg of seeds from the consortium was used as an inoculant. Following sowing, the seeds were incorporated into the soil through light harrowing. At the beginning of tillering, 50 kg of N ha-1 was applied to create treatments with and without nitrogen. Plant emergence, plant height, number of leaves and tillers were evaluated. In the first year, an inoculation response was observed for the number of black oat plants emerged m-2. In the second year (corn residue), an inoculation response was observed for the number of leaves and height of black oats. Under soybean straw for oat leaf number and ryegrass tiller number. The current research highlights the effectiveness of inoculation with Azospirillum brasilense in promoting significant improvements in the morphological components of forage crops. Following corn, an increase in the number of leaves and tillers was observed, as well as in the height of black oats. Similarly, following soybeans, there was an increase in the number of black oat tillers and the number of ryegrass leaves. The results emphasize the importance of inoculation as an effective practice to enhance crop development and performance in agricultural systems, improving sustainability and productivity under soybean and corn residues.

The use of cold plasma (CP) seed treatment is an emerging agricultural technology that exhibits the potential to enhance nodulation and symbiotic nitrogen fixation (SNF) in legumes. CP is composed of a diverse mixture of excited atoms, molecules, ions, and radicals that have the potential to affect the physical properties of the seed and influence gene expressions that could have a lasting impact on the nodulation, SNF, growth, and yield of legumes. The direct participation of the CP in the nodulation process and its correlation with the escalation of nodules and SNF is still not fully understood. This review discussed four areas in the nodulation and SNF process that can directly or indirectly affect CP seed treatments: root–rhizobia signal exchange pathways, root/shoot growth and development, phytohormone production, and the nitrogen fixation process. We also discuss the potential challenges and future research requirements associated with plasma technology to enhance SNF in legumes.

The nitrogen (N) requirement for barley cultivation can vary from 90 to 130 kg ha−1 cycle−1, representing 30% of production costs. Furthermore, obtaining mineral N is difficult due to constant conflicts involving producing countries. Excess N will increase the cost of production and contaminate the soil and groundwater, unbalancing the fauna, flora, and microbiology of the environment. The objective of this research was to evaluate the management of inoculation and N fertilization with A. brasilense on the development and productivity of barley. Field experiment was carried out for two years. A randomized block design was used in a 5 (Factor A) × 2 (Factor D) factorial arrangement with four replications. In which the "factor A" included five doses of A. brasilense, whereas the "factor D" was evaluated N management (with or without N management). The inoculant used had approximately of 2.0 × 108 viable cells mL−1 (Ab-V5 and Ab-V6 strains). Innoculation was carried out on the seeds 1 hour before sowing, in order to homogenize the inoculant in the seeds, to guarantee homogeneity of the distribution of microorganisms on the seeds. The best dose of A. brasilense was 12.5 mL kg−1 of seed, which provided an increase in plant height, number of ears, mass and grain productivity. The use of A. brasilense showed significant improvements in management with or without the use of mineral N. However, better results are obtained when the association between N management and the use of inoculant is made. Thus, when using up to 22.5 kg of N, the best results were observed. [ABSTRACT FROM AUTHOR]

The curriculum for undergraduate Agricultural Engineering does not usually include much laboratory practice. In order to address this, here we propose the design of a practical class on legume-rhizobia symbiosis, using an interdisciplinary approach that takes elements from botany, agriculture, and microbiology. The students perform an assay to stimulate nodulation in chickpea seedlings through inoculation with Mesorhizobium strains recommended as commercial inoculants, and evaluate improvement in plant growth as a result of nitrogen fixation. The experiment could serve as a springboard to discuss the importance of plant-bacteria interactions in the cultivation of a regionally relevant crop. It may also encourage the use of abilities of different kinds, including the appropriate manipulation of lab materials, the active engagement of scientific thought, the application of knowledge to specific real world scenarios and collaborative teamwork, all of which will be of paramount importance throughout the students' careers. The class can be easily adapted for other degrees connected to environmental microbiology, as well as for other symbiotic pairs that may include significant crops from other regions around the world. [ABSTRACT FROM AUTHOR]

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the efficiency and agronomic practicality of pre-inoculating soybean seeds with Bradyrhizobium elkanii (Lalfix Nitro) and a cell protector (Add-it) under field conditions. The trials were conducted in different Brazilian soil and climate regions (Brotas-SP, Caibaté-RS, Lavras-MG and Unaí-MG). The experimental design was randomized blocks, with six treatments and 10 replications. The treatments consisted of different inoculation times of the liquid inoculant Lalfix Nitro (4 mL kg-1 of seeds) + cell bioprotector Add-it (2 mL kg-1 of seeds): on the day of planting (LNT0), 25 days (LNT25) and 30 days (LNT30) before planting. In addition, a standard control was used consisting of a peat inoculant (Brasiliec TS IN BOX®; 60 g per 50 kg of seeds) (PI); an N-mineral control, with the recommended nitrogen fertilizer for the crop (NI+N); and a treatment with no fertilizer added and no inoculation (Control). In general, the pre-inoculation of LN + Add bioprotector up to 30 days before planting promoted a significant increase in nodulation. In addition, pre-inoculation ensured yield equal to or greater than peat inoculant and mineral N application. [ABSTRACT FROM AUTHOR]

This editorial published in the journal Frontiers in Agronomy discusses the importance of maximizing nitrogen fixation in legumes for sustainable agriculture intensification. It addresses the challenges faced by agricultural systems in providing food for a growing population while maintaining soil and water quality and mitigating climate change. The editorial explores the potential of biological nitrogen fixation in legumes as an alternative strategy to increase nitrogen availability and reduce the need for synthetic fertilizers. It also presents various research topics related to the symbiotic relationship between legumes and rhizobia and its application in sustainable agricultural systems. The article concludes by emphasizing the importance of inoculating legumes with efficient rhizobia strains in areas where inefficient strains are likely to be present. The authors also discuss the use of elite microorganisms to improve the growth of alfalfa crops in acid soils and suggest that more research is needed to fully understand plant-microbe interactions and develop effective strategies for replacing synthetic nitrogen fertilizers. [Extracted from the article]

Information on how bacteria in plants and soil, along with extracellular enzymes, affect nutrient cycling in Encephalartos villosus growing in phosphorus deficient and acidic scarp forests is lacking. Bacteria in coralloid roots, rhizosphere, and non-rhizosphere soils were isolated to determine the potential role of soil bacterial communities and their associated enzyme activities in nutrient contributions in rhizosphere and non-rhizosphere soils. The role of soil characteristics and associated bacteria on E. villosus nutrition and nitrogen source reliance was investigated. Encephalartos villosus leaves, coralloid roots, rhizosphere, and non-rhizosphere soils were collected at two scarp forests. Leaf nutrition, nitrogen source reliance, soil nutrition, and extracellular enzyme activities were assayed. A phylogenetic approach was used to determine the evolutionary relationship between identified bacterial nucleotide sequences. The clustering pattern of isolated bacterial strains was primarily dictated by the ecological niches from which they originated (rhizosphere soil, non-rhizosphere soil, and coralloid roots), thus indicating that host-microbe interactions may be a key driver of this pattern, in line with the hologenome theory. There were insignificant differences in the phosphorus and nitrogen cycling enzyme activities in E. villosus rhizosphere and non-rhizosphere soils in both localities. Significantly positive correlations were recorded between nitrogen and phosphorus cycling enzymes and phosphorus and nitrogen concentrations in rhizosphere and non-rhizosphere soils. Additionally, more than 70% of the leaf nitrogen was derived from the atmosphere. This study challenged the conventional expectation that environmental filters alone dictate microbial community composition in similar habitats and revealed that host-microbe interactions, as proposed by the hologenome theory, are significant drivers of microbial community structuring. The isolated bacteria and their plant growth promoting traits play a role in E. villosus nutrition and nitrogen source reliance and secrete nutrient cycling enzymes that promote nutrient availability in rhizosphere and non-rhizosphere soils. [ABSTRACT FROM AUTHOR]

Background and aims: Invasive plants often alter soil abiotic and biotic conditions which can benefit their own growth while harming native species. The impacts on native species may persist as legacy effects after the invasive species has been controlled and removed. This study focused on the Critically Endangered Cumberland Plain Woodland (CPW) vegetation in Australia, where we examined the soil impacts, and their associated legacy effects, associated with the invasion of African olive (Olea europaea subsp. cuspidata). Methods: Seedlings of O. europaea subsp. cuspidata and two native legume species, Acacia implexa and Indigofera australis, were grown in different sterilised and unsterilised soils: uninvaded CPW soil, restored CPW soil where O. europaea subsp. cuspidata was removed approximately 20 years ago, and soil from sites still under O. europaea subsp. cuspidata invasion. We characterised nitrogen-fixing rhizobia in the root nodules of seedlings of the two legumes using next-generation sequencing (NGS) barcoding. Results: Olea europaea subsp. cuspidata did not appear to condition the soil to favour its own growth and grew best in uninvaded CPW soil. The performance of both native legume species, however, was negatively impacted when grown in sterilised and unsterilised invaded soils, relative to their growth in CPW and restored soils. The soils from invaded sites affected the associations between both legume species and their rhizobium mutualists. Nodulation was higher in CPW and restored soils than in invaded soils, indicating that the availability of rhizobia was negatively impacted by O. europaea subsp. cuspidata. This was confirmed by a negative link between nodulation and the abundance of rhizobia that were characteristic of invaded soils. Conclusion: Our findings demonstrate that O. europaea subsp. cuspidata invasion affects the availability of microbial mutualists for native legumes in the CPW. The soil conditions created by O. europaea subsp. cuspidata do not benefit its own performance and these impacts do not persist as legacy effects 20 years after the removal of the invader. [ABSTRACT FROM AUTHOR]

The use of cold plasma (CP) seed treatment is an emerging agricultural technology that exhibits the potential to enhance nodulation and symbiotic nitrogen fixation (SNF) in legumes. CP is composed of a diverse mixture of excited atoms, molecules, ions, and radicals that have the potential to affect the physical properties of the seed and influence gene expressions that could have a lasting impact on the nodulation, SNF, growth, and yield of legumes. The direct participation of the CP in the nodulation process and its correlation with the escalation of nodules and SNF is still not fully understood. This review discussed four areas in the nodulation and SNF process that can directly or indirectly affect CP seed treatments: root–rhizobia signal exchange pathways, root/shoot growth and development, phytohormone production, and the nitrogen fixation process. We also discuss the potential challenges and future research requirements associated with plasma technology to enhance SNF in legumes. [ABSTRACT FROM AUTHOR]

Millions of small scale legume farmers lack access to rhizobia bacterial inoculants that improve crop protein and yield, and minimize fossil-fuel based nitrogen fertilizers, through biological nitrogen fixation (BNF). BNF converts atmospheric nitrogen gas into ammonia, required to synthesize chlorophyll and amino acids. BNF is catalyzed by rhizobia that inhabit nodule organs in legume roots (e.g., soybean, cowpea, chickpeas, lentil, fava, peas, beans). Rhizobia inoculant access in remote communities is limited by centralized facilities to grow bacteria, inadequate transportation networks and refrigeration. Recently, we proposed that rhizobia inoculants can be diffused by farmers themselves, simply, by crushing nodules onto seeds, and demonstrated its efficacy under field conditions. A concern was whether nodules remain viable between growing seasons. Here we provide preliminary evidence that bean nodules, dried and stored at room temperature after 6 months, retain nodulation potential. We discuss: (1) the feasibility, constraints and risks of nodule crushing; (2) scaling up strategies; (3) entrepreneurship that benefits women farmers (e.g., nodule-selling microenterprises); and (4) empowering farmers to directly select, evolve and indigenize rhizobia for the first time. Finally, we prioritize research questions and encourage the formation of a global participatory research network, with the goal of decentralizing and democratizing rhizobia inoculants.

The aim of this study was to evaluate the contribution of inoculation with microorganisms, mainly rhizobia, on pea (Pisum sativum) production using a systematic literature review approach and a comparative analysis of grain yield to provide information to fill gaps in nontraditional regions of cultivation for this legume. A systematic search strategy was implemented, targeting papers published in scientific journals accessible through the Web of Science® (WoS) database spanning from January 1990 to April 2022. The search terms "Pisum sativum," "inoculation," and "strain" were used. The differences between the pea grain yields from plants inoculated with nitrogen-fixing microorganisms and those from noninoculated plants (control) were analyzed using thirteen field-scale studies. Overall, pea inoculation/coinoculation promoted a greater grain yield (3118 ±210 kg ha-1) than noninoculation (2338± 127 kg ha-1), showing the importance of biological nitrogen fixation for legume crop production. In the pursuit of reducing pea production costs, emphasis has been placed on inoculation, highlighting the importance of comprehending the symbiotic relationship between peas and Rhizobium. Furthermore, this research aimed to explore the interaction between Rhizobium and selected microorganisms known to enhance plant growth to identify optimal combinations to boost pea productivity.

Abstract The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF.

Abstract Perennial tall grasses show promise as bioenergy crops due to high productivity and efficient nutrient use. Ongoing research on bioenergy grasses seeks to reduce their reliance on synthetic nitrogen (N) fertilizer, the manufacture of which relies on fossil fuel combustion. Excessive use of fertilizers also causes adverse environmental consequences and leads to the evolutionary loss of plant traits beneficial to sustainable N cycle. Notably, perennial tall grasses have exhibited the potential to maintain high biomass yield without the need for N fertilizer or causing soil N depletion. Perennial grasses can be adept at interacting with their microbial partners to facilitate N acquisition and retention via mechanisms such as biological N fixation and nitrification inhibition. These inherent N management traits should be preserved and optimized at the this early stage of bioenergy grass breeding programs. This review examines the impact of external N on bioenergy grass production and explores the potential of leveraging advantageous N‐cycling attributes of perennial tall grasses, laying groundwork for future management and research efforts. With minimized dependency on external N input, the cultivation of perennial energy grasses will pave the way toward more resilient agricultural systems and play a significant role in addressing key global energy and environmental challenges.

Improving biological nitrogen fixation (BNF) in cereal crops is a long-sought objective; however, no successful modification of cereal crops showing increased BNF has been reported. Here, we described a novel approach in which rice plants were modified to increase the production of compounds that stimulated biofilm formation in soil diazotrophic bacteria, promoted bacterial colonization of plant tissues and improved BNF with increased grain yield at limiting soil nitrogen contents. We first used a chemical screening to identify plant-produced compounds that induced biofilm formation in nitrogen-fixing bacteria and demonstrated that apigenin and other flavones induced BNF. We then used CRISPR-based gene editing targeting apigenin breakdown in rice, increasing plant apigenin contents and apigenin root exudation. When grown at limiting soil nitrogen conditions, modified rice plants displayed increased grain yield. Biofilm production also modified the root microbiome structure, favouring the enrichment of diazotrophic bacteria recruitment. Our results support the manipulation of the flavone biosynthetic pathway as a feasible strategy for the induction of biological nitrogen fixation in cereals and a reduction in the use of inorganic nitrogen fertilizers.

It is believed that nitrogen-fixing eukaryotes do not exist in nature, and constructing such eukaryotes is extremely challenging. Coale et al. , however, have identified the first eukaryote capable of fixing nitrogen through a nitroplast organelle. Understanding the eukaryotic nitrogen-fixing machinery may advance the development of artificial nitrogen-fixing crops and industrial yeasts. [ABSTRACT FROM AUTHOR]