Amelioration of phytotoxic impact of biosynthesized zinc oxide nanoparticles: Plant growth promoting rhizobacteria facilitates the growth and biochemical responses of Eggplant (Solanum melongena) under nanoparticles stress.

The consistently increasing use of zinc oxide nanoparticles (ZnONPs) in crop optimization practices and their persistence in agro-environment necessitate expounding their influence on sustainable agro-environment. Attempts have been made to understand nanoparticle-plant beneficial bacteria (PBB)- plant interactions; the knowledge of toxic impact of nanomaterials on soil-PBB-vegetable systems and alleviating nanotoxicity using PBB is scarce and inconsistent. This study aims at bio-fabrication of ZnONPs from Rosa indica petal extracts and investigates the impact of PBB on growth and biochemical responses of biofertilized eggplants exposed to phyto-synthesized nano-ZnO. Microscopic and spectroscopic techniques revealed nanostructure, triangular shape, size 32.5 nm, and different functional groups of ZnONPs and petal extracts. Inoculation of Pseudomonas fluorescens and Azotobacter chroococcum improved germination efficiency by 22% and 18% and vegetative growth of eggplants by 14% and 15% under NPs stress. Bio-inoculation enhanced total chlorophyll content by 36% and 14 %, increasing further with higher ZnONP concentrations. Superoxide dismutase and catalase activity in nano-ZnO and P. fluorescens inoculated eggplant shoots reduced by 15-23% and 9-11%. Moreover, in situ experiment unveiled distortion and accumulation of NPs in roots revealed by scanning electron microscope and confocal laser microscope. The present study highlights the phytotoxicity of biosynthesized ZnONPs to eggplants and demonstrates that PBB improved agronomic traits of eggplants while declining phytochemicals and antioxidant levels. These findings suggest that P. fluorescens and A. chroococcum, with NPs ameliorative activity, can be cost-effective and environment-friendly strategy for alleviating NPs toxicity and promoting eggplant production under abiotic stress, fulfilling vegetable demands.

Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.

The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log10 CFU/ml and 12.63 ± 0.04 Log10 CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log10 CFU/ml) after 72 h of incubation. RB51 and WB52liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.

Structural basis for the minimal bifunctional alginate epimerase AlgE3 from Azotobacter chroococcum.

Among the epimerases specific to alginate, some of them in Azotobacter genera convert ß-d-mannuronic acid to α-l-guluronic acid but also have lyase activity to degrade alginate. The remarkable characteristics of these epimerases make it a promising enzyme for tailoring alginates to meet specific demands. Here, we determined the structure of the bifunctional mannuronan C-5 epimerase AlgE3 from Azotobacter chroococcum (AcAlgE3) in complex with several mannuronic acid oligomers as well as in apo form, which allowed us to elucidate the binding manner of each mannuronic acid oligomer, and the structural plasticity, which is dependent on calcium ions. Moreover, a comprehensive analysis of the lyase activity profiles of AcAlgE3 combined with structural characteristics explained the preference for different chain length oligomers.

Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties.

Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient availability. The high content of CaCO3 leads to the increment of the soil alkalinity, which results in a lowering of the nutrient availability causing a challenge for the agriculture in these soils. In this study, the calcite-solubilizing potential of the diazotrophic Azotobacter salinestris YRNF3 was investigated in vitro as a probable bio-agent for enhancing the calcareous soils properties such as soil pH and nutrient availability. Twelve diazotrophic bacterial strains were isolated from wheat rhizosphere collected from different wheat-cultivated fields in five Egyptian governorates. Using Nessler's reagent, all isolated bacterial strains were found to have the ability to produce ammonia. By amplification of nifH gene, a PCR product of 450 bp was obtained for all isolated bacterial strains. For each isolate, three biological and three technical replicates were applied. All isolated diazotrophic bacteria were qualitatively screened for their calcite-solubilizing ability. To quantitatively investigate the calcite-solubilizing potential of A. salinestris YRNF3 in vitro, changes in the contents of soluble calcium (Ca2+), bicarbonate (HCO3-), total nitrogen (TN), total protein (TP), and pH were daily measured in its culture filtrate along 10 days of incubation. The results showed that the pH values in the culture filtrate ranged from 5.73 to 7.32. Concentration of Ca2+ and HCO3- in the culture filtrate significantly decreased with the increment in the incubation time, while concentration of TN increased along the time. The highest TN concentration (0.0807 gL-1) was observed on days 4 and 5, compared to that of the day 0 (0.0014 gL-1). Content of TP in the culture filtrate also significantly increased along the incubation period. The highest TP content was recorded in day 4 (0.0505%), while no TP content was recorded on day 0. Furthermore, data obtained revealed that A. salinestris YRNF3 produced acid phosphatase at low activity (5.4 U mL-1). HPLC analysis of the culture filtrate indicated production of different organic acids, namely lactic acid (82.57 mg mL-1), formic acid (46.8 mg mL-1), while acetic acid was detected in a low quantity (3.11 mg mL-1). For each analysis, three replicates of each treatment were analyzed. Means of the tested treatments were compared using Tukey's HSD test at p ≤ 0.05. In conclusion, findings of this work suggested that A. salinestris YRNF3 has the potential to be a probable bioagent to be used for the reclamation of the calcareous soils by solubilizing CaCO3, improving soil fertility, and promoting plant growth. However, further studies are needed to investigate its field application and their long-term effects on the soil properties and plant productivity. To the best of the author's knowledge, this is the first study reporting the calcite-solubilizing ability of a nitrogen-fixing bacteria. Having these two abilities by one microorganism is a unique feature, which qualifies it as a promising bioagent for reclamation of the calcareous soils.

Effects on the yield and fiber quality components of Bt cotton inoculated with Azotobacter chroococcum under elevated CO2.

Background: The raising trend of cultivation of Bacillus thuringiensis (Bt)-transgenic cotton is faced with a new challenge what effects on the growth and yield of Bt cotton under elevated CO2. Methods: Rhizobacteria is the significant biological regulator to increase environmental suitability and ameliorate soil-nitrogen utilization efficiency of crops, especially Bt cotton. Pot-culture experiments investigated the effects on the yield and fiber quality components of Bt cotton (transgenic Line SCRC 37) inoculated with Azotobacter chroococcum (AC) under elevated CO2. Results: The findings indicated that the inoculation of azotobacter significantly improved the yield and fiber quality components of Bt cotton, the elevated CO2 significantly increased the soil density of A. chroococcum and the partial yield indexes (as cottonweightper 20 bolls, lint yield per 20 bolls and boll number per plant), and non-significant decrease the fiber quality components of Bt cotton except uniform. Discussion: Overall results obviously depicted that the inoculation of azotobacter and the elevated CO2 had positive effects on the yield and fiber quality components of Bt cotton. Presumably, azotobacter inoculation can be used to stimulate plant soil-nitrogen uptake and promote plant growth for Bt cotton under elevated CO2 in the future.

Characterization and bioactivities of exopolysaccharide produced from Azotobacter salinestris EPS-AZ-6.

This study involved the extraction of an exopolysaccharide (EPS) from Azotobacter salinestris AZ-6, which was isolated from soil cultivated with leguminous plants. In a medium devoid of nitrogen, the AZ-6 strain displayed a maximum EPS yield of 1.1 g/l and the highest relative viscosity value of 3.4. The homogeneity of the polymer was demonstrated by the average molecular weight of 1.61 × 106 Da and a retention time of 17.211 min for levan. The presence of characteristic functional groups and structural units of carbohydrate polymers has been confirmed through spectroscopic analyses utilizing Fourier-transform infrared (FT-IR) and nuclear magnetic resonance (NMR) techniques. Thermogravimetric analysis (TGA) revealed a noteworthy decrease in weight (74 %) in the temperature range spanning from 260 to 350 °C. X-ray diffraction (XRD) was utilized to verify the crystalline and amorphous characteristics of EPS-AZ-6. The EPS-AZ-6 exhibited significant cytotoxicity against the MCF-7 tumor cell line, as evidenced by an IC50 value of 6.39 ± 0.05 µg/ml. It also demonstrated a moderate degree of cytotoxicity towards HepG-2 cell line, as indicated by an IC50 value of 29.79 ± 0.41 µg/ml. EPS-AZ-6 exhibited potent antioxidant and in vitro antibacterial properties. These characteristics suggest the potential application value of EPS-AZ-6 in the food industry and pharmaceutical applications.

Alginate: Microbial production, functionalization, and biomedical applications.

Alginates are natural polysaccharides widely participating in food, pharmaceutical, and environmental applications due to their excellent gelling capacity. Their excellent biocompatibility and biodegradability further extend their application to biomedical fields. The low consistency in molecular weight and composition of algae-based alginates may limit their performance in advanced biomedical applications. It makes microbial alginate production more attractive due to its potential for customizing alginate molecules with stable characteristics. Production costs remain the primary factor limiting the commercialization of microbial alginates. However, carbon-rich wastes from sugar, dairy, and biodiesel industries may serve as potential substitutes for pure sugars for microbial alginate production to reduce substrate costs. Fermentation parameter control and genetic engineering strategies may further improve the production efficiency and customize the molecular composition of microbial alginates. To meet the specific needs of biomedical applications, alginates may need functionalization, such as functional group modifications and crosslinking treatments, to achieve enhanced mechanical properties and biochemical activities. The development of alginate-based composites incorporated with other polysaccharides, gelatin, and bioactive factors can integrate the advantages of each component to meet multiple requirements in wound healing, drug delivery, and tissue engineering applications. This review provided a comprehensive insight into the sustainable production of high-value microbial alginates. It also discussed recent advances in alginate modification strategies and alginate-based composites for representative biomedical applications.

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera.

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight; pupation rate; adult longevity; fecundity; relative growth rate, RGR; efficiency of conversion of digested food, ECD; and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.

The impact of novel azotobacter Bacillus sp. T28 combined sea buckthorn pomace on microbial community structure in paddy soil.

Nitrogen (N) fertilizer application is an essential part of agricultural production in order to improve rice yields. However, long-term irrational application and low utilization of N fertilizer have caused a series of environmental problems. Biofertilizer is considered an effective alternative to N fertilizer. In this study, the effect of biofertilizer made of diazotrophic bacteria Bacillus sp. T28 combined with sea buckthorn pomace on the soil N changes and microbial community structure was conducted. Compared to CK, NO3--N content decreased 33.1%-43.8% and the rate of N2O release decreased 8-26 times under different fertilizer treatments during incubation of 0-7 days. On the contrary, NH4+-N in T28 with or without sea buckthorn pomace treatments increased by 56.5-118.8% during incubation of 7-14 days. The results indicated that this biofertilizer reduced the environmental risk associated with the accumulation of NO3--N in paddy soil and the release of N2O to the atmosphere and maintained the soil available N supply capacity. Besides, applying Bacillus T28 with sea buckthorn pomace increased the abundance of soil N functional genes such as nifH, narG, nirS, nirK, and nosZ. The 13C-PLFAs results demonstrated that this biofertilizer improves soil microbial community diversity, nutrient turnover rate and ecosystem stability by altering soil pH and total carbon (TC). In conclusion, Bacillus sp. T28 combined with sea buckthorn pomace regulated the indigenous soil microbial community structure and mitigated the environmental risk of conventional N fertilization in agroecosystems.

The effects of temperature, salt, and phosphate on biofilm and exopolysaccharide production by Azotobacter spp.

Inoculation of agriculturally important biofilms to plants under stress conditions has been of great interest in recent years. Therefore, in this study, biofilm- and exopolysaccharide (EPS)-forming ability of Azotobacter spp. was examined under different temperatures, NaCl concentrations, and phosphate levels. Azotobacter strains formed varying levels of biofilm and EPS depending on the tested factors. The pattern of biofilm formation was similar to that of EPS production under the conditions tested. Biofilm and EPS production at 28 °C was consistently higher than at either 18 or 37 °C. Biofilm production significantly increased in A. chroococcum strains (SBS2, SBS4, and SBS12) and A. vinelandii SBS6 with increasing salinity. Furthermore, a strong negative correlation was observed between biofilm or EPS production and increasing phosphate concentrations. Higher phosphate concentrations decreased biofilm and EPS production. In conclusion, contrary to temperature and phosphate effect, salinity differently affected biofilm and EPS production by Azotobacter strains.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

Evaluation of metabolites in Iranian Licorice accessions under salinity stress and Azotobacter sp. inoculation.

Licorice (Glycyrrhiza glabra L.) is an industrial medicinal plant that is potentially threatened by extinction. In this study, the effects of salinity (0 and 200 mM sodium chloride (NaCl)) and Azotobacter inoculation were evaluated on 16 licorice accessions. The results showed that salinity significantly reduced the fresh and dry biomass (FW and DW, respectively) of roots, compared to plants of the control group (a decrease of 15.92% and 17.26%, respectively). As a result of bacterial inoculation, the total sugar content of roots increased by 21.56% when salinity was applied, but increased by 14.01% without salinity. Salinity stress increased the content of glycyrrhizic acid (GA), phenols, and flavonoids in licorice roots by 104.6%, 117.2%, and 56.3%, respectively. Integrated bacterial inoculation and salt stress significantly increased the GA content in the accessions. Bajgah and Sepidan accessions had the highest GA contents (96.26 and 83.17 mg/g DW, respectively), while Eghlid accession had the lowest (41.98 mg/g DW). With the bacterial application, the maximum amounts of glabridin were obtained in Kashmar and Kermanshah accessions (2.04 and 1.98 mg/g DW, respectively). Bajgah and Kashmar accessions had higher amounts of rutin in their aerial parts (6.11 and 9.48 mg/g DW, respectively) when their roots were uninoculated. In conclusion, these results can assist in selecting promising licorice accessions for cultivation in harsh environments.

Microbial amelioration of salinity stress in endangered accessions of Iranian licorice (Glycyrrhiza glabra L.).

BACKGROUND: Glycyrrhiza glabra L. is a medicinal and industrial plant that has gone extinct due to different abiotic stress caused by climate change. To understand how the plant-associated microorganism can support this plant under salinity, we collected sixteen Iranian accessions of G. glabra L., inoculated their rhizomes with Azotobacter sp. (two levels, bacterial treatments, and no-bacterial treatments, and grown them under salinity stress (NaCl levels; 0, and 200 mM). RESULTS: Two accessions of Bardsir and Bajgah significantly showed higher resistant to salinity, for example by increasing crown diameter (11.05 and 11 cm, respectively) compared to an average diameter of 9.5 in other accessions. Azotobacter inoculation caused a significant increase in plant height and crown diameter. Among studied accessions, Kashmar (46.21%) and Ilam (44.95%) had the highest rate of membrane stability index (MSI). Evaluation of enzyme activity represented that bacterial application under salinity, increased polyphenol oxidase (PPO) (0.21 U mg-1 protein), peroxidase (POD) (3.09 U mg-1 protein U mg-1 protein), and phenylalanine ammonia-lyase (PAL) (17.85 U mg-1 protein) activity. Darab accession showed the highest increase (6.45%) in antioxidant potential compared with all studied accessions under Azotobacter inoculation. According to principal component analysis (PCA), it was found that the accession of Meshkinshahr showed a more remarkable ability to activate its enzymatic defense system under salt stress. Also, three accessions of Meshkinshahr, Eghlid, and Ilam were categorized in separated clusters than other accessions regarding various studied treatments. CONCLUSION: Analysis indicated that five accessions of Meshkinshahr, Rabt, Piranshahr, Bardsir, and Kermanshah from the perspective of induced systematic resistance are the accessions that showed a greater morphophysiological and biochemical outcome under salinity. This study suggested that, inoculation of with Azotobacter on selected accession can relieve salt stress and support industrial mass production under abiotic condition.

Antifungal potential of Azotobacter species and its metabolites against Fusarium verticillioides and biodegradation of fumonisin.

AIMS: In the study, seven Plant Growth Promoting Rhizobacteria (PGPR) Azotobacter species were screened against three strains of Fusarium verticillioides to test its antifungal activity. Azotobacter strains were tested for the degradation of fumonisin produced by F. verticillioides. Secondary metabolites were isolated and characterized from the Azotobacter strains for the first time. METHODS AND RESULTS: Potential seven Azotobacter species antifungal activity was tested following the dual culture assay against three strains of Fusarium verticillioides namely FVM-42, FVM-86 and MTCC156 estimating the substantial zone of inhibition. Azotobacter species AZT-31 and AZT-50 strains significantly inhibited the growth of F. verticillioides recording drastic growth enhancement of maize under in-vitro conditions by calculating the infection incidence, vigour index and germination percentage. As confirmation, dereplication studies were conducted for the reconfirmation of Azotobacter strains by isolating from rhizoplane. Azotobacter strains played a key role in the degradation of fumonisin produced by F. verticillioides reporting 98% degradation at 2 h of incubation with the pathogen. Furthermore, in the study first time, we have tried to isolate and characterize the secondary metabolites from the Azotobacter strains exhibiting six compounds from the species AZT-31 (2) and AZT-50 (4). Preliminary in-vitro experiments were carried out using the compounds extracted to check the reduction of infection incidence (90%) and increase in germination percentage upto 50 to 70% when compared to the test pathogen. CONCLUSION: Azotobacter strains referred as PGPR on influencing the growth of plant by producing certain substances that act as stimulators on inhibiting the growth of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The future perspective would be the production of an active combination of carboxamide compound and Azotobacter species for preventively controlling the phytopathogenic fungi of plants and crops and also towards the treatment of seeds.

Azotobacter chroococcum and Pseudomonas putida enhance pyrroloquinazoline alkaloids accumulation in Adhatoda vasica hairy roots by biotization.

Adhatoda vasica is used in the treatment of cold, cough, chronic bronchitis, asthma, diarrhea, and dysentery. The biological activities of this species are attributed with the presence of alkaloids, triterpenoids, and flavonoids. Agrobacterium rhizogenes-mediated transformation of A. vasica, produces pyrroloquinazoline alkaloids, was achieved by infecting leaf discs with strain ATCC15834. The bacterial strain infected 82.7% leaf discs and 5-7 hairy root initials were developed from the cut edges of leaf discs. In this study, seven strains of Azotobacter chroococcum and five strains of Pseudomonas putida were used for the biotization of hairy roots. Plant growth-promoting rhizobacteria (PGPR) develops symbiotic association with roots of plants and increases the growth parameters of plants. PGPR (A. chroococcum and P. putida) increased the profiles of nitrogenase and acid phosphatase enzymes, biomass, dry matter contents, anthranilate synthase activity and accumulation of pyrroloquizoline alkaloids in the biotized hairy roots. Both enzymes (nitrogenase and acid phosphatase) maintain sufficient supply of nitrogen and dissolved phosphorus to the cells of hairy roots therefore, the levels of anthranilate synthase activity and pyrroloquinazoline alkaloids are increased. Total seven pyrroloquinazoline alkaloids (vasicine, vasicinone, vasicine acetate, 2-acetyl benzyl amine, vasicinolone, deoxyvasicine and vasicol) were identified from the biotized hairy roots of A. vasica. In our study, biotization increased the profiles of pyrroloquinazoline alkaloids therefore, this strategy may be used in increasing the production of medicinally important secondary metabolites in other plant species also. Our hypothetical model demonstrates that P. putida cell surface receptors receive root exudates by attaching on hairy roots. After attachment, the bacterial strain penetrates in the biotized hairy roots. This endophytic interaction stimulates acid phosphatase activity in the cells of biotized hairy roots. The P. putida plasmid gene (ppp1) expression led to the synthesis of acid phosphatase in cytosol. The enzyme enhances phosphorus availability as well as induces the formation of phosphoribosyl diphosphate. Later, phosphoribosyl diphosphate metabolizes to tryptophan and finally tryptophan converts to anthranilic acid. The synthesized anthranilic acid used in the synthesis of alkaloids in A. vasica.

Symbiotic interplay of Piriformospora indica and Azotobacter chroococcum augments crop productivity and biofortification of Zinc and Iron.

In the present study Piriformospora indica (Pi) a phyto-promotional fungus and Azotobacter chroococcumWR5 (AzWR5) a rhizobacterium, were symbiotically evaluated for their role in improving the nutritional quality of wheat (Triticum aestivum L.). Co-inoculation of Pi+AzWR5 modified root system architecture of host and along with increasing the proportion of finer roots by 88% and 92% in C306 and Hd2967 respectively. Furthermore, the synergistic impact of Pi+AzWR5 interplayed for enhanced accumulation of Zn and Fe in different plant parts including grains (3.12 and 1.33 fold respectively). Pi+AzWR5 increased the transfer factor of Zn (62%, 94%, 91% and 213%) and Fe (31%, 54%, 68% and 32%) in root, stem, leaves and grains, respectively, and translocation factor of Zn (20%, 18% and 63%) and Fe (18%, 29% and 29%) for root-stem, root-leaves and root-grains, respectively. In addition to these co-inoculation of endophytes led to several fold increase in expression of four ZIP transporter genes in roots and shoot. In addition to these symbiotic association of endophytes with host led to 3 fold increase in grain yield. We thereby conclude that co-inoculation of Pi+AzWR5 substantially improves mobilization of Zn and Fe from soil and increase its concentration in grains as well as improves crop yield.

Characterization and diversity of native Azotobacter spp. isolated from semi-arid agroecosystems of Eastern Kenya.

Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby's agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant.

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli.

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

Stimulating effects of reduced graphene oxide on the growth and nitrogen fixation activity of nitrogen-fixing bacterium Azotobacter chroococcum.

Graphene has found important applications in various areas and hundred tons of graphene materials are annually produced. It is crucial to investigate both the negative and positive environmental effects of graphene materials to ensure the safe applications and develop environmental applications. In this study, we reported the stimulating effects of reduced graphene oxide (RGO) to nitrogen-fixing bacterium Azotobacter chroococcum. RGO stimulated the cell growth of A. chroococcum at 0.010-0.500 mg/mL according to the growth curves and the colony-forming unit (CFU) increases. RGO wrapped over the A. chroococcum cells without inducing ultrastructural changes. RGO decreased the leakage of cell membrane, but slight oxidative stress was observed in A. chroococcum. RGO promoted the nitrogen fixation activity of A. chroococcum at 0.5 mg/mL according to both isotope dilution method and acetylene reduction activity measurements. Consequently, the increases of soil nitrogen contents were evidenced, in particular about 30% increase of organic nitrogen occurred at 0.5 mg/mL of RGO. In addition, RGO might possibly benefit the plant growth through enhancing the indoleacetic acid production of A. chroococcum. These results highlighted the positive environmental effects of graphene materials to nitrogen-fixing bacteria in nitrogen cycle.

Combined application of ascorbic acid and endophytic N-fixing Azotobacter chroococcum Avi2 modulates photosynthetic efficacy, antioxidants and growth-promotion in rice under moisture deficit stress.

This group has previously reported the role of ascorbic acid (AA) as an antioxidant for survivability and ability to enhancing diazotrophic efficacy in Azotobacter chroococcum Avi2 under hydrogen peroxide (H2O2) stress. However, the present study showed the combined application of AA and Avi2 in drought-susceptible (IR64 and Naveen) and drought-tolerant (Ankit and Satyabhama) rice cultivars to determine their photosynthetic efficacy (chlorophyll fluorescence-imaging), antioxidants, and plant growth-promotion (PGP) under moisture deficit stress (MS, -60 kPa). The results indicated that combined application of AA and Avi2 significantly (p < 0.05) increased the total chlorophyll, relative water content, electrolytic leakage, super oxide dismutase, and catalase activities in all rice cultivars as compared to other MS treatments, whereas stress indicators like proline and H2O2 contents were proportionally increased under MS and their concentration were normalized under combined application of AA and Avi2. Photochemical quenching, non-photochemical quenching, photosynthetic electron transport rate, and the effective quantum efficiency were found to be increased significantly (p < 0.05) in Avi2 + AA as compared to other MS treatments. Moreover, rice roots harbored significantly (p < 0.05) higher copy number of nifH gene in Avi2 + AA treatment followed by Avi2 compared to flooded control and other MS treatments. Combined application of AA and Avi2 also increased the grain yield significantly (p < 0.05) by 7.09 % and 3.92 % in drought-tolerant (Ankit and Satyabhama, respectively) and 31.70 % and 34.19 % in drought-susceptible (IR64 and Naveen, respectively) rice cultivars compared to MS treatment. Overall, the present study indicated that AA along with Avi2 could be an effective formulation to alleviate MS vis à vis enhances PGP traits in rice.