Maximizing growth and productivity of onion (Allium cepa L.) by Spirulina platensis extract and nitrogen-fixing endophyte Pseudomonas stutzeri.

The study focuses on the impact of foliar spraying cyanobacterium Spirulina platensis extract and the inoculation with the endophyte N2-fixing Pseudomonas stutzeri, and their mixture in the presence of different nitrogen doses on growth and yield of onion under field conditions. Bioactive compounds of Spirulina and Pseudomonas were analyzed by GC-MC and amino acid production of Spirulina by the amino acid analyzer. Hydrogen cyanide (HCN), indole acetic acid (IAA), ammonia (NH3), pectinase activity, and N2-fixation of Pseudomonas were measured. Plant height (cm), leaf length (cm), number of green leaves, bulb diameter (cm), fresh and dry weight of plant (g), chlorophyll a, b of leaves, bulb weight (g), marketable bulb yield (t. ha-1), cull bulb weight (t. ha-1), total bulb yield (t. ha-1), bulb diameter (cm), total soluble solids (TSS%), dry matter content (DM%), evaluation of storage behavior, and economic feasibility were estimated. Spirulina extract has several bioactive compounds. Pseudomonas can produce HCN, NH3, IAA, pectinase, and nitrogen fixation. The application of mixture with recommended dose of nitrogen increases the onion plant parameters, marketable yield, total bulb yield, bulb weight, bulb diameter, TSS%, DM%, net return, benefit-cost ratio (B:C), lowest cumulative weight loss% of bulbs during storage, and reduce culls weight compared with other treatments in two seasons. Application of S. platensis extract and inoculation with endophyte nitrogen-fixing P. stutzeri enhance the growth and productivity of the onion under different doses of nitrogen fertilizer.

Anthracene and Pyrene Biodegradation Performance of Marine Sponge Symbiont Bacteria Consortium.

Every petroleum-processing plant produces sewage sludge containing several types of polycyclic aromatic hydrocarbons (PAHs). The degradation of PAHs via physical, biological, and chemical methods is not yet efficient. Among biological methods, the use of marine sponge symbiont bacteria is considered an alternative and promising approach in the degradation of and reduction in PAHs. This study aimed to explore the potential performance of a consortium of sponge symbiont bacteria in degrading anthracene and pyrene. Three bacterial species (Bacillus pumilus strain GLB197, Pseudomonas stutzeri strain SLG510A3-8, and Acinetobacter calcoaceticus strain SLCDA 976) were mixed to form the consortium. The interaction between the bacterial consortium suspension and PAH components was measured at 5 day intervals for 25 days. The biodegradation performance of bacteria on PAH samples was determined on the basis of five biodegradation parameters. The analysis results showed a decrease in the concentration of anthracene (21.89%) and pyrene (7.71%), equivalent to a ratio of 3:1, followed by a decrease in the abundance of anthracene (60.30%) and pyrene (27.52%), equivalent to a ratio of 2:1. The level of pyrene degradation was lower than that of the anthracene due to fact that pyrene is more toxic and has a more stable molecular structure, which hinders its metabolism by bacterial cells. The products from the biodegradation of the two PAHs are alcohols, aldehydes, carboxylic acids, and a small proportion of aromatic hydrocarbon components.

Pseudomonas stutzeri MJL19, a rhizosphere-colonizing bacterium that promotes plant growth under saline stress.

AIMS: The aim of this study was to find and use rhizobacteria able to confer plants advantages to deal with saline conditions. METHODS AND RESULTS: We isolated 24 different bacterial species from the rhizosphere of halophyte plants growing in Santiago del Estero, Argentina salt flat. Four strains were selected upon their ability to grow in salinity and their biochemical traits associated with plant growth promotion. Next, we tested the adhesion on soybean seeds surface and root colonization with the four selected isolates. Isolate 19 stood out from the rest and was selected for further experiments. This strain showed positive chemotaxis towards soybean root exudates and a remarkable ability to form biofilm both in vitro conditions and on soybean roots. Interestingly, this trait was enhanced in high saline conditions, indicating the extremely adapted nature of the bacterium to high salinity. In addition, this strain positively impacted on seed germination, plant growth and general plant health status also under saline stress. CONCLUSIONS: A bacterium isolate with outstanding ability to promote seed germination and plant growth under saline conditions was found. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental approach allowed us to find a suitable bacterial candidate for a biofertilizer intended to alleviate saline stress on crops. This would allow the use of soil now considered inadequate for agriculture and thus prevent further advancement of agriculture frontiers into areas of environmental value.

Hi-tech restoration by two-steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy).

AIMS: In this work, the 'hi-tech' complex biocleaning and restoration of the 14th-century fresco Triumph of Death (5·6 × 15·0 m) at the Camposanto Monumental Cemetery (Pisa, Italy) is reported. Since 2000, the restoration based on the biological cleaning of noble medieval frescoes, has been successfully utilized in this site. METHODS AND RESULTS: The novelty of this study is the two-steps biocleaning process using Pseudomonas stutzeri A29 viable cells, previously applied for recovering other valuable frescoes. In this case, after the fresco detachment from the asbestos-cement support (eternity), both the animal glue and the residues of calcium caseinate were biologically removed respectively from the front and from the back of the fresco in 3 h as indicated by GC-MS and PY/GC-MS analyses. The data obtained during the monitoring of the biorestoration process confirmed that the adopted procedure does not leave residual cells on the fresco surfaces as showed by plate count method, ATP determination and also SEM observation. In addition, to avoid the risk of condensation phenomena after the relocation of the restored fresco sections onto the original walls, the use of a new support has been set up together with the design of a control system that allows a continuous monitoring of environmental parameters for prevention and conservation purposes. CONCLUSIONS: This large-scale biorestoration work clearly shows and confirms that this biotechnology is highly efficient, safe, noninvasive, risk-free and very competitive compared to the traditional cleaning methods, offering an unusual 'resurrection' of the degraded artworks also in very complicated and delicate conditions such as the Triumph of Death fresco, defined for its dimension and artistic importance the 'Pisa's Sistina frescoes'. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings can be of significant importance for other future new restoration activities and they are crucial for determining preservation strategies in this field.

The plant growth-promoting effect of the nitrogen-fixing endophyte Pseudomonas stutzeri A15.

The use of plant growth-promoting rhizobacteria as a sustainable alternative for chemical nitrogen fertilizers has been explored for many economically important crops. For one such strain isolated from rice rhizosphere and endosphere, nitrogen-fixing Pseudomonas stutzeri A15, unequivocal evidence of the plant growth-promoting effect and the potential contribution of biological nitrogen fixation (BNF) is still lacking. In this study, we investigated the effect of P. stutzeri A15 inoculation on the growth of rice seedlings in greenhouse conditions. P. stutzeri A15 induced significant growth promotion compared to uninoculated rice seedlings. Furthermore, inoculation with strain A15 performed significantly better than chemical nitrogen fertilization, clearly pointing to the potential of this bacterium as biofertilizer. To assess the contribution of BNF to the plant growth-promoting effect, rice seedlings were also inoculated with a nitrogen fixation-deficient mutant. Our results suggest that BNF (at best) only partially contributes to the stimulation of plant growth.

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri.

UNLABELLED: Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu(2+) and Zn(2+) resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu(2+) and Zn(2+) resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu(2+) and Zn(2+) resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu(2+) resistance systems do not show decreased fitness relative to the wild type when exposed to Cu(2+) In addition, new genes are identified that have no known connection to Zn(2+) (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu(2+) resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu(2+)- and Zn(2+)-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu(2+) and Zn(2+) efflux and resistance and their effects on denitrifying metabolism. IMPORTANCE: In this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca(2+)/H(+) antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg(2+) transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.

[Plasmids of Salt-tolerant Rhizosphere Microorganisms].

Five plasmid DNAs were detected in three of microorganisms, which formed microbial composition «Ð—амин-М¼. One plasmid (23.1 kb) was found in Bacillus megaterium СКБ-310 cells and another one (55.0 kb) was found in cells of Pseudomonas stutzeri СКБ-308, but cells of Bacillus subtilis СКБ-309 contained 3 plasmids (48.5 kb, 30.0 kb and 13.3 kb). It was assumed that these plasmids may carry genes of resistance to adverse environmental conditions, including the high content (10 %) of ions Cl- and SO4(2-).

Isolation and characterization of Pseudomonas stutzeri and its potential application in biological nitrification.

Ammonia-oxidizing bacteria (AOB) were isolated from sediment samples of fishponds with an aim to use them for application in biological nitrification of water. Isolation of AOB was done in an inorganic medium and nitrite-producing bacterial isolates were selected. These isolates were further screened by polymerase chain reaction using specific primers forAOB. Out of 119 nitrate positive isolates, only 12 showed positive amplification and yielded a PCR product of ~465 bp. Treatment of aquaculture pond and riverwaterwith one of the bacterial isolate (HC-5) resulted in lowering of soluble ammonia level from 3.50 to 0.05 mgl(-1) and 7.5 to 0.01 mgl(-1), respectively. Partial 16S rRNA gene sequencing of isolate HC-5 identified the microorganism as Pseudomonasstutzeri.

Tolerance of an aerobic denitrifier (Pseudomonas stutzeri) to high O2 concentrations.

An aerobic denitrifier was isolated from activated sludge and the isolate possessed an average removal rate of 5.7 mg NO3 (-)-N l(-1) h(-1) without accumulation of NO2 (-)-N (less than 2.1 mg l(-1)). The average removal efficiency of nitrate was 93.7 % in 24 h, when the dissolved oxygen (DO) concentrations ranged from 3.2 to 17.5 mg l(-1). The activity of both nap (periplasmic nitrate reductase) and nir (nitrite reductase), whose corresponding genes (napA and nirS) were amplified by touchdown PCR, could be responsible for the tolerance of DO concentrations. Other three genes relating to narG, norB and nosZ were noted to involve in isolate strain.

Immigrant Pantoea agglomerans embedded within indigenous microbial aggregates: a novel spatial distribution of epiphytic bacteria.

Immigrant bacteria located on leaf surfaces are important to the health of plants as well as to people who consume fresh fruits and vegetables. However, the spatial distribution and organization of these immigrant bacteria on leaf surfaces are still poorly understood. To examine the spatial organization of these strains, two bacterial strains on tobacco leaves: (1) an indigenous strain, Pseudomonas stutzeri Nov. Y2011 labeled with green fluorescent protein, and (2) an immigrant strain Pantoea agglomerans labeled with cyan fluorescent protein isolated from pear, were studied. Under moist conditions, P. agglomerans cells quickly disappeared from direct observation by laser-scanning confocal microscopy, although elution results indicated that large amounts of live cells were still present on the leaves. Following exposure to desiccation stress, particles of cyan fluorescent protein-labeled P. agglomerans were visible within cracked aggregates of P. stutzeri Nov. Y2011. Detailed observation of sectioned aggregates showed that colonies of immigrant P. agglomerans were embedded within aggregates of P. stutzeri Nov. Y2011. Furthermore, carbon-resource partitioning studies suggested that these two species could coexist without significant nutritional competition. This is the first observation of an immigrant bacterium embedding within aggregates of indigenous bacteria on leaves to evade harsh conditions in the phyllosphere.

Genome sequence of a highly efficient aerobic denitrifying bacterium, Pseudomonas stutzeri T13.

Pseudomonas stutzeri T13 is a highly efficient aerobic denitrifying bacterium. Information about the genome of this aerobic denitrifying bacterium has been limited until now. We present the draft genome of P. stutzeri T13. The results could provide further insight into the aerobic denitrification mechanism in strain T13.

Adaptation mechanisms of bacteria during the degradation of polychlorinated biphenyls in the presence of natural and synthetic terpenes as potential degradation inducers.

In this study, we examined the effect of polychlorinated biphenyls (PCBs) in the presence of natural and synthetic terpenes and biphenyl on biomass production, lipid accumulation, and membrane adaptation mechanisms of two PCB-degrading bacterial strains Pseudomonas stutzeri and Burkholderia xenovorans LB400. According to the results obtained, it could be concluded that natural terpenes, mainly those contained in ivy leaves and pine needles, decreased adaptation responses induced by PCBs in these strains. The adaptation processes under investigation included growth inhibition, lipid accumulation, composition of fatty acids, cis/trans isomerization, and membrane saturation. Growth inhibition effect decreased upon addition of these natural compounds to the medium. The amount of unsaturated fatty acids that can lead to elevated membrane fluidity increased in both strains after the addition of the two natural terpene sources. The cells adaptation changes were more prominent in the presence of carvone, limonene, and biphenyl than in the presence of natural terpenes, as indicated by growth inhibition, lipid accumulation, and cis/trans isomerization. Addition of biphenyl and carvone simultaneously with PCBs increased the trans/cis ratio of fatty acids in membrane fractions probably as a result of fluidizing effects of PCBs. This stimulation is more pronounced in the presence of PCBs as a sole carbon source. This suggests that PCBs alone have a stronger effect on bacterial membrane adaptation mechanisms than when added together with biphenyl or natural or synthetic terpenes.

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.

Type IV Pilus Shapes a 'Bubble-Burst' Pattern Opposing Spatial Intermixing of Two Interacting Bacterial Populations.

Microbes are social organisms that commonly live in sessile biofilms. Spatial patterns of populations within biofilms can be important determinants of community-level properties. Spatial intermixing emerging from microbial interaction is one of the best-studied characteristics of spatial patterns. The specific levels of spatial intermixing critically contribute to how the dynamics and functioning of such communities are governed. However, the precise factors that determine spatial patterns and intermixing remain unclear. Here, we investigated the spatial patterning and intermixing of an engineered synthetic consortium composed of two mutualistic Pseudomonas stutzeri strains that degrade salicylate via metabolic cross-feeding. We found that the consortium self-organizes across space to form a previously unreported spatial pattern (here referred to as a 'bubble-burst' pattern) that exhibits a low level of intermixing. Interestingly, when the genes encoding type IV pili were deleted from both strains, a highly intermixed spatial pattern developed and increased the productivity of the entire community. The intermixed pattern was maintained in a robust manner across a wide range of initial ratios between the two strains. Our findings show that the type IV pilus plays a role in mitigating spatial intermixing of different populations in surface-attached microbial communities, with consequences for governing community-level properties. These insights provide tangible clues for the engineering of synthetic microbial systems that perform highly in spatially structured environments. IMPORTANCE When growing on surfaces, multispecies microbial communities form biofilms that exhibit intriguing spatial patterns. These patterns can significantly affect the overall properties of the community, enabling otherwise impermissible metabolic functions to occur as well as driving the evolutionary and ecological processes acting on communities. The development of these patterns is affected by several drivers, including cell-cell interactions, nutrient levels, density of founding cells, and surface properties. The type IV pilus is commonly found to mediate surface-associated behaviors of microorganisms, but its role on pattern formation within microbial communities is unclear. Here, we report that in a cross-feeding consortium, the type IV pilus affects the spatial intermixing of interacting populations involved in pattern formation and ultimately influences overall community productivity and robustness. This novel insight assists our understanding of the ecological processes of surface-attached microbial communities and suggests a potential strategy for engineering high-performance synthetic microbial communities.

Complete genome sequence of the type strain Pseudomonas stutzeri CGMCC 1.1803.

Here we report the complete genome sequence of Pseudomonas stutzeri strain CGMCC 1.1803 (equivalent to ATCC 17588), the type strain of P. stutzeri, which encodes 4,138 open reading frames on a 4,547,930-bp circular chromosome. The CGMCC 1.1803 genome contains genes involved in denitrification, benzoate/catechol degradation, chemotaxis, and other functions.

Differential effects of dissolved organic carbon upon re-entrainment and surface properties of groundwater bacteria and bacteria-sized microspheres during transport through a contaminated, sandy aquifer.

Injection-and-recovery studies involving a contaminated, sandy aquifer (Cape Cod, Massachusetts) were conducted to assess the relative susceptibility for in situ re-entrainment of attached groundwater bacteria (Pseudomonas stuzeri ML2, and uncultured, native bacteria) and carboxylate-modified microspheres (0.2 and 1.0 µm diameters). Different patterns of re-entrainment were evident for the two colloids in response to subsequent injections of groundwater (hydrodynamic perturbation), deionized water (ionic strength alteration), 77 µM linear alkylbenzene sulfonates (LAS, anionic surfactant), and 76 µM Tween 80 (polyoxyethylene sorbitan monooleate, a very hydrophobic nonionic surfactant). An injection of deionized water was more effective in causing detachment of micrsopheres than were either of the surfactants, consistent with the more electrostatic nature of microsphere's attachment, their extreme hydrophilicity (hydrophilicity index, HI, of 0.99), and negative charge (zeta potentials, ζ, of -44 to -49 mv). In contrast, Tween 80 was considerably more effective in re-entraining the more-hydrophobic native bacteria. Both the hydrophilicities and zeta potentials of the native bacteria were highly sensitive to and linearly correlated with levels of groundwater dissolved organic carbon (DOC), which varied modestly from 0.6 to 1.3 mg L(-1). The most hydrophilic (0.52 HI) and negatively charged (ζ -38.1 mv) indigenous bacteria were associated with the lowest DOC. FTIR spectra indicated the latter community had the highest average density of surface carboxyl groups. In contrast, differences in groundwater (DOC) had no measurable effect on hydrophilicity of the bacteria-sized microspheres and only a minor effect on their ζ. These findings suggest that microspheres may not be very good surrogates for bacteria in field-scale transport studies and that adaptive (biological) changes in bacterial surface characteristics may need to be considered where there is longer-term exposure to contaminant DOC.

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

Biology of Pseudomonas stutzeri.

Pseudomonas stutzeri is a nonfluorescent denitrifying bacterium widely distributed in the environment, and it has also been isolated as an opportunistic pathogen from humans. Over the past 15 years, much progress has been made in elucidating the taxonomy of this diverse taxonomical group, demonstrating the clonality of its populations. The species has received much attention because of its particular metabolic properties: it has been proposed as a model organism for denitrification studies; many strains have natural transformation properties, making it relevant for study of the transfer of genes in the environment; several strains are able to fix dinitrogen; and others participate in the degradation of pollutants or interact with toxic metals. This review considers the history of the discovery, nomenclatural changes, and early studies, together with the relevant biological and ecological properties, of P. stutzeri.

Spatial organization in microbial range expansion emerges from trophic dependencies and successful lineages.

Evidence suggests that bacterial community spatial organization affects their ecological function, yet details of the mechanisms that promote spatial patterns remain difficult to resolve experimentally. In contrast to bacterial communities in liquid cultures, surface-attached range expansion fosters genetic segregation of the growing population with preferential access to nutrients and reduced mechanical restrictions for cells at the expanding periphery. Here we elucidate how localized conditions in cross-feeding bacterial communities shape community spatial organization. We combine experiments with an individual based mathematical model to resolve how trophic dependencies affect localized growth rates and nucleate successful cell lineages. The model tracks individual cell lineages and attributes these with trophic dependencies that promote counterintuitive reproductive advantages and result in lasting influences on the community structure, and potentially, on its functioning. We examine persistence of lucky lineages in structured habitats where expansion is interrupted by physical obstacles to gain insights into patterns in porous domains.

Chlorpyrifos inhibits nitrogen fixation in rice-vegetated soil containing Pseudomonas stutzeri A1501.

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.