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Nickel oxide nanoparticles (NiO-NPs) pose potential threats to agricultural production. Bacillus subtilis has emerged as a stress-mitigating microbe that alleviates the phytotoxicity caused by NiO-NPs. However, the mechanisms underlying its effectiveness, particularly in root-nodule symbiosis and biological N2-fixation (BNF), remain unclear. Here, we tested the combined exposure of NiO-NPs (50 mg kg-1) and B. subtilis on soybean (Glycine max L.) growth and BNF. Combined exposure increased root length, shoot length, root biomass, and shoot biomass by 19% to 26%, while Ni (200 mg kg-1) reduced them by 38% to 53% compared to the control. NiO-NPs at 100 and 200 mg kg-1 significantly (P < 0.05) reduced nodule formation by 16% and 58% and Nitrogen assimilation enzyme activities levels (urease, nitrate reductase, glutamine synthetase, and glutamate synthetase) by 13% to 57%. However, co-exposure with B. subtilis improved nodule formation by 22% to 44%. Co-exposure of NiO-NPs (200 mg kg-1) with B. subtilis increased peroxidase, catalase, and glutathione peroxidase activity levels by 20%, 16%, and 14% while reducing malondialdehyde (14%) and hydrogen peroxide (12%) levels compared to NiO-NPs alone. Additionally, co-exposure of NiO-NPs (100 and 200 mg kg-1) with B. subtilis enhanced the relative abundance of Stenotrophomonas, Gemmatimonas, and B. subtilis, is associated with N2-cycling and N2-fixation potential. This study confirms that B. subtilis effectively mitigates NiO-NP toxicity in soybean, offering a sustainable method to enhance BNF and crop growth and contribute to addressing global food insecurity., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mulching has been demonstrated to improve the soil environment and promote plant growth. However, the effects of mulching and mulch-derived microplastics (MPs) on nitrogen fixation by root nodules remain unclear. In this study, we investigated the effects of polyethylene (PE) and polylactic acid-polybutylene adipate-co-terephthalate (PLA-PBAT) film mulching on nitrogen fixation by root nodules after 4 years of continuous mulching using 15 N tracer technology. Additionally, we examined the relationship between nitrogen fixation and MPs. We found a reduction in the proportion of nitrogen fixation by nodules (54.3 %-58.7 %) due to mulching. This decrease may be attributed to reduced dinitrogenase activity and flavonoid content at the seedling stage caused by mulching, and mulching with PLA-PBAT films significantly decreased the abundance of Bradyrhizobium at maturity. Furthermore, combined analysis of nitrogen-fixing bacteria (nifH) and metabolomes indicated that N-lauroylethanolamine may act as a regulatory signal influencing the root nodule nitrogen fixation process and that mulching resulted in significant changes in its content. The mantel test and PLS-PM suggest that microplastic from mulching may harm root nodule nitrogen fixation. This study reveals the influence of mulching on plant nitrogen uptake and the potential threat of mulch-derived microplastics, with a special focus on root nodule nitrogen fixation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 N 2 -fixation have been largely overlooked. The current findings highlighted that NiO-NMs at 50 mg kg -1 improved nodule formation and N 2 -fixation potential, leading to enhanced N 2 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 %), N 2 -related enzymes (12-17 %) that ultimately affected the N 2 -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 N 2 -cycling and N 2 -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 N 2 -fixation potential., Competing Interests: Declaration of Competing Interest The authors declare that they have no financial or personal relationships that could influence the research work presented in this manuscript. There are no conflicts of interest regarding the publication of this research., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Recent studies have indicated that soil contamination with microplastics (MPs) can negatively affect agricultural productivity, although these effects vary greatly depending on the context. Furthermore, the mechanisms behind these effects remain largely unknown. In this study, we examined the impact of two concentrations of polypropylene (PP) fibers in the soil (0.4 % and 0.8 % w/w) on soybean growth, nitrogen uptake, biological nitrogen fixation (BNF), and water use efficiency by growing plants in two soil types, with and without arbuscular mycorrhizal fungi (AMF). PP contamination consistently reduced vegetative growth (-12 %, on average compared to the control), with the severity of this effect varying significantly by soil type (more pronounced in Alfisol than in Vertisol). The extent of BNF progressively reduced with the increase in PP contamination level in both soils (on average, -17.1 % in PP0.4 and -27.5 % in PP0.8 compared to the control), which poses clear agro-environmental concerns. Water use efficiency was also reduced due to PP contamination but only in the Alfisol (-9 %, on average). Mycorrhizal symbiosis did not seem to help plants manage the stress caused by PP contamination, although it did lessen the negative impact on BNF. These findings are the first to demonstrate the effect of PP on BNF in soybean plants, underscoring the need to develop strategies to reduce PP pollution in the soil and to mitigate the impact of PP on the functionality and sustainability of agroecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Heavy metal contamination poses an escalating global challenge to soil ecosystems, with hyperaccumulators playing a crucial role in environmental remediation and resource recovery. The enrichment of diazotrophs and resulting nitrogen accumulation promoted hyperaccumulator growth and facilitated phytoremediation. Nonetheless, the regulatory mechanism of hyperaccumulator biological nitrogen fixation has remained elusive. Here, we report the mechanism by which arsenic regulates biological nitrogen fixation in the arsenic-hyperaccumulator Pteris vittata. Field investigations and greenhouse experiments, based on multi-omics approaches, reveal that elevated arsenic stress induces an enrichment of key diazotrophs, enhances plant nitrogen acquisition, and thus improves plant growth. Metabolomic analysis and microfluidic experiments further demonstrate that the upregulation of specific root metabolites plays a crucial role in recruiting key diazotrophic bacteria. These findings highlight the pivotal role of nitrogen-acquisition mechanisms in the arsenic hyperaccumulation of Pteris vittata, and provide valuable insights into the plant stress resistance., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Symbiotic nitrogen fixation (SNF) in legume-rhizobia serves as a sustainable source of nitrogen (N) in agriculture. However, the addition of inorganic N fertilizers significantly inhibits SNF, and the underlying mechanisms remain not-well understood. Here, we report that inorganic N disrupts iron (Fe) homeostasis in soybean nodules, leading to a decrease in SNF efficiency. This disruption is attributed to the inhibition of the Fe transporter genes Natural Resistance-Associated Macrophage Protein 2a and 2b (GmNRAMP2a&2b) by inorganic N. GmNRAMP2a&2b are predominantly localized at the tonoplast of uninfected nodule tissues, affecting Fe transfer to infected cells and consequently, modulating SNF efficiency. In addition, we identified a pair of N-signal regulators, nitrogen-regulated GARP-type transcription factors 1a and 1b (GmNIGT1a&1b), that negatively regulate the expression of GmNRAMP2a&2b, which establishes a link between N signaling and Fe homeostasis in nodules. Our findings reveal a plausible mechanism by which soybean adjusts SNF efficiency through Fe allocation in response to fluctuating inorganic N conditions, offering valuable insights for optimizing N and Fe management in legume-based agricultural systems., (© 2024. The Author(s).)

Symbiotic nitrogen fixation can reduce the impact of agriculture on the environment by reducing fertilizer input. The rapid development of nanomaterials in agriculture provides a new prospect for us to improve the biological nitrogen fixation ability of leguminous crops. Molybdenum is an important component of nitrogenase, and the potential application of MoO 3 NPs in agriculture is largely unexplored. In this study, on the basis of verifying that MoO 3 NPs can improve the nitrogen fixation ability of soybean, the effects of MoO 3 NPs on the symbiotic nitrogen fixation process of soybean were investigated by using dynamic transcriptome and targeted metabolome techniques. Here we showed that compared with conventional molybdenum fertilizer, minute concentrations of MoO 3 NPs (0.01-0.1 mg kg -1 ) could promote soybean growth and nitrogen fixation efficiency. The nodules number, fresh nodule weight and nitrogenase activity of 0.1 mg kg -1 were increased by 17 %, 14 % and 27 %, and plant nitrogen accumulation increased by 17 %. Compared with conventional molybdenum fertilizer, MoO 3 NPs had a greater effect on apigenin, kaempferol and other flavonoid, and the expression of nodulation related genes such as ENOD93, F3'H. Based on WGCNA analysis, we identified a core gene GmCHS9 that was positively responsive to molybdenum and was highly expressed during MoO 3 NPs induced nodulation. MoO 3 NPs could improve the nitrogen fixation ability of soybean by promoting the secretion of flavonoids and the expression of key genes. This study provided a new perspective for the nano-strengthening strategy of nodules development and flavonoid biosynthesis by molybdenum., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Cadmium (Cd)-contamination impairs biological nitrogen fixation in legumes (BNF), threatening global food security. Innovative strategies to enhance BNF and improve plant resistance to Cd are therefore crucial. This study investigates the effects of graphitic carbon nitride nanosheets (g-C 3 N 4 NSs) on soybean (Glycine max L.) in Cd contaminated soil, focusing on Cd distribution, chemical forms and nitrogen (N) fixation. Soybean plants were treated with 100 mg kg -1  g-C 3 N 4 NSs, with or without 10 mg kg -1 Cd for 4 weeks. Soil addition of g-C 3 N 4 NSs alleviated Cd toxicity and promote soybean growth via scavenging Cd-mediated oxidative stress and improving photosynthesis. Compared to Cd treatment, g-C 3 N 4 NSs increased shoot and root dry weights under Cd toxicity by 49.5% and 63.4%, respectively. g-C 3 N 4 NSs lowered Cd content by 35.7%-54.1%, redistributed Cd subcellularly by increasing its proportion in the cell wall and decreasing it in soluble fractions and organelles, and converted Cd from high-toxicity to low-toxicity forms. Additionally, g-C 3 N 4 NSs improved the soil N cycle, stimulated nodulation, and increased the N-fixing capacity of nodules, thus increasing N content in shoots and roots by 12.4% and 43.2%, respectively. Mechanistic analysis revealed that g-C 3 N 4 NSs mitigated Cd-induced loss of endogenous nitric oxide in nodules, restoring nodule development. This study highlights the potential of g-C 3 N 4 NSs for remediating Cd-contaminated soil, reducing Cd accumulation, and enhancing plant growth and N fixation, offering new insights into the use of carbon nanomaterials for soil improvement and legume productivity under metal(loid)s stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The actinobacterium Arthrobacter sp. UMCV2 promotes plant growth through the emission of N,N-dimethylhexadecilamine (DMHDA). The Medicago-Sinorhizobium nodulation has been employed to study symbiotic nitrogen fixation by rhizobia in nodulating Fabaceae. Herein, we isolated three Sinorhizobium medicae strains that were used to induce nodules in Medicago truncatula. The co-inoculation of M. truncatula with Arthrobacter sp. strain UMCV2 produced a higher number of effective nodules than inoculation with only Sinorhizobium strains. Similarly, the exposure of inoculated M. truncatula to DMHDA produced a greater number of effective nodules compared to non-exposed plants. Thus, we conclude that Arthrobacter sp. UMCV2 promotes nodulation, and propose that this effect is produced, at least partly, via DMHDA emission., (Copyright © 2024 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Nanoplastics pollution is a growing environmental problem worldwide. Recent research has demonstrated the toxic effects of nanoplastics on various marine organisms. However, the influences of nanoplastics on marine nitrogen-fixing cyanobacteria, a critical nitrogen source in the ocean, remained unknown. Here, we report that nanoplastics exposure significantly reduced growth, photosynthetic, and nitrogen fixation rates of Crocosphaera watsonii (a major marine nitrogen-fixing cyanobacterium). Transcriptomic analysis revealed that nanoplastics might harm C. watsonii via downregulation of photosynthetic pathways and DNA damage repair genes, while genes for respiration, cell damage, nitrogen limitation, and iron (and phosphorus) scavenging were upregulated. The number and size of starch grains and electron-dense vacuoles increased significantly after nanoplastics exposure, suggesting that C. watsonii allocated more resources to storage instead of growth under stress. We propose that nanoplastics can damage the cell (e.g., DNA, cell membrane, and membrane-bound transporters), inhibit nitrogen and carbon fixation, and hence lead to nutrient limitation and impaired growth. Our findings suggest the possibility that nanoplastics pollution could reduce the new nitrogen input and hence affect the productivity in the ocean. The impact of nanoplastics on marine nitrogen fixation and productivity should be considered when predicting the ecosystem response and biogeochemical cycling in the changing ocean., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fertilization with nickel (Ni) can positively affect plant development due to the role of this micronutrient in nitrogen (N) metabolism, namely, through urease and NiFe-hydrogenase. Although the application of Ni is an emerging practice in modern agriculture, its effectiveness strongly depends on the chosen application method, making further research in this area essential. The individual and combined effects of different Ni application methods-seed treatment, leaf spraying and/or soil fertilization-were investigated in soybean plants under different edaphoclimatic conditions (field and greenhouse). Beneficial effects of the Soil, Soil + Leaf and Seed + Leaf treatments were observed, with gains of 7 to 20% in biological nitrogen fixation, 1.5-fold in ureides, 14% in shoot dry weight and yield increases of up to 1161 kg ha -1 . All the Ni application methods resulted in a 1.1-fold increase in the SPAD index, a 1.2-fold increase in photosynthesis, a 1.4-fold increase in nitrogenase, and a 3.9-fold increase in urease activity. Edaphoclimatic conditions exerted a significant influence on the treatments. The integrated approaches, namely, leaf application in conjunction with soil or seed fertilization, were more effective for enhancing yield in soybean cultivation systems. The determination of the ideal method is crucial for ensuring optimal absorption and utilization of this micronutrient and thus a feasible and sustainable management technology. Further research is warranted to establish official guidelines for the application of Ni in agricultural practices., (© 2024. The Author(s).)

The present study was undertaken to investigate the arsenite (As III)-induced changes in the diazotrophic cyanobacterium Anabaena PCC 7120. It was observed that the growth of cyanobacterial decreased with increase in As (III) concentration. The cells exposed to As (III) showed morphological variation (deformity) due to the formation of deeper constrictions in vegetative cells. Strain showed increased heterocyst differentiation (1.6-fold higher) whereas decreased nitrogenase activity at the concentration of 40 ppm As (III). The activities of NR, NiR, urease and GS decreased with increase in As (III) concentrations and attained their minimum levels at 40 ppm of As (III). The Ca 2+ -dependent ATPase activity increased with increase in As (III) concentration and attained its about 2.72-fold higher level at 40 ppm of As (III). In contrast, sharp decline in Mg 2+ -dependent ATPase activity (28%) was recorded at 1 ppm of As (III) over untreated control. The rates of photosynthetic O 2 evolution and respiration decreased with increase in As (III) concentration and attained its minimal level at 40 ppm of As (III). Therefore, this study highlighted arsenite regimes efficiently correlated with behavioral changes in consort with strain., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N 2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N 2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms' ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA's action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected-due to their notable expression differences-for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.

Soybean ( Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

Soybean [Glycine max (L.) Merr.] has great economic and nutritional importance mainly due to its high protein content. All plant's N needs can be met by the symbiosis with elite Bradyrhizobium strains applied as inoculants to the seeds at sowing time; however, the increasing use of pesticides in seed treatments can impair the contribution of the biological nitrogen fixation. In this study, we report decreases in cell survival of two strains, B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 in seeds inoculated and treated with StandakTop™, composed of the fungicides pyraclostrobin and thiophanate-methyl and the insecticide fipronil, the pesticides most used in soybean seed treatment in several countries. Cell death was enhanced with the time of exposure to the pesticides, and B. elkanii was less tolerant, with almost no detectable viable cells after 15 days. Change in colony morphology with smaller colonies was observed in the presence of the pesticides, being more drastic with the time of exposure, and attributed to an adaptive response towards survival in the presence of the abiotic stress. However, morphological changes were reversible after elimination of the stressing agent and symbiotic performance under controlled greenhouse conditions was similar between strains that had been or not exposed to the pesticides. In addition, no changes in DNA profiles (BOX-PCR) of both strains were observed after the contact with the pesticides. In two field experiments, impacting effects of the pesticides were observed mainly on the total N accumulated in grains of plants relying on both N 2 -fixation and N-fertilizer. Our data indicate that StandakTop® affects parameters never reported before, including colony morphology of Bradyrhizobium spp. and N metabolism and/or N remobilization to soybean grains.

Chlorpyrifos, a common organophosphorus pesticide, is widely used for agricultural pest control and can inhibit nitrogen-fixing bacteria biomass in paddy. In this study, the additions of chlorpyrifos (1 and 8 mg kg -1 ) to soil, with or without Pseudomonas stutzeri A1501, resulted in a significant decrease in nitrogen fixation, despite insignificant effects on the abundances of P. stutzeri A1501 and bacteria in soil. Toxic effect of chlorpyrifos on P. stutzeri A1501 nitrogenase activity in medium was also observed, accompanied by a significant reduction in the expression of nitrogen-fixing related genes (nifA and nifH). Furthermore, rhizosphere colonization and biofilm formation by P. stutzeri A1501 were repressed by chlorpyrifos, leading to decreased nitrogenase activity in the rhizosphere. Biofilm formation in medium was inhibited by bacterial hyperkinesis and reduction of extracellular polymeric substance, including exopolysaccharides and proteins. Together, these findings showed that chlorpyrifos-induced production of reactive oxygen species (ROS) which was directly responsible for reduced nitrogenase activity in the medium, soil, and rhizosphere by inhibiting the expressions of nitrogen-fixing related genes. Furthermore, the inhibition of biofilm formation by chlorpyrifos or ROS likely aggravated the reduction in rhizospherere nitrogenase activity. These findings provide potentially valuable insights into the toxicity of chlorpyrifos on nitrogen-fixing bacteria and its mechanisms. Furthermore, for sustainable rice production, it is necessary to evaluate whether other pesticides affect nitrogen fixation and select pesticides that do not inhibit nitrogen fixation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cyanobacteria are an important component in the rice field ecosystem and are a well known source of natural biofertilizer. Pesticidal application for the control of pests in rice field soil has led to several environmental problems, and poses a great threat to these beneficial microorganisms. Studies on the impact of pesticides on the diazotrophic growth and survivability of these microorganisms have recently gained much attention. The present paper describes the effects of an iterated use of the insecticide deltamethrin (2.8% EC) on the growth and nitrogen fixation capacity of the filamentous cyanobacterium Calothrix sp. (strain GUEco 1002). This organism has shown a varying degree of sensitivity to the insecticide. For evaluating the deltamethrin toxicity, the test organism was subjected to varying concentrations of deltamethrin i.e. 17.5 ppm, 35 ppm, 70 ppm and 140 ppm based upon LC 50 for 20 days. The data obtained in the laboratory revealed that the treatment of the test organism with deltamethrin (17.5-140 ppm) negatively affected its growth, pigments, protein and nitrogen content in a time dose dependent manner. In contrast, carbohydrate content significantly increased with increasing concentrations of deltamethrin, this effect being more prominent at 140 ppm treatment (38%). At this high level (140 ppm), the test organism showed a significant decrease in dry weight biomass (46%), chlorophyll-a (72%), carotenoids (57%), phycocyanin (67%), protein (69%) and nitrogen content (61%) over the control. A little, but insignificant, stimulatory effect on nitrogen content was recorded at 17.5 ppm of the insecticide which however, was the opposite in the case of growth, pigments, carbohydrate and protein content.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena ) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Large amounts of aluminum (Al) enter the ocean through atmospheric dust deposition and river runoffs. However, few studies have reported the effects of Al on marine phytoplankton, especially nitrogen-fixing cyanobacteria. By using the isotope tracer method and quantitative reverse transcription PCR (RT-qPCR), we examined the physiological effect of Al (0.2, 2 and 20 μM) on the unicellular marine nitrogen-fixing cyanobacterium Crocosphaera watsonii in Aquil* medium. We show that Al has an inhibitory physiological effect on C. watsonii, including changes in growth rate, nitrogen fixation rate, carbon fixation rate, cell size, fast rise chlorophyll fluorescence kinetics, cellular photosynthetic pigment and C/N/P content, the same as that of the phosphorus deficient treatment. The ratio of cellular elements C:N:P showed that phosphorus was deficient in the cell of C. watsonii after Al treatment (2 and 20 μM). In addition, Al stimulated the expression of phosphorus-related genes pstS, phoH, phoU, ppK and ppX in C. watsonii. All these results suggest that Al-treated C. watsonii is phosphorus-limited, and that the phosphorus deficiency induced by Al may be one mechanism behind aluminum's toxicity., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is finally approved by all authors for publication., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Soybean (Glycine max (L.) Merrill) is one of the most important crops worldwide providing dietary protein and vegetable oil. Most of the nitrogen required by the crop is supplied through biological N 2 fixation. Non-thermal plasma is a fast, economical, and environmental-friendly technology that can improve seed quality, plant growth, and crop yield. Soybean seeds were exposed to a dielectric barrier discharge plasma operating at atmospheric pressure air with superimposed flows of O 2 or N 2 as carrying gases. An arrangement of a thin phenolic sheet covered by polyester films was employed as an insulating barrier. We focused on the ability of plasma to improve soybean nodulation and biological nitrogen fixation. The total number of nodules and their weight were significantly higher in plants grown from treated seeds than in control. Plasma treatments incremented 1.6 fold the nitrogenase activity in nodules, while leghaemoglobin content was increased two times, indicating that nodules were fixing nitrogen more actively than control. Accordingly, the nitrogen content in nodules and the aerial part of plants increased by 64% and 23%, respectively. Our results were supported by biometrical parameters. The results suggested that different mechanisms are involved in soybean nodulation improvement. Therefore, the root contents of isoflavonoids, glutathione, auxin and cytokinin, and expansin (GmEXP1) gene expression were determined. We consider this emerging technology is a suitable pre-sowing seed treatment.

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) resources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N2 fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium behaves mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes., (© FEMS 2020.)

Bradyrhizobium sp. strain SUTN9-2 is a symbiotic and endophytic diazotrophic bacterium found in legume and rice plants and has the potential to promote growth. The present results revealed that SUTN9-2 underwent cell enlargement, increased its DNA content, and efficiently performed nitrogen fixation in response to rice extract. Some factors in rice extract induced the expression of cell cycle and nitrogen fixation genes. According to differentially expressed genes (DEGs) from the transcriptomic analysis, SUTN9-2 was affected by rice extract and the deletion of the bclA gene. The up-regulated DEGs encoding a class of oxidoreductases, which act with oxygen atoms and may have a role in controlling oxygen at an appropriate level for nitrogenase activity, followed by GroESL chaperonins are required for the function of nitrogenase. These results indicate that following its exposure to rice extract, nitrogen fixation by SUTN9-2 is induced by the collective effects of GroESL and oxidoreductases. The expression of the sensitivity to antimicrobial peptides transporter (sapDF) was also up-regulated, resulting in cell differentiation, even when bclA (sapDF) was mutated. This result implies similarities in the production of defensin-like antimicrobial peptides (DEFs) by rice and nodule-specific cysteine-rich (NCR) peptides in legume plants, which affect bacterial cell differentiation.

The Deepwater Horizon (DWH) oil spill contaminated coastlines from Louisiana to Florida, burying oil up to 70 cm depth in sandy beaches, posing a potential threat to environmental and human health. The dry and nutrient-poor beach sand presents a taxing environment for microbial growth, raising the question how the biodegradation of the buried oil would proceed. Here we report the results of an in-situ experiment that (i) characterized the dominant microbial communities contained in sediment oil agglomerates (SOAs) of DWH oil buried in a North Florida sandy beach, (ii) elucidated the long-term succession of the microbial populations that developed in the SOAs, and (iii) revealed the coupling of SOA degradation to nitrogen fixation. Orders of magnitude higher bacterial abundances in SOAs compared to surrounding sands distinguished SOAs as hotspots of microbial growth. Blooms of bacterial taxa with a demonstrated potential for hydrocarbon degradation (Gammaproteobacteria, Alphaproteobacteria, Actinobacteria) developed in the SOAs, initiating a succession of microbial populations that mirrored the evolution of the petroleum hydrocarbons. Growth of nitrogen-fixing prokaryotes or diazotrophs (Rhizobiales and Frankiales), reflected in increased abundances of nitrogenase genes (nifH), catalyzed biodegradation of the nitrogen-poor petroleum hydrocarbons, emphasizing nitrogen fixation as a central mechanism facilitating the recovery of sandy beaches after oil contamination.

Nitrogen (N 2 ) fixation by moss-associated cyanobacteria is one of the main sources of new N input in pristine ecosystems such as boreal forests and arctic tundra. Given the non-vascular physiology of mosses, they are especially sensitive to e.g. increased N input and heavy metal deposition. While the effects of increased N input on moss-associated N 2 fixation has been comprehensively assessed, hardly any reports exist on the effects of increased heavy metal load on this key ecosystem function. To address this knowledge gap, we made use of an extreme metal pollution gradient in boreal forests of Northern Sweden originating from a metal mine and its associated smelters. We collected the common moss Pleurozium schreberi, known to host cyanobacteria, along a distance gradient away from the metal source of pollution and measured moss-metal content (Fe, Cu, Zn, Pb) as well as N 2 fixation. We found a strong distance gradient in moss-metal content for all investigated metals: a sharp decline in metal content with distance away from the metal pollution source. However, we found a similarly steep gradient in moss-associated N 2 fixation, with highest activity closest to the metal source of pollution. Hence, while mosses may be sensitive to increased heavy metal inputs, the activity of colonising cyanobacteria seem to be unaffected by heavy metals, and consequently, ecosystem function may not be compromised by elevated metal input.

The objective of this work was to evaluate the effect of nitrogen (N) fertilization and the biological nitrogen fixation efficiency in soybean crops grown in unfavorable environments (high temperature, low fertility, and areas with sandy soil). Six field experiments were conducted between 2015 and 2018. Two experiments were performed per year. They were conducted in two separate areas. One was previously covered with degraded pasture (post-pasture area). The other was previously used to plant soybean (post-soybean crop area). The treatments consisted of inoculations with N-fixing bacteria (NFB) (0, 4, 8, and 12 doses ha -1 ) and N fertilization with rates of 0, 25, 50, and 100 kg ha -1 . N fertilization and inoculation with NFB increased soil N, though the levels were still low. Among the tested groups, those with the application of eight doses of inoculant recorded the highest grain yields in post-soybean areas. They showed 10% (237 kg ha -1 ) and 15% (336 kg ha -1 ) higher grain yields when compared to crops treated without inoculant and crops with four doses of inoculant, respectively. N fertilization with 25 and 100 kg ha -1 decreased the root nodules of soybean plants grown in the post-soybean and post-pasture areas, respectively. Soybean crops grown on degraded pasture areas also showed good response to N fertilization (50 kg ha -1 ) when combined with NFB inoculation (12 doses ha -1 ). These showed grain yields 22% (439 kg ha -1 ) higher than those of plants treated with just 12 doses of inoculant and no N fertilization.

Unicellular nitrogen fixer Crocosphaera contributes substantially to nitrogen fixation in oligotrophic subtropical gyres. They fix nitrogen even when significant amounts of ammonium are available. This has been puzzling since fixing nitrogen is energetically inefficient compared with using available ammonium. Here we show that by fixing nitrogen, Crocosphaera can increase their population and expand their niche despite the presence of ammonium. We have developed a simple but mechanistic model of Crocosphaera based on their growth in steady state culture. The model shows that the growth of Crocosphaera can become nitrogen limited despite their capability to fix nitrogen. When they fix nitrogen, the population increases by up to 78% relative to the case without nitrogen fixation. When we simulate a simple ecological situation where Crocosphaera exists with non-nitrogen-fixing phytoplankton, the relative abundance of Crocosphaera increases with nitrogen fixation, while the population of non-nitrogen-fixing phytoplankton decreases since a larger fraction of fixed nitrogen is consumed by Crocosphaera. Our study quantitatively supports the benefit of nitrogen fixation despite the high electron/energy costs, even when an energetically efficient alternative is available. It demonstrates a competitive aspect of Crocosphaera, permitting them to be regionally significant nitrogen fixers.

Cultivation on selective media revealed that the oil-sorbents, wheat straw, corncobs and sugarcane bagasse harbor hydrocarbonoclastic, diazotrophic and heavy metal-resistant microorganisms. Nitrogen-free media containing 1.0% crude oil lost between 32.2 and 37.5% of this oil, after 8 months when they have been inoculated with such microorganism-loaded sorbents. The used wheat straw, corncobs and sugarcane bagasse samples, 1.0 g each, absorbed respectively, 1.9, 1.1 and 2.5 g oil samples, and lost 24.3-39.2% of these amounts, after they had been incubated for 8 months. Total genomic DNA's from culture media and sorbents revealed various nitrogenase-coding nifH-genes. Pure hydrocarbonoclastic microbial isolates tolerated certain concentrations of, Hg 2+ , Cd 2+ , Pb 2+ , AsO 4 3- and AsO 3 3- . Some of those isolates even grew excellently with up to 1000 ppm of Pb 2+ and 36,000 ppm of AsO 4 3- also in the presence of oil. Tested strains removed the tested heavy metals, Hg 2+ , Cd 2+ and Pb 2+ from the media and thus, reduced their toxicity against the hydrocarbon-degraders. It was concluded that plant-based sorbents, not only remove oil physically, but also harbor microbial communities effective in spilled oil-bioremediation under multiple stresses. Although each community consisted of one to three species only, the consortia which reached in numbers millions of CFU ml -1 enrich the oily media with fixed nitrogen, and remove heavy metals which otherwise inhibit the oil-degrading microorganisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pseudomonas stutzeri A1501 is a versatile nitrogen-fixing bacterium capable of living in diverse environments and coping with various oxidative stresses. NfiS, a regulatory noncoding RNA (ncRNA) involved in the control of nitrogen fixation in A1501, was previously shown to be required for optimal resistance to H 2 O 2 ; however, the precise role of NfiS and the target genes involved in the oxidative stress response is entirely unknown. In this work, we systematically investigated the NfiS-based mechanisms underlying the response of this bacterium to H 2 O 2 at the cellular and molecular levels. A mutant strain carrying a deletion of nfiS showed significant downregulation of oxidative stress response genes, especially katB , a catalase gene, and oxyR , an essential regulator for transcription of catalase genes. Secondary structure prediction revealed two binding sites in NfiS for katB mRNA. Complementation experiments using truncated nfiS genes showed that each of two sites is functional, but not sufficient, for NfiS-mediated regulation of oxidative stress resistance and nitrogenase activities. Microscale thermophoresis assays further indicated direct base pairing between katB mRNA and NfiS at both sites 1 and 2, thus enhancing the half-life of the transcript. We also demonstrated that katB expression is dependent on OxyR and that both OxyR and KatB are essential for optimal oxidative stress resistance and nitrogenase activities. H 2 O 2 at low concentrations was detoxified by KatB, leaving O 2 as a by-product to support nitrogen fixation under O 2 -insufficient conditions. Moreover, our data suggest that the direct interaction between NfiS and katB mRNA is a conserved and widespread mechanism among P. stutzeri strains. IMPORTANCE Protection against oxygen damage is crucial for survival of nitrogen-fixing bacteria due to the extreme oxygen sensitivity of nitrogenase. This work exemplifies how the small ncRNA NfiS coordinates oxidative stress response and nitrogen fixation via base pairing with katB mRNA and nifK mRNA. Hence, NfiS acts as a molecular link to coordinate the expression of genes involved in oxidative stress response and nitrogen fixation. Our study provides the first insight into the biological functions of NfiS in oxidative stress regulation and adds a new regulation level to the mechanisms that contribute to the oxygen protection of the MoFe nitrogenase., (Copyright © 2019 Zhang et al.)

The rhizobium-legume symbiotic system is crucial for nitrogen cycle balance in agriculture. Hydrogen sulfide (H 2 S), a gaseous signaling molecule, may regulate various physiological processes in plants. However, whether H 2 S has regulatory effect in this symbiotic system remains unknown. Herein, we investigated the possible role of H 2 S in the symbiosis between soybean ( Glycine max ) and rhizobium ( Sinorhizobium fredii ). Our results demonstrated that an exogenous H 2 S donor (sodium hydrosulfide [NaHS]) treatment promoted soybean growth, nodulation, and nitrogenase (Nase) activity. Western blotting analysis revealed that the abundance of Nase component nifH was increased by NaHS treatment in nodules. Quantitative real-time polymerase chain reaction data showed that NaHS treatment upregulated the expressions of symbiosis-related genes nodA , nodC , and nodD of S. fredii . In addition, expression of soybean nodulation marker genes, including early nodulin 40 ( GmENOD40 ), ERF required for nodulation ( GmERN ), nodulation signaling pathway 2b ( GmNSP2b ), and nodulation inception genes ( GmNIN1a , GmNIN2a , and GmNIN2b ), were upregulated. Moreover, the expressions of glutamate synthase ( GmGOGAT ), asparagine synthase ( GmAS ), nitrite reductase ( GmNiR ), ammonia transporter ( GmSAT1 ), leghemoglobin ( GmLb ), and nifH involved in nitrogen metabolism were upregulated in NaHS-treated soybean roots and nodules. Together, our results suggested that H 2 S may act as a positive signaling molecule in the soybean-rhizobia symbiotic system and enhance the system's nitrogen fixation ability.

Copper-based fungicides have been used for a long time in viticulture and have accumulated in many vineyard soils. In this study, incrementing Cu(OH) 2 -based fungicide application from 0.05 to 5 g Cu kg -1 on two agricultural soils (an acidic sandy loam (L, pH 4.95) and an alkaline silt loam (D, pH 7.45)) resulted in 5 times more mobile Cu in the acidic soil. The most sensitive parameters of alfalfa (Medicago sativa) growing in these soils were the root nodule number, decreasing to 34% and 15% of the control at 0.1 g Cu kg -1 in soil L and at 1.5 g Cu kg -1 in soil D, respectively, as well as the nodule biomass, decreasing to 25% and 27% at 0.5 g Cu kg -1 in soil L and at 1.5 g Cu kg -1 in soil D, respectively. However, the enzymatic N 2 -fixation was not directly affected by Cu in spite of the presence of Cu in the meristem and the zone of effective N 2 -fixation, as illustrated by chemical imaging. The strongly different responses observed in the two tested soils reflect the higher buffering capacity of the alkaline silt loam and showed that Cu mitigation and remediation strategies should especially target vineyards with acidic, sandy soils.

Cyanothece sp., a coccoid, unicellular, nitrogen-fixing and hydrogen-producing cyanobacterium, has been used in this study to biosynthesize customized gold nanoparticles under certain chemical conditions. The produced gold nanoparticles had a characteristic absorption band at 525-535 nm. Two types of gold nanoparticle, the purple and blue, were formed according to the chemical environment in which the cyanobacterium was grown. Dynamic light scattering was implemented to estimate the size of the purple and blue nanoparticles, which ranged from 80 ± 30 nm and 129 ± 40 nm in diameter, respectively. The highest scattering of laser light was recorded for the blue gold nanoparticles, which was possibly due to their larger size and higher concentration. The appearance of anodic and cathodic peaks in cyclic voltammetric scans of the blue gold nanoparticles reflected the oxidation into gold oxide, followed by the subsequent reduction into the nano metal state. The two produced forms of gold nanoparticles were used to treat isoproterenol-induced myocardial infarction in experimental rats. Both forms of nanoparticles ameliorated myocardial infarction injury, with a slight difference in their curative activity with the purple being more effective. Mechanisms that might explain the curative effect of these nanoparticles on the myocardial infarction were proposed. The morphological, physiological, and biochemical attributes of the Cyanothece sp. cyanobacterium were fundamental for the successful production of "tailored" nanoparticles, and complemented the chemical conditions for the differential biosynthesis process. The present research represents a novel approach to manipulate cyanobacterial cells towards the production of different-sized gold nanoparticles whose curative impacts vary accordingly. This is the first report on that type of manipulated gold nanoparticles biosynthesis which will hopefully open doors for further investigations and biotechnological applications.

Oxidative stress generates reactive oxygen species which causes cell damage of living organisms and are normally detoxified by antioxidants. Indirect reports signify the damages caused by reactive oxygen species and neutralized by antioxidant, but the direct evidence to confirm this hypothesis is still unclear. To validate our hypothesis, an attempt was made in a diazotrophic bacterium (Azotobacter chroococcum Avi2) as a biological system, and hydrogen peroxide (H 2 O 2 ) and ascorbic acid were used as oxidative stress and antioxidant supplement, respectively. Additionally, rice plant-growth attributes by Avi2 was also assessed under H 2 O 2 and ascorbic acid. Results indicated that higher concentration of H 2 O 2 (2.5 mM-4.5 mM) showed the complete mortality of Avi2, whereas one ppm ascorbic acid neutralized the effect of H 2 O 2 . Turbidity, colony forming unit, DNA quantity, nifH gene abundance, indole acetic acid and ammonia productions were significantly (p < 0.5) increased by 11.93%, 17.29%, 19.80%, 74.77%, 71.89%, and 42.53%, respectively in Avi2-treated with 1.5 mM H 2 O 2 plus ascorbic acid compared to 1.5 mM H 2 O 2 alone. Superoxide dismutase was significantly (p < 0.5) increased by 60.85%, whereas catalase and ascorbate peroxidase activities were significantly (p < 0.05) decreased by 64.28% and 68.88% in Avi2-treated with 1.5 mM H 2 O 2 plus ascorbic acid compared to 1.5 mM H 2 O 2 alone. Germination percentage of three rice cultivars (FR13a, Naveen and Sahbhagi dhan) were significantly (p < 0.5) increased by 20%, 13.33%, and 4%, respectively in Avi2-treated with 0.6 mM H 2 O 2 plus ascorbic acid compared with uninoculated control. Overall, this study indicated that ascorbic acid formulation neutralizes the H 2 O 2 -oxidative stress and enhances the survivability and plant growth-promoting efficacy of A. chroococcum Avi2 and therefore, it may be used as an effective formulation of bio-inoculants in rice under oxidative stress., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Microelements play important roles in improving crop productivity and quality. Two traditional methods of providing micronutrients are soil and foliar application, yet the cost involved and phytotoxicity risks poses a major challenge in most global agricultural areas. Nutri-priming represents thus a very promising and viable alternative to ensure that the plants' requirements for micronutrients are met. This paper explores the Mo-priming effects on the germination, growth and yield components of common bean (Phaseolus vulgaris L.). In order to accomplish this research objective, our study is divided in two phases. The first phase seeks to determine the effects of two durations (5 and 10 h) of Mo-priming on the germination parameters. Then, a greenhouse pot experiment was conducted with primed seeds showing better germination performance to assess the effects of Mo-priming on certain parameters related to biological nitrogen fixation and crop yield. Five seed pretreatments were studied, namely, 0, 0.025, 0.05, 0.1 and 0.2% of ammonium heptamolybdate ((NH 4 ) 6 Mo 7 O 24 .4H 2 O). Unprimed seeds treated with or without the recommended dose through a soil application were used as controls. Hydropriming is sufficient to improve seed germination performance. However, 0.1% Mo-priming for 10 h improved the net CO 2 assimilation rate, chlorophyll content and biological nitrogen fixation. Six times more nitrogen was fixed (65.8 kg ha -1 ) in these plants than in the controls. Additionally, grain yield increased by 115% compared to the negative control. Thus, compared to soil intake, nutri-priming was a very efficient experimental method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Traffic derived nitrogen (N) and heavy metal pollution is a well-known phenomenon, but little explored in otherwise pristine ecosystems such as subarctic tundra. Here, the main source of N input to the ecosystem is via N 2 fixation by moss- and lichen-associated bacteria. While inhibitory effects of N deposition on moss-associated N 2 fixation have been reported, we still lack understanding of the effects of traffic derived N and heavy metal deposition on this ecosystem function in an otherwise pristine setting. To test this, we established a distance gradient (0-1280 m) away from a metal pollution source -a railway transporting iron ore that passes through a subarctic birch forest. We assessed the effects of railway-derived pollution on N 2 fixation associated with two moss species Pleurozium schreberi, Hylocomium splendens and with the lichen Peltigera aphthosa. Deposition and availability of N and heavy metals (Fe, Cu, Zn, Pb) as well as the respective contents in moss, lichen and soil was assessed. While we found a steep gradient in metal concentration in moss, lichen and soil with distance away from the pollution source, N deposition did not change, and with that, we could not detect a distance gradient in moss- or lichen-associated N 2 fixation. Hence, our results indicate that N 2 fixing bacteria are either not inhibited by heavy metal deposition, or that they are protected within the moss carpet and lichen tissue., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigated the effects of 35 years of application of inorganic fertilizers containing nitrogen (N), phosphate (P), or potassium (K) alone or in combination on the abundance and composition of diazotrophic community in a black soil (Mollisol) in northeast China. The abundance and composition of diazotrophic community were analyzed using qPCR and Illumina MiSeq sequencing targeting nifH genes. Nitrogen fertilization decreased the abundance and Shannon diversity of nifH genes. The diazotrophic community was dominated by Alphaproteobacteria (Bradyrhizobium at the genus level), with relatively higher abundance in the N fertilization treatments than in the non-N fertilization treatments. All diazotrophic communities were clustered into two groups with and without N fertilization history, and the soil pH, total C, total N, and NO 3 - -N significantly influenced the structure of the whole diazotrophic community. Moreover, random matrix theory analysis elucidated a clear difference in network structures between the N and non-N fertilization treatments, with N fertilization causing a less stable network structure. These results highlighted that it was N fertilizer, but not P and K fertilizers, contributed to great changes in the diazotrophic community in this black agricultural soil., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Plant communities play an important role in the C-sink function of peatlands. However, global change and local perturbations are expected to modify peatland plant communities, leading to a shift from Sphagnum mosses to vascular plants. Most studies have focused on the direct effects of modification in plant communities or of global change (such as climate warming, N fertilization) in peatlands without considering interactions between these disturbances that may alter peatlands' C function. We set up a mesocosm experiment to investigate how Greenhouse Gas (CO 2 , CH 4 , N 2 O) fluxes, and dissolved organic carbon (DOC) and total dissolved N (TN) contents are affected by a shift from Sphagnum mosses to Molinia caerulea dominated peatlands combined with N fertilization. Increasing N deposition did not alter the C fluxes (CO 2 exchanges, CH 4 emissions) or DOC content. The lack of N effect on the C cycle seems due to the capacity of Sphagnum to efficiently immobilize N. Nevertheless, N supply increased the N 2 O emissions, which were also controlled by the plant communities with the presence of Molinia caerulea reducing N 2 O emissions in the Sphagnum mesocosms. Our study highlights the role of the vegetation composition on the C and N fluxes in peatlands and their responses to the N deposition. Future research should now consider the climate change in interaction to plants community modifications due to their controls of peatland sensitivity to environmental conditions., (Copyright © 2018. Published by Elsevier B.V.)

Molybdenum (Mo) deficiency in the farmland of China may limit biological nitrogen fixation (BNF), however, the impact of Mo application on BNF capacities and diazotrophic communities in rice-soil systems is unclear. In this experiment, treatments in a 6.7 atom% 15 N 2 -labelling field-based growth chamber for 74 days and treatments in a 99 atom% 15 N 2 -labelling microcosm experiment for 40 days combined with 16S rRNA gene sequencing and DNA-stable isotope probing (SIP) were used to investigate the impacts of Mo application on BNF and diazotrophic communities. Our results showed that under the condition that no nitrogen (N) fertilizer was applied, Mo application (500 g sodium molybdate ha -1 ) significantly increased N 2 fixation in a rice-Inceptisol system, from 22.3 to 53.1 kg N ha -1 . Mo application significantly increased the number of nifH gene copies and the relative abundance of cyanobacteria in both growth chamber and microcosm experiments. Among cyanobacteria, the relative abundances of the most abundant genera Leptolyngbya and Microcoleus were significantly increased by Mo application. 15 N 2 -DNA-SIP further demonstrated that Leptolyngbya and Microcoleus incorporated 15 N 2. Mo application greatly increased BNF in Mo-deficient paddy field (≤0.068 mg kg -1 ) and stimulated the growth of cyanobacteria. These results indicated that Mo application in Mo-deficient paddy field could be a useful measure to increase soil N input under no N fertilization., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Globally, eutrophication and warming of aquatic ecosystems has increased the frequency and intensity of cyanobacterial blooms and their associated toxins, with the simultaneous detection of multiple cyanotoxins often occurring. Despite the co-occurrence of cyanotoxins such as microcystins and anatoxin-a (ATX) in water bodies, their effects on phytoplankton communities are poorly understood. The individual and combined effects of microcystin-LR (MC-LR) and ATX on the cyanobacteria Microcystis spp., and Anabaena variabilis (a.k.a. Trichormus variabilis ), and the chlorophyte, Selenastrum capricornutum were investigated in the present study. Cell density, chlorophyll-a content, and the maximum quantum efficiency of photosystem II (Fv/Fm) of Microcystis cells were generally lowered after exposure to ATX or MC-LR, while the combined treatment with MC-LR and ATX synergistically reduced the chlorophyll-a concentration of Microcystis strain LE-3. Intracellular levels of microcystin in Microcystis LE-3 significantly increased following exposure to MC-LR + ATX. The maximum quantum efficiency of photosystem II of Anabaena strain UTEX B377 declined during exposure to the cyanotoxins. Nitrogen fixation by Anabaena UTEX B377 was significantly inhibited by exposure to ATX, but was unaffected by MC-LR. In contrast, the combination of both cyanotoxins (MC-LR + ATX) caused a synergistic increase in the growth of S. capricornutum . While the toxins caused an increase in the activity of enzymes that scavenge reactive oxygen species in cyanobacteria, enzyme activity was unchanged or decreased in S. capricornutum . Collectively this study demonstrates that MC-LR and ATX can selectively promote and inhibit the growth and performance of green algae and cyanobacteria, respectively, and that the combined effect of these cyanotoxins was often more intense than their individual effects on some strains. This suggests that the release of multiple cyanotoxins in aquatic ecosystems, following the collapse of blooms, may influence the succession of plankton communities.

The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of nanomaterial toxicity on microorganisms. Herein, we studied the effect of two different sizes of nanosilver (10 nm and 50 nm) on the soil nitrogen fixation by the model bacteria Azotobacter vinelandii. Smaller size AgNPs correlated with higher toxicity, which was evident from reduced cell numbers. Flow cytometry analysis further confirmed this finding, which was carried out with the same concentration of 10 mg/L for 12 h, the apoptotic rates were20.23% and 3.14% for 10 nm and 50 nm AgNPs, respectively. Structural damage to cells were obvious under scanning electron microscopy. Nitrogenase activity and gene expression assays revealed that AgNPs could inhibit the nitrogen fixation of A. vinelandii. The presence of AgNPs caused intracellular reactive oxygen species (ROS) production and electron spin resonance further demonstrated that AgNPs generated hydroxyl radicals, and that AgNPs could cause oxidative damage to bacteria. A combination of Ag content distribution assays and transmission electron microscopy indicated that AgNPs were internalized in A. vinelandii cells. Overall, this study suggested that the toxicity of AgNPs was size and concentration dependent, and the mechanism of antibacterial effects was determined to involve damage to cell membranes and production of reactive oxygen species leading to enzyme inactivation, gene down-regulation and death by apoptosis., Competing Interests: The authors have declared that no competing interests exist.

Nitrogen (N) is an essential macronutrient for plant development and growth, and the deposition of N has increased in recent decades. Legumes that fix N can also provide N for nearby species. However, N in soil inhibits N fixation. We tested the effects of N fertilisation on one N-fixing (Robinia pseudoacacia) and two non-N-fixing (Sophora japonica and Senna surattensis) woody legume species, which were subjected to five different N levels (0, 1.5, 2.9, 5.9 and 11.4 mg N per plant day -1 ) under greenhouse conditions. The growth of the two non-N-fixing species was promoted by N supply, while that of R. pseudoacacia was unaffected. Among the three species, R. pseudoacacia had the largest specific leaf area and chlorophyll concentration, S. japonica had the largest root-to-shoot ratio and main root-to-lateral root ratio, and S. surattensis had the largest leaf N and phosphorus concentrations. The N-fixing species was mostly unaffected by N supply. The growth, leaf chlorophyll concentration, and leaf number in the non-N-fixing species were promoted by N supply. The N-fixing species showed better growth in low-N environments, while under increased N deposition, its growth was similar to that of the non-N-fixing species.

Azospirillum sp. B510, a free-living nitrogen-fixing bacterium isolated from the stems of rice (Oryza sativa cv. Nipponbare), was investigated to establish effective conditions for the colonization of rice plants. We analyzed the effects of the nitrogen sources KNO 3 , NH 4 Cl, urea (CO[NH 2 ] 2 ), and NH 4 NO 3 at different concentrations (0.01-10 mM) on this colonization. Nitrogen promoted plant growth in a concentration-dependent manner, with minor differences being observed among the different nitrogen sources. Bacterial colonization was markedly suppressed on media containing NH 4 + concentrations higher than 1 mM. Since concentrations of up to and including 10 mM NH 4 + did not exhibit any antibacterial activity, we analyzed several factors affecting the NH 4 + -dependent inhibition of endophytic colonization, including the accumulation of the reactive oxygen species H 2 O 2 and the secretion of the chemotactic substrate malic acid. The accumulation of H 2 O 2 was increased in rice roots grown on 1 mM NH 4 Cl. The amounts of malic acid secreted from NH 4 -grown rice plants were lower than those secreted from plants grown without nitrogen or with KNO 3 . Although the bacterium exhibited chemotactic activity, moving towards root exudates from plants grown without nitrogen and KNO 3 -grown plants, this activity was not observed with root exudates from NH 4 + -grown plants. NH 4 + , but not NO 3 - , caused the acidification of growth media, which inhibited plant bacterial colonization. These NH 4 + -dependent phenomena were markedly suppressed by the stabilization of medium pH using a buffer. These results demonstrate that the type and concentration of nitrogen fertilizer affects the colonization of rice plants by Azospirillum sp. B510.

Rhodopseudomonas palustris strains PS3 and YSC3 are purple non-sulfur phototrophic bacteria isolated from Taiwanese paddy soils. PS3 has beneficial effects on plant growth and enhances the uptake efficiency of applied fertilizer nutrients. In contrast, YSC3 has no significant effect on plant growth. The genomic structures of PS3 and YSC3 are similar; each contains one circular chromosome that is 5,269,926 or 5,371,816 bp in size, with 4,799 or 4,907 protein-coding genes, respectively. In this study, a large class of genes involved in chemotaxis and motility was identified in both strains, and genes associated with plant growth promotion, such as nitrogen fixation-, IAA synthesis- and ACC deamination-associated genes, were also identified. We noticed that the growth rate, the amount of biofilm formation, and the relative expression levels of several chemotaxis-associated genes were significantly higher for PS3 than for YSC3 upon treatment with root exudates. These results indicate that PS3 responds better to the presence of plant hosts, which may contribute to the successful interactions of PS3 with plant hosts. Moreover, these findings indicate that the existence of gene clusters associated with plant growth promotion is required but not sufficient for a bacterium to exhibit phenotypes associated with plant growth promotion.

Aluminum (Al) toxicity is a major problem affecting soil fertility, microbial diversity, and nutrient uptake of plants. Rhizobia response and legume interaction under Al conditions are still unknown; it is important to understand how to develop and improve legume cultivation under Al stress. In this study, rhizobia response was recorded under different Al concentrations. Al effect on rhizobial cells was characterized by combination with different two pH conditions. Symbiosis process was compared between α- and β-rhizobia inoculated onto soybean varieties. Rhizobial cell numbers was decreased as Al concentration increased. However, induced Al tolerance considerably depended on rhizobia types and their origins. Accordingly, organic acid results were in correlation with growth rate and cell density which suggested that citric acid might be a positive selective force for Al tolerance and plant interaction on rhizobia. Al toxicity delayed and interrupted the plant-rhizobia interaction and the effect was more pronounced under acidic conditions. Burkholderia fungorum VTr35 significantly improved plant growth under acid-Al stress in combination with all soybean varieties. Moreover, plant genotype was an important factor to establish an effective nodulation and nitrogen fixation under Al stress. Additionally, tolerant rhizobia could be applied as an inoculant on stressful agroecosystems. Furthermore, metabolic pathways have still been unknown under Al stress.

A study was undertaken to test the effects of molybdenum (Mo) and phosphorus (P) amendments on biological nitrogen (N) fixation (BNF) by boreal forest moss-associated cyanobacteria. Feather moss (Pleurozium schreberi) samples were collected on five sites, on two dates and at different roadside distances (0-100m) corresponding to an assumed gradient of reactive N deposition. Potential BNF of Mo and P amended moss samples was measured using the acetylene reduction assay. Total N, P and heavy metal concentrations of mosses collected at 0 and 100m from roadsides were also measured. Likewise, the needles from Norway spruce trees (Picea abies) at different roadside distances were collected in late summer and analyzed for total N, P and heavy metals. There was a significant increase in BNF with roadside distance on 7-of-10 individual Site×Date combinations. We found no clear evidence of an N gradient across roadside distances. Elemental analyses of feather moss and Norway spruce needle tissues suggested decreasing deposition of heavy metals (Mo-Co-Cr-Ni-V-Pb-Ag-Cu) as well as P with increasing distance from the roadside. The effects of Mo and P amendments on BNF were infrequent and inconsistent across roadside distances and across sites. One particular site, however, displayed greater concentrations of heavy metals near the roadside, as well as a steeper P fertility gradient with roadside distance, than the other sites. Here, BNF increased with roadside distance only when moss samples were amended with P. Also at this site, BNF across all roadside distances was higher when mosses were amended with both Mo and P, suggesting a co-limitation of these two nutrients in controlling BNF. In summary, our study showed a potential for car emissions to increase heavy metals and P along roadsides and underscored the putative roles of these anthropogenic pollutants on BNF in northern latitudes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Various species of cyanobacteria, diatoms and dinoflagellates are capable of synthesizing the non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA), which is known to be a causative agent of human neurodegeneration. Similar to most cyanotoxins, the biological and ecological functions of BMAA in cyanobacteria are unknown. In this study, we show for the first time that BMAA, in micromolar amounts, inhibits the formation of heterocysts (specialized nitrogen-fixing cells) in heterocystous, diazotrophic cyanobacteria [Anabaena sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), Nostoc sp. strain 8963] under conditions of nitrogen starvation. The inhibitory effect of BMAA is abolished by the addition of glutamate. To understand the genetic reason for the observed phenomenon, we used qPCR to study the expression of key genes involved in cell differentiation and nitrogen metabolism in the model cyanobacterium Anabaena sp. PCC 7120. We observed that in the presence of BMAA, Anabaena sp. PCC 7120 does not express two essential genes associated with heterocyst differentiation, namely, hetR and hepA. We also found that addition of BMAA to cyanobacterial cultures with mature heterocysts inhibits nifH gene expression and nitrogenase activity., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Compared to extensive studies reporting the aluminum (Al) toxicity to terrestrial plants and freshwater organisms, very little is known about how marine phytoplankton responds to Al in the field. Here we report the marine phytoplankton responses to Al enrichment in the South China Sea (SCS) using on-deck bottle incubation experiments during eight cruises from May 2010 to November 2013. Generally, Al addition alone enhanced the growth of diatom and Trichodesmium, and nitrogen fixation, but it inhibited the growth of dinoflagellates and Synechococcus. Nevertheless, Al addition alone did not influence the chlorophyll a concentration of the entire phytoplankton assemblages. By adding nitrate and phosphate simultaneously, Al enrichment led to substantial increases in chlorophyll a concentration (especially that of the picophytoplankton<3μm), and cell abundances of diatom and photosynthetic picoeukaryotes. These results indicate varied responses of phytoplankton in different size fractions and taxonomic groups to Al enrichment. Further, by simultaneously adding different macronutrients and/or sufficient trace metals including iron, we found that the phytoplankton responses to Al enrichment were relevant to nutrients coexisting in the environment. Al enrichment may give some phytoplankton a competitive edge over using nutrients, especially the limited ones. The possible influences of Al on the competitors and grazers (predators) of some phytoplankton might indirectly contribute to the positive responses of the phytoplankton to Al enrichment. Our results indicate that Al may influence marine carbon cycle by impacting phytoplankton growth and structure in natural seawater., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Few studies focus on the effects of aluminum (Al) on marine nitrogen-fixing cyanobacteria, which play important roles in the ocean nitrogen cycling. To examine the effects of Al on the nitrogen-fixing cyanobacteria, bioassay experiments in the oligotrophic South China Sea (SCS) and culture of Crocosphaera watsonii in the laboratory were conducted. Field data showed that 200 nM Al stimulated the growth and the nitrogenase gene expression of Trichodesmium and unicellular diazotrophic cyanobacterium group A, and the nitrogen fixation rates of the whole community. Laboratory experiments demonstrated that Al stimulated the growth and nitrogen fixation of C. watsonii under phosphorus limited conditions. Both field and laboratory results indicated that Al could stimulate the growth of diazotrophs and nitrogen fixation in oligotrophic oceans such as the SCS, which is likely related to the utilization of phosphorus, implying that Al plays an important role in the ocean nitrogen and carbon cycles by influencing nitrogen fixation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Copper is an essential nutrient for symbiotic nitrogen fixation. This element is delivered by the host plant to the nodule, where membrane copper (Cu) transporter would introduce it into the cell to synthesize cupro-proteins. COPT family members in the model legume Medicago truncatula were identified and their expression determined. Yeast complementation assays, confocal microscopy and phenotypical characterization of a Tnt1 insertional mutant line were carried out in the nodule-specific M. truncatula COPT family member. Medicago truncatula genome encodes eight COPT transporters. MtCOPT1 (Medtr4g019870) is the only nodule-specific COPT gene. It is located in the plasma membrane of the differentiation, interzone and early fixation zones. Loss of MtCOPT1 function results in a Cu-mitigated reduction of biomass production when the plant obtains its nitrogen exclusively from symbiotic nitrogen fixation. Mutation of MtCOPT1 results in diminished nitrogenase activity in nodules, likely an indirect effect from the loss of a Cu-dependent function, such as cytochrome oxidase activity in copt1-1 bacteroids. These data are consistent with a model in which MtCOPT1 transports Cu from the apoplast into nodule cells to provide Cu for essential metabolic processes associated with symbiotic nitrogen fixation., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)