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Introduction: Plant growth-promoting bacteria (PGPB) have been primarily studied for atmospheric nitrogen (N) fixation but they also have the capacity to improve nutrition and yield of crop plants. Methods: Therefore, the objective of this research was to investigate the effects of inoculation with PGPB in association with different N rates on N uptake, grain yield, and oil concentration of dwarf castor beans in succession to legumes and grasses in Ilha Solteira, Brazil. The treatments consisted of N rates (0 to 180 kg ha-1 of N) and inoculation with three plant growth-promoting bacteria (Azospirillum brasiliense, Bacillus subtilis, and Pseudomonas fluorescens, applied by leaf) and a control with no-inoculation. Results: The grain and oil yields of castor beans were increased by 20 and 40% at a rate of 103 kg ha-1 of N in succession to grasses as compared to without N application. In addition, the grain yield of castor bean after legumes was increased by 28, 64, and 40% with estimated rates of 97, 113, and 92 kg ha-1 of N in combination with inoculations of A. brasilense, B. subtilis, and P. fluorescens as compared to without N application, respectively. Shoot, grain, and total N uptake were improved with foliar inoculation of A. brasilense, B. subtilis, and P. fluorescens at the N rates of 45, 90, and 135 kg ha-1, respectively. Discussion and conclusions: Topdressing of N at the rate of 103 kg ha-1 and foliar inoculation in succession to grasses and 180 kg ha-1 of N without the effect of foliar inoculation in succession to legumes are recommended for higher grain and oil yield of castor beans. Foliar inoculations with A. brasilense, B. subtilis, and P. fluorescens increased grain yield under reduced use of N fertilizer by 44, 37, and 49% in dwarf castor cultivation in succession to legumes, potentially contributing to sustainable agriculture.

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Arsenic (As) toxicity is a serious hazard to agricultural land due to growing industrialization, which has a negative effect on wheat crop yields. To address this issue, using seaweed extract and Azospirillum brasilense has emerged as an effective strategy for improving yield under stress conditions. However, the combined application of A. brasilense and seaweed extract in wheat crops under As toxicity has not been fully explored. The effectiveness of combining A. brasilense and seaweed extract in reducing As toxicity in wheat production was examined in this study through a 2-year pot experiment with nine treatments. These treatments included a control with no additives and two As concentrations (50 and 70 µM). At 50 and 70 µM, As was tested alone, with seaweed extract, with A. brasilense, and both. Significant results were achieved in reducing As toxicity in wheat crops. Arsenic at 70 µM proved more harmful than at 50 µM. The application of A. brasilense and seaweed extract was more effective in improving crop growth rates, chlorophyll levels, and stomatal conductance. The combined application notably decreased As concentration in wheat plants. It was concluded that applying A. brasilense and seaweed extract not only improves wheat growth but can also improve soil parameters under As toxicity conditions by increasing organic matter contents, boosting nutrient availability, and increasing the production of antioxidant enzymes.

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Seven auxin-producing endophytic bacterial strains (Azospirillum spp., Methylobacterium symbioticum, Bacillus spp.), and two different combinations of these strains were used to verify their influence on tomato during germination and development in hydroponic conditions where, as a novelty for Canestrino di Lucca cultivar, endophytic bacteria were inoculated. To emphasize the presence of bacterial auxins in roots and stems of seedlings, both in situ staining qualitative assessment and quantitative analysis were carried out. Moreover, hypogeal and epigeal growth of the plantlets were measured, and correlation analyses were conducted to examine the relationship between the amount of indolacetic acid (IAA) produced by the bacterial strains and root and stem parameters. Plantlets treated with microbial inoculants showed a significant increase in the survival rate compared to the control treatment. The best results as IAA producers were from Azospirillum baldaniorum Sp245 and A. brasilense Cd, which also induced significant root growth. On the other hand, Bacillus amyloliquefaciens and B. licheniformis induced the best rates in stem growth. These findings highlight the potential for using endophytic bacterial strains in a hydroponic co-cultivation system that enables inoculating plantlets, at an early stage of growth (5 days old).

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BACKGROUND: Azospirillum baldaniorum Sp245 produces poly-ß-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-ß-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release. METHODS: A study was conducted using in silico analysis of the two phbC genes of A. baldaniorum Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from Azospirillum baldaniorum (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV-Vis assay was performed. To confirm the presence of the poly-ß-hydroxybutyrate product, an NMR assay was performed. RESULTS: In silico analyses, it is revealed that AbPhbC1 and the PhbC2 protein from Azospirillum baldaniorum (AbPhbC2) retain the poly-ß-hydroxybutyrate polymerase and α/ß hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyß-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-ß-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV-Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-ß-hydroxybutyrate samples suggested their presence. CONCLUSION: In silico analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-ß-hydroxyalcanoate synthase. UV-Vis scanning and NMR spectroscopy revealed the synthesis of poly-ß-hydroxybutyrate by the A. baldaniorum enzyme AbPhbC1, indicating that the enzyme is functional.

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Microorganisms are known to be a promising source of biopigments because they are easy to obtain, can be produced on a commercial scale, and are environmentally friendly. Therefore, the aim of this work was to characterize a brown pigment (BP) produced by HM053 in NFbHPN-lactate medium. The BP was extracted from the pellet (BPP) or supernatant (BPS), in the presence (BPPTrp, BPSTrp) or absence (BPPw, BPSw) of tryptophan (Trp). The UV-vis results were similar among all BP samples and compared with commercial melanin used as a standard, and the maximum absorption was observed around 200-220 nm. FTIR spectra showed that BP and commercial melanin had slight differences, with a small band between 3000-2840 cm- 1, related to C-H in the CH2 and CH3 aliphatic groups, which is not observed in the commercial melanin. Between BPP and BPS showed a different structure with bands in the region 1230-1070 cm- 1 related to groups C-O. The thermogravimetric curves for BPSw and BPSTrp showed similar behavior, with 4 stages of mass loss. The similarity between BPPw and BPPTrp with 2 stages of mass loss was also observed. Scanning electron microscopy results showed morphological differences between BPP and BPS, where BPP had a physical structure more homogeneous and a regular flat surface, while the BPS physical structure did not seem homogeneous and the surface was uneven with some spherical structures as commercial melanin.

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Azospirillum brasilense is a non-photosynthetic α-Proteobacteria, belongs to the family of Rhodospirillaceae and produces carotenoids to protect itself from photooxidative stress. In this study, we have used Resonance Raman Spectra to show similarity of bacterioruberins of Halobacterium salinarum to that of A. brasilense Cd. To navigate the role of genes involved in carotenoid biosynthesis, we used mutational analysis to inactivate putative genes predicted to be involved in carotenoid biosynthesis in A. brasilense Cd. We have shown that HpnCED enzymes are involved in the biosynthesis of squalene (C30), which is required for the synthesis of carotenoids in A. brasilense Cd. We also found that CrtI and CrtP desaturases were involved in the transformation of colorless squalene into the pink-pigmented carotenoids. This study elucidates role of some genes which constitute very pivotal role in biosynthetic pathway of carotenoid in A. brasilense Cd.

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Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.

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BACKGROUND: Considering the challenges posed by nitrogen (N) pollution and its impact on food security and sustainability, it is crucial to develop management techniques that optimize N fertilization in croplands. Our research intended to explore the potential benefits of co-inoculation with Azospirillum brasilense and Bacillus subtilis combined with N application rates on corn plants. The study focused on evaluating corn photosynthesis-related parameters, oxidative stress assay, and physiological nutrient use parameters. Focus was placed on the eventual improved capacity of plants to recover N from applied fertilizers (AFR) and enhance N use efficiency (NUE) during photosynthesis. The two-year field trial involved four seed inoculation treatments (control, A. brasilense, B. subtilis, and A. brasilense + B. subtilis) and five N application rates (0 to 240 kg N ha-1, applied as side-dress). RESULTS: Our results suggested that the combined effects of microbial consortia and adequate N-application rates played a crucial role in N-recovery; enhanced NUE; increased N accumulation, leaf chlorophyll index (LCI), and shoot and root growth; consequently improving corn grain yield. The integration of inoculation and adequate N rates upregulated CO2 uptake and assimilation, transpiration, and water use efficiency, while downregulated oxidative stress. CONCLUSIONS: The results indicated that the optimum N application rate could be reduced from 240 to 175 kg N ha-1 while increasing corn yield by 5.2%. Furthermore, our findings suggest that replacing 240 by 175 kg N ha-1 of N fertilizer (-65 kg N ha-1) with microbial consortia would reduce CO2 emission by 682.5 kg CO2 -e ha-1. Excessive N application, mainly with the presence of beneficial bacteria, can disrupt N-balance in the plant, alter soil and bacteria levels, and ultimately affect plant growth and yield. Hence, highlighting the importance of adequate N management to maximize the benefits of inoculation in agriculture and to counteract N loss from agricultural systems intensification.

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BACKGROUND: Water stress is a major danger to crop yield, hence new approaches to strengthen plant resilience must be developed. To lessen the negative effects of water stress on wheat plants, present study was arranged to investigate the role of synergistic effects of biochar, trans-zeatin riboside (t-ZR), and Azospirillum brasilense on soil improvement and enzymatic activity in water-stressed wheat. RESULTS: In a three-replication experiment comprising of four treatments (T0: Control, T1: Drought stress (DS), T2: DS + t-ZR with biochar, T3: DS + A. brasilense with biochar), we observed notable improvements in soil quality and enzymatic activities in water-stressed wheat plants with the application of t-ZR and A. brasilense with biochar. In drought stress, Treatment having the application of A. brasilense with biochar performs best as compared to the other and significant increased the enzymatic activities such as peroxidase (7.36%), catalase (8.53%), superoxide dismutase (6.01%), polyphenol oxidase (14.14%), and amylase (16.36%) in wheat plants. Different enzymatic activities showed different trends of results. Soil organic C, dissolved organic C, dissolved organic N also enhanced 29.46%, 8.59%, 22.70% respectively with the application of A. brasilense with biochar under drought stress condition. CONCLUSIONS: The synergistic action of A. brasilense and biochar creates an effective microbiological environment that supports essential plant physiological processes during drought stress. This enhancement is attributed to improved soil fertility and increased organic matter content, highlighting the potential of these novel strategies in mitigating water stress effects and enhancing crop resilience.

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The utilization of a novel (systemic) biofertilizer containing Pseudomonas fluorescens, Azospirillum brasilense, and Bacillus subtilis and possessing the technology to facilitate the entry of bacteria through the stomata, was evaluated at three localities in Mexico (Potrero Nuevo, Veracruz; Ameca, Jalisco; and Champotón, Campeche) in two sugarcane varieties (NCO-310 and Mex 57-473) at different time scales. Inoculation of the systemic biofertilizer was imposed over the local agricultural management of the sugarcane; chemical fertilization of the experimental parcels at Potrero Nuevo was done using 70-20-20 and 120-80-80 at Ameca and Champotón. Three doses of the biofertilizer per hectare were applied during the annual productive cycle of sugarcane at each site; one year at Potrero Nuevo and Champotón; and six years at Ameca. The annual sugarcane yield was evaluated at each site. Additionally, sugar quality (°Brix or sucrose content) was evaluated at the three localities, while different variables of stalk performance were also measured at Ameca and Champotón. Our data provide evidence that this systemic biofertilizer consistently and reliably increased the sugarcane yield at all localities during the time of evaluation, ranging from 73.7 tons ha-1 at Potrero Nuevo (2.5 times increase; P < 0.05) and 77.7 tons ha-1 at Ameca (1.9 times increase; P < 0.05) to 23.8 tons ha-1 at Champotón (1.4 times increase; P < 0.05). This increase in sugarcane biomass was related to increased tillering rather than increased stalk height or diameter. This novel biological product improved the sugarcane quality in terms of °Brix (P < 0.05, 2.6° difference) and sucrose content (P < 0.5, 0.7% difference).

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Edaphic factors can modulate the effects of microbial inoculants on crop yield promotion. Given the potential complexity of microbial inoculant responses to diverse soil management practices, we hypothesize that sustainable management of soil and water irrigation may improve soil quality and enhance the effects of plant growth-promoting bacteria (PGPB). Consequently, the primary objective was to assess the effectiveness of microbial inoculants formulated with Herbaspirillum seropedicae (Hs) and Azospirillum brasilense (Ab) on maize growth in soils impacted by different historical conservation management systems. We evaluated two soil management systems, two irrigation conditions, and four treatments: T0 - without bioinoculant and 100% doses of NPK fertilization; T1 - Hs + humic substances and 40% of NPK fertilization; T2 - Ab and 40% of NPK fertilization; T3 - co-inoculation (Hs + Ab) and 40% of NPK fertilization. Using a reduced fertilization dose (40% NPK) associated with microbial inoculants proved efficient in increasing maize shoot dry mass : on average, there was a 16% reduction compared to the treatment with 100% fertilization. In co-inoculation (Hs + Ab), the microbial inoculants showed a mutualistic effect on plant response, higher than isolate ones, especially increasing the nitrogen content in no-tillage systems irrigated by swine wastewater. Under lower nutrient availability and higher biological soil quality, the microbial bioinputs positively influenced root development, instantaneous water use efficiency, stomatal conductance, and nitrogen contents.

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Azospirillum brasilense Sp7 produces PHB, which is covered by granule-associated proteins (GAPs). Phasins are the main GAPs. Previous studies have shown phasins can regulate PHB synthesis. When A. brasilense grows under stress conditions, it uses sigma factors to transcribe genes for survival. One of these factors is the σ24 factor. This study determined the possible interaction between phasins and the σ24 factor or phasin-σ24 factor complex and DNA. Three-dimensional structures of phasins and σ24 factor structures were predicted using the I-TASSER and SWISS-Model servers, respectively. Subsequently, a molecular docking between phasins and the σ24 factor was performed using the ClusPro 2.0 server, followed by molecular docking between protein complexes and DNA using the HDOCK server. Evaluation of the types of ligand-receptor interactions was performed using the BIOVIA Discovery Visualizer for three-dimensional diagrams, as well as the LigPlot server to obtain bi-dimensional diagrams. The results showed the phasins (Pha4Abs7 or Pha5Abs7)-σ24 factor complex was bound near the -35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5Abs7 and the σ24 factor, with DNA, both proteins were bound to the -35 box. This did not occur with PhaP4Abs7, which was bound to the -10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

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Diazotrophic bacteria of the genus Azospirillum are known widely, because they are ubiquitous in the rhizosphere and can promote the growth and performance of nonlegume plants. Recently, more Azospirillum species have been isolated from sources other than plants or soil. We report the structures of the O polysaccharides (OPSs) from the lipopolysaccharides of the type strains A. thiophilum BV-ST (1) and A. griseum L-25-5w-1T (2), isolated from aquatic environments. Both structures have a common tetrarhamnan in the repeating-unit, which is decorated with a side xylose in the OPS of A. thiophilum BV-ST.

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This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

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Azospirillum sp. is a plant growth-promoting rhizobacteria largely recognized for its potential to increase the yield of different important crops. In this work, we present a thorough genomic and phenotypic analysis of A. argentinense Az39T to provide new insights into the beneficial mechanisms of this microorganism. Phenotypic analyses revealed the following in vitro abilities: growth at 20-38 °C (optimum, 28 °C), pH 6.0-8.0 (optimum, pH 6.8), and in the presence of 1% (w/v) NaCl; production of variable amounts of PHB as intracellular granules; nitrogen fixation under microaerophilic conditions; IAA synthesis in the presence of L-tryptophan. Through biochemical (API 20NE) and carbon utilization profiling (Biolog) assays, we proved that A. argentinense Az39T is able to use 15 substrates and metabolize 19 different carbon substrates. Lipid composition indicated a predominance of medium and long-chain saturated fatty acids. A total of 6 replicons classified as one main chromosome, three chromids, and two plasmids, according to their tRNA and core essential genes contents, were identified. Az39T genome includes genes associated with multiple plant growth-promoting (PGP) traits such as nitrogen fixation and production of auxins, cytokinin, abscisic acid, ethylene, and polyamines. In addition, Az39T genome harbor genetic elements associated with physiological features that facilitate its survival in the soil and competence for rhizospheric colonization; this includes motility, secretion system, and quorum sensing genetic determinants. A metadata analysis of Az39T agronomic performance in the pampas region, Argentina, demonstrated significant grain yield increases in wheat and maize, proving its potential to provide better growth conditions for dryland cereals. In conclusion, our data provide a detailed insight into the metabolic profile of A. argentinense Az39T, the strain most widely used to formulate non-legume inoculants in Argentina, and allow a better understanding of the mechanisms behind its field performance.

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BACKGROUND: The plant growth-promoting bacteria (PGPB) Azospirillum brasilense is widely used as an inoculant for important grass crops, providing numerous benefits to the plants. However, one limitation to develop viable commercial inoculants is the control of PGPB survival, requiring strategies that guarantee their survival during handling and field application. The application of sublethal stress appears to be a promising strategy to increase bacterial cells tolerance to adverse environmental conditions since previous stress induces the activation of physiological protection in bacterial cell. In this work, we evaluated the effects of thermal and salt stresses on the survival of inoculant containing A. brasilense Ab-V5 and Ab-V6 strains and we monitored A. brasilense viability in inoculated maize roots after stress treatment of inoculant. RESULTS: Thermal stress application (> 35 °C) in isolated cultures for both strains, as well as salt stress [sodium chloride (NaCl) concentrations > 0.3 mol L-1], resulted in growth rate decline. The A. brasilense enumeration in maize roots obtained by propidium monoazide quantitative polymerase chain reaction (PMA-qPCR), for inoculated maize seedlings grown in vitro for 7 days, showed that there is an increased number of viable cells after the salt stress treatment, indicating that A. brasilense Ab-V5 and Ab-V6 strains are able to adapt to salt stress (0.3 mol L-1 NaCl) growth conditions. CONCLUSION: Azospirillum brasilense Ab-V5 and Ab-V6 strains had potential for osmoadaptation and salt stress, resulting in increased cell survival after inoculation in maize plants. © 2024 Society of Chemical Industry.

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MicroRNA (miRNA) is a class of non-coding RNAs. They play essential roles in plants' physiology, as in the regulation of plant development, response to biotic and abiotic stresses, and symbiotic processes. This work aimed to better understand the importance of maize's miRNA during Azospirillum-plant interaction when the plant indole-3-acetic acid (IAA) production was inhibited with yucasin, an inhibitor of the TAM/YUC pathway. Twelve cDNA libraries from a previous Dual RNA-Seq experiment were used to analyze gene expression using a combined analysis approach. miRNA coding genes (miR) and their predicted mRNA targets were identified among the differentially expressed genes. Statistical differences among the groups indicate that Azospirillum brasilense, yucasin, IAA concentration, or all together could influence the expression of several maize's miRNAs. The miRNA's probable targets were identified, and some of them were observed to be differentially expressed. Dcl4, myb122, myb22, and morf3 mRNAs were probably regulated by their respective miRNAs. Other probable targets were observed responding to the IAA level, the bacterium, or all of them. A. brasilense was able to influence the expression of some maize's miRNA, for example, miR159f, miR164a, miR169j, miR396c, and miR399c. The results allow us to conclude that the bacterium can influence directly or indirectly the expression of some of the identified mRNA targets, probably due to an IAA-independent pathway, and that they are somehow involved in the previously observed physiological effects.

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A Gram-negative and rod-shaped bacterium, designated C340-1T, was isolated and screened from paddy soil in Zhongshan County, Guangxi Province, PR China. This strain grew at 20-42 °C (optimum, 37 °C), pH 5.0-9.0 (optimum, pH 7.0) and 0-4 % (w/v) NaCl (optimum, 0-1 %) on Reasoner's 2A medium. The strain could fix atmospheric nitrogen and acetylene reduction activity was recorded up to 120.26 nmol ethylene h-1 (mg protein)-1. Q-10 was the only isoprenoid quinone component; phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and an unidentified polar lipid were the major polar lipids. Summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) were the primary cellular fatty acids. The genome of strain C340-1T was 6.18 Mb, and the G+C content was 69.0 mol%. Phylogenetic tree analysis based on 16S rRNA gene and 92 core genes showed that strain C340-1T was closely related to and clustered with the type strains Azospirillum brasilense JCM 1224T, Azospirillum argentinense Az39T, Azospirillum baldaniorum Sp245T and Azospirillum formosense JCM 17639T. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values between strain C340-1T and the closely related type strains mentioned above were significantly lower than the threshold values for species classification (95-96 %, 95-96 % and 70 %, respectively). Based on phylogenetic, genomic, phenotypic, physiological and biochemical data, we have reason to believe that C340-1T represents a new species of the genus Azospirillum, for which the name Azospirillum isscasi sp. nov. is proposed. The type strain is C340-1T(=CCTCC AB 2023105T=KCTC 8126T).

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The enhancing effect of N2-fixing bacterial strains in the presence of mineral N doses on maize plants in pots and field trials was investigated. The OT-H1 of 10 isolates maintained the total nitrogen, nitrogenase activities, IAA production, and detection of NH3 in their cultures. In addition, they highly promoted the germination of maize grains in plastic bags compared to the remainder. Therefore, OT-H1 was subjected for identification and selected for further tests. Based on their morphological, cultural, and biochemical traits, they belonged to the genera Azotobacter. The genomic sequences of 16S rRNA were, thus, used to confirm the identification of the genera. Accordingly, the indexes of tree and similarity for the related bacterial species indicated that genera were exactly closely linked to Azotoacter salinestris strain OR512393. In pot (35 days) and field (120 days) trials, the efficiencies of both A. salinestris and Azospirillum oryzea SWERI 111 (sole/dual) with 100, 75, 50, and 25% mineral N doses were evaluated with completely randomized experimental design and three repetitions. Results indicated that N2-fixing bacteria in the presence of mineral N treatment showed pronounced effects compared to controls. A high value of maize plants was also noticed through increasing the concentration of mineral N and peaked at a dose of 100%. Differences among N2-fixing bacteria were insignificant and were observed for A. oryzea with different mineral N doses. Thus, the utilization of A. oryzea and A. salinestris in their dual mix in the presence of 75 followed by 50% mineral N was found to be the superior treatments, causing the enhancement of vegetative growth and grain yield parameters of maize plants. Additionally, proline and the enzyme activities of both polyphenol oxidase (PPO) and peroxidase (PO) of maize leaves were induced, and high protein contents of maize grains were accumulated due to the superior treatments. The utilization of such N2-fixing bacteria was, therefore, found to be effective at improving soil fertility and to be an environmentally safe strategy instead, or at least with low doses, of chemical fertilizers.

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Azospirillum alphaproteobacteria, which live in the rhizosphere of many crops, are used widely as biofertilizers. Two-component signal transduction systems (TCSs) mediate the bacterial perception of signals and the corresponding adjustment of behavior facilitating the adaptation of bacteria to their habitats. In this study, we obtained the A. baldaniorum Sp245 mutant for the AZOBR_150176 gene, which encodes the TCS of the hybrid histidine kinase/response sensory regulator (HSHK-RR). Inactivation of this gene affected bacterial morphology and motility. In mutant Sp245-HSHKΔRR-Km, the cells were still able to synthesize a functioning polar flagellum (Fla), were shorter than those of strain Sp245, and were impaired in aerotaxis, elaboration of inducible lateral flagella (Laf), and motility in semiliquid media. The mutant showed decreased transcription of the genes encoding the proteins of the secretion apparatus, which ensures the assembly of Laf, Laf flagellin, and the repressor protein of translation of the Laf flagellin's mRNA. The study examined the effects of polyethylene glycol 6000 (PEG 6000), an agent used to simulate osmotic stress and drought conditions. Under osmotic stress, the mutant was no longer able to use collective motility in semiliquid media but formed more biofilm biomass than did strain Sp245. Introduction into mutant cells of the AZOBR_150176 gene as part of an expression vector led to recovery of the lost traits, including those mediating bacterial motility under mechanical stress induced by increased medium density. The results suggest that the HSHK-RR under study modulates the response of A. baldaniorum Sp245 to mechanical and osmotic/water stress.

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