Leaf blight is one of the diseases prevalent in zucchini fields in Iraq. This study investigated the etiological agent of zucchini leaf blight in agricultural fields across the Erbil, Diyala and Salah Al-Din governorates of Iraq. The fungus Alternaria cucumerina was found to be the predominant cause, with a percentage of appearance reaching 70.7 % in the samples and a frequency rate of 55 %. The Eac-12 strain showed the highest virulence among the 44 fungal isolates, with disease severity reaching 80.5 % under greenhouse conditions. Copper sulphate and neem oil showed 100 % efficacy in inhibiting pathogenic fungal growth in vitro, followed by plant growth-promoting bacteria (PGB), Azotobacter vinelandii and Azospirillum brasilense. In the greenhouse, the dual inoculum treatment of A. brasilense and A. vinelandii isolates significantly reduced disease incidence and severity. The quadruple combination treatment (copper sulphate, neem oil, A. brasilense and A. vinelandii) achieved the highest disease control rate, with disease incidence and severity recorded at 9 % and 4.3 % respectively. This study demonstrated that the rhizobacterial isolates of A. brasilense and A. vinelandii induced disease resistance in zucchini plants through elevated peroxidase levels, as well as the effectiveness of neem oil and copper sulphate in controlling the pathogenic fungus A. cucumerina.

Nitrogen-fixing microbes are a primary contributor of this important nutrient to the global nitrogen cycle. Biological nitrogen fixation (BNF) through the enzyme nitrogenase requires extensive energy that in whole cells is generally studied during the oxidation of carbohydrates such as sugars. The nitrogen-fixing bacterium Azotobacter vinelandii is a model diazotroph for the study of aerobic BNF. Much is known about metabolism in A. vinelandii when cultured on a simple medium where energy is provided primarily in the form of sucrose or glucose. Outside of the laboratory, this soil bacterium grows on metabolites primarily derived from plant root exudates or from the degradation of dead plant matter. In this work, we expand on previous studies looking at genes that are essential to BNF in A. vinelandii when grown on sucrose medium using transposon sequencing (Tn-seq). We applied Tn-seq to determine the genes essential to growth when the medium was shifted to acetate, succinate or glycerol as the primary carbon and energy source to fuel both growth and BNF. A global overview of the genes of central metabolism and those directing substrates toward central metabolism, along with a selection of unexpected genes that were essential for specific growth substrates, is provided.

This study evaluated the performance of a carrier-based Azotobacter vinelandii biofertilizer on the growth parameters of selected cereal crops—maize (Zea mays), rice (Oryza sativa), millet (Pennisetum glaucum), and guinea corn (Sorghum bicolor)—under pot experimental conditions. Soil physicochemical properties were determined using standard analytical procedures. Azotobacter vinelandii was isolated from soil using Jensen’s medium and identified based on morphological, biochemical, and molecular characteristics. Rice husk was used as the carrier material and inoculated with A. vinelandii at a concentration of 1 × 10⁷ CFU g⁻¹, followed by incubation for 28 days. The experiment was conducted in triplicate, and growth parameters—including plant height, number of leaves, leaf area, chlorophyll content, and root length—were measured at 5, 10, and 15 days after germination. Data were analyzed using one-way analysis of variance (ANOVA) with SPSS version 20. Plants treated with the Azotobacter biofertilizer exhibited significantly higher plant height, leaf area, chlorophyll content, and root length compared to the uninoculated control (p < 0.05), while no significant difference was observed in leaf number at early growth stages. These findings demonstrate the effectiveness of carrier-based A. vinelandii biofertilizer in enhancing early growth performance of cereal crops and support its potential use as a sustainable alternative to chemical fertilizers.

Poly-3-hydroxybutyrate (P3HB) is a biodegradable thermoplastic polyester with mechanical and thermal properties comparable to those of petrochemical-based plastics. In this study, the synthesis of P3HB by Bacillus cereus ATCC 14579 and Azotobacter vinelandii OP ATCC 13705 in complex media under different agitation conditions and cultivation times was evaluated. The growth kinetics of each microorganism responded differently to changes in agitation patterns. Maximum cell concentrations of 2.4 g L−1 and 4.3 g L−1 were achieved at 200 rpm (24 h) for B. cereus and 150 rpm (48 h) for A. vinelandii, respectively. While B. cereus reached an accumulation of 31.3% (0.37 g P3HB L−1), A. vinelandii OP achieved 55.8% (2.3 g P3HB L−1). The biopolymer was characterized by ATR-FTIR, with a prominent carbonyl (C = O) stretching vibration observed at 1724 cm−1. SEC-HPLC analysis revealed mean molecular weights (MMW) weights of 80,050 g mol−1 to 116,960 g mol−1 for B. cereus and from 75,805 to 111,000 g mol−1 for A. vinelandii OP. TGA/DSC analysis revealed that higher agitation rates decrease crystallinity and thermal stability by altering polymer chain alignment. The volumetric oxygen transfer coefficient (kLa) confirmed the role of oxygen availability on P3HB. These results highlight two promising strains with distinct metabolic behaviors and strong potential for scale-up in P3HB production.Graphical abstract

In 1955, the enzyme polynucleotide phosphorylase was discovered in the bacterium Azotobacter vinelandii, and thus the enzymatic synthesis of oligonucleotides in 2025 formally marks the 70th anniversary, and the discovery of terminal deoxynucleotidyltransferase (TdT) isolated from the calf thymus, sometimes also called the Bollum’s enzyme (by the author's surname), is 65 years old. However, it is only in the last decade that serious progress has been made in the enzymatic synthesis of extended oligonucleotides with a given sequence using this enzyme. In fairness, it should be noted that F.J.Bollum himself, back in 1986 in a review article, well ahead of his time, he described a method for such enzymatic synthesis of oligonucleotides with defined sequence. Currently, the synthesis of oligonucleotides using TdT with the desired sequence can be carried out in various ways, providing a temporary stop of polymerization after the insertion of one modified dNMP carrying a blocking group at the 3’-end, and then its resumption after the removal of the blocker, which has already been proposed a lot. Another approach is to form a complex of conventional dNTP with TdT, which leads to the incorporation of dNMP bound to TdT into the growing DNA chain, but in order to continue synthesis with a new similar complex, the enzyme from the previous conjugate must be removed from the DNA chain. Another approach is based on the competition of TdT with other enzymes, for example, with apyrase, which destroys dNTPs unused at a last stage. For effective enzymatic synthesis of oligonucleotides, it is necessary to improve the properties of TdT, including increasing its thermal stability, which is given increased attention in many studies. If for many years TdT, in addition to the calf's thymus, has also been isolated from mice, then in recent years similar enzymes have been described from birds, which, by the way, are warmer-blooded than mammals and, therefore, their enzymes are a priori more resistant to elevated temperatures. At the same time, many genetically engineered TdTs have already been created that have improved properties compared to native enzymes. There are examples of commercial custom synthesis of extended-range oligonucleotides produced by various companies (including up to 600 nucleotides), as well as a specialized "TdT synthesizer".

The year 2025 marks several anniversaries directly or indirectly related to oligonucleotide synthesis. Seventy years ago, in 1955, a paper was published describing the synthesis of the dinucleotides d(TpT) and d(pTpT). In that year, a paper was published reporting the isolation of the enzyme polynucleotide phosphorylase, which polymerizes RNA in vitro, from the bacterium Azotobacter vinelandii. In the 1970s, this enzyme was used for some time to synthesize short oligonucleotides with a given sequence. The chemical synthesis of oligonucleotides has undergone significant evolution over the years, from phosphotriester, through phosphonate, phosphodiester, phosphitetriester, and then converted to phosphoramidite. The latter is currently the primary method for producing oligonucleotides using automated DNA synthesizers. In fact, the phosphoramidite method of oligonucleotide synthesis currently provides a solution to the relevant scientific and practical problems. However, the need for long-term storage of non-biological information in DNA molecules (oligonucleotides) necessitates their production in significant quantities and of a different quality (longer), which the existing approach is no longer able to handle. A solution could be provided by the enzymatic production of oligonucleotides using terminal deoxynucleotidyl transferase, an enzyme isolated from calf thymus in 1960 that enables non-template DNA synthesis.

Carbon monoxide chemistry of α-V70I Mo-nitrogenase: Evidence from EPR- and IR-monitored photolysis - or, what a difference a methyl makes.

The urgent need for sustainable agriculture places biological nitrogen fixation at the forefront of current biotechnological research. Plant growth-promoting rhizobacteria play crucial roles in agriculture by enhancing nutrient absorption, regulating hormonal balance, and providing reduced nitrogen to plants. Among these, diazotrophic bacteria, such as Azotobacter vinelandii, stand out for their ability to fix atmospheric nitrogen and release it in bioavailable forms. In this issue of The FEBS Journal, scientists mapped the interaction between NifL and NifA proteins, which regulate nitrogen fixation in A. vinelandii and many other Proteobacteria. This understanding will allow for engineering bacteria to enhance nitrogen delivery to plants by improving nitrogen fixation.

Enhanced electron microscopy imaging for a detailed structural study of alginate hydrogel containing the encapsulated cells.

ABSTRACTPolyhydroxybutyrate (PHB) is a natural polyester synthesized and stored intracellularly as cytoplasmic granules, serving as a carbon and energy reserve in certain bacteria. These granules are surrounded by a variety of proteins, including those directly involved in PHB metabolism—both in its synthesis and degradation—as well as a group of non‐enzymatic proteins known as phasins, which constitute the major protein component of the PHB granule. Although phasins lack enzymatic activity, some, such as PhbP1 in Azotobacter vinelandii, significantly influence granule size and number, while others have been shown to modulate enzymes involved in PHB synthesis or degradation in PHB‐producing bacteria. This study aimed to elucidate the roles of phasins PhbP2 and PhbP3 in PHB metabolism in A. vinelandii. Our findings indicate that PhbP3 belongs to a novel family of phasin proteins and demonstrate that, although PhbP2 and PhbP3 do not contribute to granule structure, they are involved in PHB degradation. Gene inactivation of phbP2 and phbP3 led to a decrease in PHB depolymerase activity. Additionally, SDS‐PAGE analysis revealed alterations in the relative abundance of some granule‐associated proteins in the mutants, suggesting that PhbP2 and PhbP3 may play a role in stabilizing proteins on the PHB granule surface.

Wheat (Triticum aestivum L.) is the major grain crop that ensures global food security. Intensive farming often involves overuse of mineral fertilizers and pesticides, which leads to soil degradation and environmental pollution. Microorganisms and natural sorbents, e.g., zeolite, offer an alternative solution to the crop yield problem. Zeolite improves the soil structure while helping to retain moisture and nutrients. Growth-stimulating bacteria increase the availability of nutrients for plants and stimulate their growth. This research featured the effect of the combined use of zeolite and bacteria on different wheat varieties and growth indicators in laboratory conditions. The experiment involved spring wheat varieties of Sibirskiy Alyans, Pamyati Afrodity, and Nadezhda Kuzbassa. The list of growthstimulating bacteria included Azotobacter chroococcum B-4148, Azotobacter vinelandii B-932, and Pseudomonas chlororaphis subsp. aurantiaca B-548, as well as their consortium (1:3:1). The indicators to be checked included the solubilizing activity of the strains and the effect of zeolite (1 t/ha) and bacterial preparations on wheat growth. All bacteria solubilized zeolite (2.5–17.7 mm). The highest activity belonged to P. chlororaphis subsp. aurantiaca B-548 (17.7 mm). The combined application of zeolite (1 t/ha) and the bacterial consortium had a positive effect on the growth and development of all wheat varieties. The Sibirskiy Alyans variety showed a germination rate of 86%, a shoot length of 183 mm, a dry weight of 42.4%, a chlorophyll content of 24.47%, a carotenoid content of 16.21%, and a nitrogen concentration of 51.83%. The Pamyati Afrodity variety demonstrated 80% germination rate, 157 mm shoot length, 31.3% dry weight, 32.07% chlorophylls, 19.40% carotenoids, and 59.35% nitrogen. The Nadezhda Kuzbassa variety had 98% germination rate, 185 mm shoot length, 41.2% dry weight, 39.74% chlorophylls, 28.47% carotenoids, and 55.26% nitrogen. The results confirmed the i ndustrial efficiency of zeolite and bacteria in wheat farming, as did other reports on their positive effect on crop yield. However, further field trials are needed to confirm the results in conditions close to reality.

Intensifying agricultural production involves an active use of agrochemicals, which results in disrupted ecological balance and poor product quality. To address this issue, we need to introduce biologized science-intensive technologies. Bacteria belonging to the genera Azotobacter and Pseudomonas have complex growth-stimulating properties and therefore can be used as a bioproduct to increase plant productivity. We aimed to create a growth-stimulating consortium based on the strains of the genera Azotobacter and Pseudomonas, as well as to select optimal cultivation parameters that provide the best synergistic effect. We studied strains Azotobacter chroococcum B-4148, Azotobacter vinelandii B-932, and Pseudomonas chlororaphis subsp. aurantiaca B-548, which were obtained from the National Bioresource Center “All-Russian Collection of Industrial Microorganisms” of Kurchatov Institute. All the test strains solubilized phosphates and produced ACC deaminase. They synthesized 0.98–1.33 mg/mL of gibberellic acid and produced 37.95–49.55% of siderophores. Their nitrogen-fixing capacity ranged from 49.23 to 151.22 μg/mL. The strain had high antagonistic activity against phytopathogens. In particular, A. chroococcum B-4148 and A. vinelandii B-932 inhibited the growth of Fusarium graminearum, Bipolaris sorokiniana, and Erwinia rhapontici, while P. chlororaphis subsp. aurantiaca B-548 exhibited antagonism against F. graminearum and B. sorokiniana. Since all the test strains were biologically compatible, they were used to create several consortia. The greatest synergistic effect was achieved by Consortium No. 6 that contained the strains B-4148, B-932, and B-548 in a ratio of 1:3:1. The optimal nutrient medium for this consortium contained 25.0 g/L of Luria-Bertani medium, 8.0 g/L molasses, 0.1 g/L magnesium sulfate heptahydrate, and 0.01 g/L of aqueous manganese sulfate. The optimal cultivation temperature was 28°C. The microbial consortium created in our study has high potential for application in agricultural practice. Further research will focus on its effect on the growth and development of plants, in particular cereal crops, under in vitro conditions and in field experiments.

P(3HB) is a biodegradable and biocompatible polymer, which can replace petroleum-derived plastics. Previous studies have shown that Azotobacter vinelandii strain OP-PhbP3+, which overexpresses the phasin protein PhbP3, produces high concentrations of P(3HB) and undergoes early autolysis, facilitating polymer release. The aim of the present study was to evaluate the performance of this strain for P(3HB) production in 3 L bioreactors and assess the feasibility of a simplified recovery process. After 36 h of cultivation, rapid cell lysis was observed, resulting in a ~50% decrease in the protein content of the cell dry weight, without reducing P(3HB) concentration, which reached 4.6 g L−1. Flow cytometry analysis revealed significant morphological changes during cultivation, which was consistent with the strain’s lytic behavior. The biomass recovered at 36 h was washed with SDS, obtaining a yield of 92.5% (respect to P(3HB) initial) and a purity of 97.6%. An alternative extraction procedure using the non-halogenated solvent cyclohexanone (CYC) resulted in an even higher yield of 97.8% with a purity of 99.3% of P(3HB). Notably, the weight average molecular weight of the polymer remained stable at 8000 kDa during the entire process. Overall, the combination of PhbP3 over-expression and environmentally friendly solvents, such as CYC, enabled efficient P(3HB) production with high yield and purity while preserving polymer quality.

Global concern over plastic pollution and fossil resource depletion has driven the development of biodegradable alternatives like polyhydroxybutyrate (PHB). PHB, synthesized by microorganisms, faces commercialization challenges due to high production and separation costs. This study evaluated an alternative platform for sustainable PHB production using air-lift bioreactors (ALRs) of up to 15L. We achieved a high concentration, enrichment and specific productivity of PHB of 4.4g ± 0.1g·L⁻¹, 87.9 ± 0.3% (w/w) and 0.43 ± 0.0g·L⁻¹·h-1, in an Azotobacter vinelandii mutant strain deficient in alginate biosynthesis, in 28h. Bacterial cells were cultured using air as the sole N source. PHB was recovered with 96% efficiency and 95% purity by osmotic shock using glycerol and soap produced from recycled spent cooking oil. This strategy not only replaces commonly used hazardous solvents for PHB extraction but also repurposes spent cooking oil, an otherwise difficult-to-manage waste, into a valuable resource for bioplastics production. Additionally, we demonstrate the feasibility of recycling glycerol recovered after PHB extraction, which partially substituted for sucrose as a carbon source in subsequent PHB production. The cells exhibited a preferential utilization of C sources in the following order: sucrose > analytical-grade glycerol > glycerol recovered from recycled spent cooking oil post-osmotic shock and PHB recovery > fresh glycerol recovered from recycled spent cooking oil. These findings provide practical opportunities for improving the environmental and economic sustainability of PHB production while aligning with circular economy objectives, supporting the gradual replacement of certain petrochemical plastics.

Azotobacter vinelandii OP is a bacterium that can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HBV), a biodegradable and biocompatible polymer with applications in the biomedical field. This study aimed to evaluate P3HBV production and its 3-hydroxyvalerate (3HV) fraction under different agitation rates and oxygen uptake rates (qO2) in chemostat cultures of A. vinelandii OP. Steady-state conditions with either oxygen or carbon limitation were established by modulating the agitation rates. Under oxygen-limited conditions (low qO2 values) biomass and P3HBV concentrations increased to 3.3 g L−1 and 2.1 g L−1, respectively. At higher qO2 values, the chemostat cultures were limited by carbon, and P3HBV content decreased from 62% to 33% (w w−1). The highest 3HV molar fractions, 33.7 and 36.4 mol %, were observed at both the lowest and highest qO2 levels, possibly linked to comparable valeric acid consumption rates. An elevated NAD(P)H/NAD(P)+ ratio was also observed under oxygen limitation, favoring polymer accumulation by indicating a more favorable intracellular redox state. These findings highlight the impact of nutrient limitation and respiratory activity on the biosynthesis of P3HBV and the 3HV composition by Azotobacter vinelandii OP. Such insights can support the development of tailored bioprocesses to modulate polymer characteristics, enabling a broader range of potential biomedical applications for P3HBV.

Abstract Key message Application of Azotobacter vinelandii is a strategy for 'Hass' avocado at various phenological stages, showing a 25% decrease in chemical fertilizer use while improving growth and physiological performance. Abstract Azotobacter-based bio-stimulants increase soil nutrient availability, provide substances for plant growth, and reduce fertilizer needs. We examined drench Azotobacter vinelandii (Av) application with two chemical fertilization levels (CF: 100% and CF 75%: 75% chemical soil fertilization) on physiological, nutritional, and fruit yield parameters. Over 20 weeks, three experiments were conducted on different avocado development stages: seedlings, post-transplantation, and mature trees. In each trial, plants received soil treatments with three commercial Av doses [2.5 (Av1), 5 (Av2), and 7.5 (Av3) mL L−1], with two fertilization levels. Soil Av and CF treatments were applied every 30 days from treatment start up to 16 weeks after treatment initiation (WAT). In the seedling trial, treatments with CF 75% combined with either Av2 or Av3 result in improved seedling quality, as indicated by the Dickson Quality Index (DQI), which measures 0.58 for CF 75% alone and approximately 0.79 for CF 75% with Av2 or Av3 at 20 WAT. In post-transplantation trees, CF 75% + Av2 or Av3 improved relative growth rate (0.021 and 0.024 cm cm−1 week−1 for CF 75% + Av2 and Av3, respectively) compared to CF 75% plants (0.013 cm cm−1 week−1) at 20 WAT. Mature trees showed CF 75% + Av2 or Av3 treatments had higher agronomic efficiency (44.7 and 38.2% CF 75% + Av2 and Av3, respectively) than CF 75% trees at 20 WAT. Av could serve as an alternative strategy for integrated plant nutrient management in sustainable 'Hass' avocado production as it reduces chemical fertilization needs by 25% without impacting crop physiology.

Previously, we identified the only dinitrogen reduction mechanism known to date as an ancient feature conserved from nitrogenase ancestors, which we directly tested by resurrecting and integrating synthetic ancestral nitrogenases into the genome of Azotobacter vinelandii (Garcia et al., 2023), a genetically tractable, nitrogen-fixing model bacterium. Here, we extend this paleomolecular approach to investigate the structural evolution of nitrogenase over billions of years of evolution by combining phylogenetics, ancestral sequence reconstruction, protein crystallography, and deep-learning based predictions. This study reveals that nitrogenase, while maintaining a conserved multimeric core, evolved novel modular features aligned with major environmental transitions, suggesting that subtle distal changes and transient regulatory adaptations were key to its long-term persistence and to shaping protein evolution over geologic time. The framework established here provides a foundation for identifying structural constraints that governed ancient proteins and for situating their sequences and structures within phylogenetic and environmental contexts across time.

Understanding the molecular basis of regulated nitrogen (N2) fixation is essential for engineering N2-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

Growth inhibition and activity stimulation of non-target organism nitrogen-fixing bacterium Azotobacter vinelandii by herbicide florasulam.

CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes.