Unveiling the Molecular Mechanism of Azospirillum in Plant Growth Promotion

Abstract

Azospirillum is a well-studied genus of plant growth-promoting rhizobacteria (PGPR) and one of the most extensively researched diazotrophs. This genus can colonize rhizosphere soil and enhance plant growth and productivity by supplying essential nutrients to the host. Azospirillum–plant interactions involve multiple mechanisms, including nitrogen fixation, the production of phytohormones (auxins, cytokinins, indole acetic acid (IAA), and gibberellins), plant growth regulators, siderophore production, phosphate solubilization, and the synthesis of various bioactive molecules, such as flavonoids, hydrogen cyanide (HCN), and catalase. Thus, Azospirillum is involved in plant growth and development. The genus Azospirillum also enhances membrane activity by modifying the composition of membrane phospholipids and fatty acids, thereby ensuring membrane fluidity under water deficiency. It promotes the development of adventitious root systems, increases mineral and water uptake, mitigates environmental stressors (both biotic and abiotic), and exhibits antipathogenic activity. Biological nitrogen fixation (BNF) is the primary mechanism of Azospirillum, which is governed by structural nif genes present in all diazotrophic species. Globally, Azospirillum spp. are widely used as inoculants for commercial crop production. It is considered a non-pathogenic bacterium that can be utilized as a biofertilizer for a variety of crops, particularly cereals and grasses such as rice and wheat, which are economically significant for agriculture. Furthermore, Azospirillum spp. influence gene expression pathways in plants, enhancing their resistance to biotic and abiotic stressors. Advances in genomics and transcriptomics have provided new insights into plant-microbe interactions. This review explored the molecular mechanisms underlying the role of Azospirillum spp. in plant growth. Additionally, BNF phytohormone synthesis, root architecture modification for nutrient uptake and stress tolerance, and immobilization for enhanced crop production are also important. A deeper understanding of the molecular basis of Azospirillum in biofertilizer and biostimulant development, as well as genetically engineered and immobilized strains for improved phosphate solubilization and nitrogen fixation, will contribute to sustainable agricultural practices and help to meet global food security demands.

1. Introduction

Agriculture is the primary source of food and provides essential nutrients for the survival of the global population. Over time, more advanced farming methods have been adopted to ensure consistent yields and meet the demands of growing populations [1]. Chemical pesticides and fertilizers are widely used to enhance agricultural productivity [2]. However, only approximately 50–60% of the inorganic compounds in mineral fertilizers are utilized for plant development, while the remainder is released into the environment, posing significant risks to human health and ecosystems [3]. Excessive use of these chemicals leads to loss of microbial diversity, soil degradation, runoff-induced water pollution, and food chain contamination. Sustainable farming practices are also threatened by long-term dependence on these chemicals, which ultimately reduce soil fertility [4].

Organic farming has emerged as a sustainable agricultural practice that employs biofertilizers to improve crop productivity and soil health. Biofertilizers are formulations containing living microorganisms that enhance nutrient availability and promote plant growth when applied to plants, seeds, roots, or soil. They can fix atmospheric nitrogen, solubilize inorganic phosphates, and produce growth-promoting substances [5,6,7]. The microorganisms used in biofertilizers include Azotobacter, Azospirillum, blue-green algae, phosphate-solubilizing microorganisms, mycorrhizae, and Rhizobium. Some aquatic ferns, like Azolla, are also used to enhance the nutritive profile of biofertilizers. Azospirillum is one of the most widely used nitrogen-fixing bacteria for biofertilizer production (Figure 1) [8].

Azospirillum is a free-living, nitrogen-fixing, plant growth-promoting bacterium (PGPB) capable of converting atmospheric nitrogen (N₂) into ammonia. It is also known to produce phytohormones, particularly auxins [9]. Additionally, it supports a healthy plant-microbe relationship and contributes to soil nutrient mineralization. According to the LPSN database reports, 37 species of Azospirillum have been identified (LPSN database accessed on 2 June 2025, available at https://lpsn.dsmz.de/search?word=Azospirillum). The genus Azospirillum has been subject to taxonomic controversies owing to its phenotypic plasticity, genetic heterogeneity, and ongoing discoveries of novel strains. Critical discussions revolve around discrepancies between classical morphological methods and modern molecular techniques, such as 16S rRNA gene sequencing and whole-genome analysis, which often yield conflicting results. These inconsistencies challenge the species delineation and strain classification. Horizontal gene transfer and environmental adaptability further complicate the taxonomic clarity. Recent attempts have highlighted the importance of metagenomic and environmental DNA (eDNA) techniques, integrating genomic, chemotaxonomic, and physiological data for accurate classification. Resolving these controversies is essential for understanding the ecological roles and exploiting Azospirillum strains in sustainable agriculture [10].

Azospirillum is recognized as one of the most effective PGPR and a valuable biofertilizer because of its ability to enhance plant growth and development [11]. The current study focused on the molecular mechanisms of Azospirillum in promoting plant growth, emphasizing nitrogen fixation, phytohormone synthesis, root architecture modification, and immobilization for enhanced crop production. As a biofertilizer and biostimulant, Azospirillum improves nutrient availability, mitigates stress, enhances plant resilience, and reduces reliance on chemical fertilizers. Advances in genomics and transcriptomics have offered insights into their roles in sustainable agriculture. Genetically engineered and immobilized strains with improved phosphate solubilization and nitrogen fixation can boost productivity. Given the global food security challenges, Azospirillum contributes to eco-friendly farming practices, ensuring higher yields and environmental sustainability.

2. Comparative Genomics and Proteomics Study Among Different Azospirillum Strains

Genomic studies of Azospirillum spp. have primarily focused on genes associated with nitrogen fixation, auxin synthesis, and rhizosphere survival [12,13,14]. Understanding the genetic diversity of multiple strains can enhance our knowledge of Azospirillum–plant interactions. The genomes of Azospirillum are highly complex and consist of several mega-replicons (chromosomes and plasmids), with sizes ranging from 4.8 to 9.7 Mbp [15,16]. Azospirillum contains both chromosomes and plasmids, which play crucial roles in various cellular functions, metabolism, and nitrogen fixation [15,16]. These genomes exhibit significant plasticity, minimal synteny between replicons, and extensive genomic rearrangements compared to rhizobia, highlighting their evolutionary adaptability [17,18].

Several Azospirillum species have gone through genomic studies. Notably, the whole genome sequencing of A. brasilense CBG497 has been isolated from maize grown on alkaline soils in northeastern Mexico. The core genome of Azospirillum consists of conserved genes shared across all strains, which are essential for cellular functions, nitrogen metabolism, and survival under normal conditions. These genes are highly conserved and are involved in fundamental biological processes such as metabolism, DNA replication, protein synthesis, and cellular structure. A total of 4216 genes were identified as common among the various Azospirillum strains and A. brasilense strains obtained from multiple databases.

The unique sequences identified included 2099 distinct sequences in A. brasilense Sp245, 1778 in A. brasilense FP2, 1140 in A. brasilense Az39, 213 in A. brasilense Sp7, and 182 in A. brasilense CBG497, all of which contribute to indole acetic acid synthesis. Comparative genomic studies revealed that A. brasilense Az39 and A. brasilense Sp245 share 5350 genes, A. brasilense Az39 and A. brasilense Sp7 share 4976 genes, A. brasilense Az39 and A. brasilense CBG497 share 4954 genes, and A. brasilense Az39 and A. brasilense FP2 share 4794 genes. A. brasilense Az39 exhibited a five-fold increase in the number of distinct genes, demonstrating substantial genetic variation within this genus [19].

Notably, over the past 50 years, A. brasilense Az39 has been one of the most successfully utilized strains in agriculture in South America. Its genome has recently been fully sequenced without gaps, making it a valuable tool for molecular research and a model for in-depth comparative genome analysis and Azospirillum plant interactions (Figure 2a) [19]. Most Azospirillum genes are chromosomally encoded, with 74% sharing an evolutionary origin with their aquatic relatives. The core genomes of non-ancestral regions contain genes involved in signal transduction, transport, glucose and amino acid metabolism, as well as surface structures that facilitate adaptation to variable environments such as soil and the rhizosphere [20]. A comparative study was performed to categorize proteins into families and analyze the genomic relationships among Azospirillum strains based on protein-coding content. Due to its incomplete genome assembly (1617 contigs and 3319 predicted coding DNA sequence (CDS), much lower than the expected 7.3 Mbp genome), A. amazonense Y2 was excluded. The remaining four genomes were grouped using the Markov Cluster Algorithm (MCL), revealing 5575 protein families. The 5575 protein families discovered among the 27,400 proteins comprising four Azospirillum genomes are a mix of core, accessory, and strain-specific proteins. Among them, 47% (2600) were shared across all strains, A. brasilense strains shared 74% (4136), A. lipoferum and Azospirillum strains shared 66% (3667), and other combinations shared 51.2–54.6%. Some families are strain-specific (Figure 2b) [20].

The study of the molecular mechanisms of Azospirillum is currently an exciting area of research, particularly in understanding its capacity for nitrogen fixation, auxin production pathways in coordination with other growth regulators, phosphate solubilization, and its role in controlling plant stress responses [21]. Additionally, immobilization studies on Azospirillum are underway to provide physical protection from adverse agricultural conditions [22]. Understanding the complex molecular pathways and signaling networks involved in Azospirillum-mediated plant growth promotion can offer novel insights into bioinoculant development and innovative agricultural solutions. Moreover, optimizing the use of Azospirillum as a biofertilizer could help mitigate the environmental impact of conventional farming practices. Based on this perspective, the present review explores the molecular mechanisms of Azospirillum in promoting plant growth, hormone and regulator production, genetics of nitrogen fixation, stress management systems, and the implications of Azospirillum immobilization for sustainable agricultural advancements.

3. Plant Growth Promoting Hormones and Regulators Produced by Azospirillum Species

The hormonal responses of Azospirillum include the production and regulation of phytohormones such as cytokinins, gibberellins, salicylic acid, indole-3-acetic acid (IAA), and ethylene. These substances stimulate root elongation and enhance nutrient and water uptake by the plants. Cytokinins promote cell division and delay leaf senescence, thereby supporting plant growth, whereas gibberellins contribute to stem elongation and seed germination. Additionally, Azospirillum spp. improve nutrient availability through nitrogen fixation and phosphate solubilization. These hormones also play crucial roles in regulating stress responses and in increasing plant resilience under challenging environmental conditions. By influencing biosynthetic pathways, Azospirillum creates a favorable environment for overall plant growth and development.

In addition to nitrogen fixation, phytohormone synthesis, phosphate solubilization, and siderophore production, Azospirillum is known for its non-pathogenic nature. However, even at low dosages, excessive concentrations of plant growth hormones can negatively impact plant maturation, alter morphology and metabolism, and disrupt overall development [23]. The association of Azospirillum species with plants influences growth at the tips, phototropism, geotropism, cell division, root initiation, and other processes regulated by IAA-stimulated plant growth-promoting rhizobacteria (PGPR) activity [24]. Inoculation with PGPR enhances mineral and water uptake, making plants more dynamic and promoting overall growth.

The stimulation of plant growth by Azospirillum depends on both direct and indirect mechanisms. Direct mechanisms involve facilitating nutrient absorption and phytohormone synthesis, whereas indirect mechanisms suppress plant diseases, thereby indirectly enhancing growth and productivity. This genus also synthesizes cadaverine, which promotes root growth and regulates stomatal activity. Nitric oxide (NO) plays a role in lateral root formation and root hair development.

A. brasilense biosynthesizes IAA in multiple pathways [indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile, indole-3-acetamide (IAM) pathway, and indole-3-acetonitrile (IAN)] where tryptophan is used as a precursor. The species converts tryptophan into indole-3-pyruvic acid (IPA) in the presence of the enzyme tryptophan aminotransferase. This IPA is converted into indole-3-acetyldehyde (IAAId) in the presence of the enzyme pyruvate decarboxylase. Finally, the enzyme aldehyde decarboxylase converts IAAId into IAA [25,26].

Both symbiotic and phytopathogenic bacteria use the indole-3-acetamide (IAM) pathway, which converts tryptophan into IAA by two steps. The enzyme tryptophan-2-monooxygenase (iaaM) first converts to IAM, then in the second step, IAM hydrolase (iaaH) converts IAM into IAA. There is a paucity of studies regarding the IAN pathway in bacteria. However, two steps have been discovered that convert Trp to IAA by the IAN pathway. Firstly, trp is converted into indole-3-acetaldoxime (IAOX) by an oxidoreductase. In the second step, the enzyme indoleacetaldoxime dehydratase converts IAOX into IAN. After the formation of IAN, it can be transformed into IAA through two distinct pathways: one involving nitrilases and the other utilizing the NHase/amidase system. The NHase enzyme connects the IAN and IAM pathways by converting IAN into IAM [27].

Apart from this, there is another pathway used only by Pseudomonas fluorescens where tryptophan (Trp) can be directly converted into IAAId by the Trp side chain oxidase (TSO) pathway [27].

3.1. Phytohormone Production

Azospirillum produces numerous phytohormones and associated plant growth regulators, including gibberellins, ethylene, abscisic acid, nitric oxide (NO), and polyamines (Table 1) [12]. Auxins, gibberellins, and indole-3-acetic acid (IAA) play major roles in plant growth [28]. Additionally, Azospirillum can synthesize other hormones in vitro, such as ethylene, salicylic acid, and abscisic acid [29]. These growth regulators contribute to a significant increase in plant yield.

Auxins, a class of plant growth regulators, influence many aspects of plant growth and development [30]. They are associated with cell division, elongation, and differentiation, and promote xylem and root development. Auxins also regulate phototropism, shoot growth, and fructification, all of which affect photosynthesis, pigment production, metabolite biosynthesis, and resistance to biotic stressors. Gibberellic acid (GA) plays a key role in breaking seed dormancy during germination, which coincides with the high GA production by Azospirillum [12].

Cytokinins facilitate cell division, shoot and root architecture, chloroplast maturation, and axillary bud formation. Auxins regulate cytokinin biosynthesis, establishing a hormonal balance that is essential for plant growth. The culture medium of Azospirillum spp. has been reported to contain various auxin-related compounds, including indole-butyric acid, phenylacetic acid, indole-3-lactic acid, indole-3-ethanol, indole-3-methanol, indole-3-acetamide, indole-3-acetaldehyde, tryptamine, and anthranilate [31].

Under both anaerobic and aerobic culture conditions, A. brasilense Sp245 and Az39 produce nitrogen oxide (NO) [32]. In strain Sp245, NO production occurred via multiple pathways, including heterotrophic nitrification and aerobic denitrification. NO participates in the IAA signaling pathway, promoting adventitious and lateral root formation. Its precise function in IAA-induced root growth is as an intermediate signaling molecule [33,34,35].

Table 1. Function of different phytohormones and molecules produced by Azospirillum.

Phytohormones	Function	Molecules Present in Phytohormones	Reference
Auxin	Growth and development of different plant tissues, Cell Division	IAA, PAA, IBA	[29,36]
Gibberellic Acid	Cell division, breaking dormancy	GA3, GA1	[37,38]
Cytokines	Leaf growth, chloroplast maturation, and shoot and root morphogenesis	iP, iPr, Z, t-Zr	[39,40]
Abscisic acid	Phytohormone production in response to environmental stress	ABA	[41,42]
Polyamines	Root growth, control stomata	Cad, Spm, Spd	[29,41]
Ethylene	Breaking of seed dormancy, growth modulation, stress responses	Et	[43,44]

Indole-3-acetic acid (IAA), phenylacetic acid (PAA), indole-3-butyric acid (IBA), isopentenyl adenine (iP), isopentenyl adenine riboside (iPr), zeatin (Z), trans-zeatin riboside (t-Zr), gibberellins (GA), ethylene (Et), abscisic acid (ABA), cadaverine (Cad), spermine (Spm), spermidine (Spd), and putrescine (Put).

Table 1. Function of different phytohormones and molecules produced by Azospirillum.

Phytohormones	Function	Molecules Present in Phytohormones	Reference
Auxin	Growth and development of different plant tissues, Cell Division	IAA, PAA, IBA	[29,36]
Gibberellic Acid	Cell division, breaking dormancy	GA3, GA1	[37,38]
Cytokines	Leaf growth, chloroplast maturation, and shoot and root morphogenesis	iP, iPr, Z, t-Zr	[39,40]
Abscisic acid	Phytohormone production in response to environmental stress	ABA	[41,42]
Polyamines	Root growth, control stomata	Cad, Spm, Spd	[29,41]
Ethylene	Breaking of seed dormancy, growth modulation, stress responses	Et	[43,44]

Indole-3-acetic acid (IAA), phenylacetic acid (PAA), indole-3-butyric acid (IBA), isopentenyl adenine (iP), isopentenyl adenine riboside (iPr), zeatin (Z), trans-zeatin riboside (t-Zr), gibberellins (GA), ethylene (Et), abscisic acid (ABA), cadaverine (Cad), spermine (Spm), spermidine (Spd), and putrescine (Put).

3.2. Siderophore Production

Iron is one of the most abundant chemical elements on Earth and is commonly found in its ionic form (Fe²⁺–Fe³⁺). It serves as a crucial enzyme cofactor in various metabolic processes, including photosynthesis, amino acid synthesis, oxygen transfer, RNA synthesis, nitrogen fixation, and respiration. As a result, iron is essential for all organisms [29].

Siderophores are low-molecular-weight, ferric-iron-chelating compounds secreted by bacteria and fungi under iron-deficient conditions. Plant growth-promoting rhizobacteria (PGPR) produce siderophores to enhance iron absorption in iron-restricted environments, thereby increasing their bioavailability to plants [45]. The quantity of siderophores produced and their site of isolation, whether in the rhizosphere or within plant tissues, are interrelated. Environmental conditions such as temperature regulate siderophore production.

Siderophores belong primarily to four chemical classes: carboxylates, pyoverdines, hydroxamates, and phenols. Azospirillum brasilense, a well-known plant growth-promoting bacterium (PGPB), requires substantial amounts of iron because several nitrogen-fixation enzymes, including nitrogenase, utilize iron as a cofactor. In A. brasilense, four high molecular mass proteins (87, 83, 78, and 72 kDa) in the outer membrane were induced under iron-deficient conditions [46]. Additionally, during iron scarcity, A. brasilense synthesizes a catechol-type siderophore, spirilobactin. The bacterium activates the biochemical pathways responsible for siderophore synthesis, leading to the release of spirilobactin, which binds to the available ferric ions to form a complex. Azospirillum recognizes and internalizes the iron–spirilobactin complex for cellular use (Figure 3) [47]. Tortora et al. [48] reported that A. brasilense (REC2 and REC3) secretes catechol-type siderophores such as salicylic acid (SA), which show antifungal activity against Colletotrichum acutatum M11 in Strawberry (Camarosa and Milsei) plants.

The production of siderophores is a biocontrol approach through which Azospirillum can protect plants from pathogen attack. However, not all Azospirillum spp. can produce siderophores. Previous reports have highlighted the production of siderophores by A. brasilense. In addition to A. brasilense, other Azospirillum species, such as A. lipoferum strain D-2, can produce the phenolate siderophores salicylic acid, 3,5-dihydroxybenzoic acid, and 2,3-dihydroxybenzoic acid (DHBA) when iron availability is reduced. The strain A. lipoferum D-2 produced 8.68 µg mL⁻¹ of 2,3-DHBA and 7.80 µg mL⁻¹ of salicylic acid under microaerophilic conditions [49]. In addition to its siderophore activity, salicylic acid is a precursor in the synthesis of catechol-type microbial siderophores, including pyoverdin, pyochelin, and yersiniabactin. Furthermore, it has been shown to be a key endogenous regulator of both systemic and localized acquired resistance to pathogen infection in a variety of plants. Thus, strains that produce SA may improve plant defense. The idea was that plant roots would recognize the bacterial SA released into the medium and trigger signals for systemic resistance. However, in certain interactions, SA may not be the main signal for inducing systemic resistance (ISR); instead, other siderophores may be involved. Notably, A. brasilense Cd and Az39, two agronomically important strains, have not been reported to produce siderophores [50]. Apart from Azospirillum spp., other bacteria such as Pseudomonas putida, which can produce siderophores, namely pseudobactin, which is responsible for boosting Pseudomonas putida plant growth and crop yield [51]. Other bacteria, such as Azotobacter vinelandii, Bacillus substilis, and Rhizobium radiobacter, can also produce siderophores that aid chelation under alkaline conditions [52]. Delaporte-Quintana et al. [53] concluded that siderophores synthesized by Gluconacetobacter diazotrophicus PAL5 and A. brasilense REC3 may support iron uptake in hydroponically cultivated strawberry plants. Bacteria that produce siderophores in the plant rhizosphere are particularly significant because they can also bind heavy metal ions and release them into the roots, improving the capacity of plants for phytoextraction [54].

3.3. Other Plant Regulators for Plant Growth

3.3.1. Nitric Oxide

Nitric oxide (NO) is a small, volatile, lipophilic free radical and diffusible gas that functions as a bioactive compound in various developmental and pathological processes in living organisms [55]. In plants, NO plays a crucial role in metabolic and signaling pathways, contributing to defence mechanisms and developmental growth processes [56]. It acts as a mediator in the indole-3-acetic acid (IAA) signaling pathway, promoting root growth and facilitating lateral and adventitious root formation [57]. Previous studies have demonstrated that NO can initiate lateral root development in tomato plants [33]. Additionally, Azospirillum brasilense Sp245 has been reported to produce nitric oxide, which induces morphological changes in tomato roots, independent of the ability of the bacterium to synthesize IAA.

3.3.2. Polyamines

The genus Azospirillum can synthesize and regulate polyamines, which directly affect plant growth. Polyamines enhance nitrogen fixation, root development, biofilm formation, and stress tolerance [58]. In a chemically defined medium, Azospirillum species produced putrescine (Put), spermidine (Spd), and spermine (Spm) [58]. It has been suggested that bacterial cadaverine (Cad) synthesis is a unique mechanism that promotes plant growth and regulates plant responses to osmotic stress [12]. The polyamines spermidine, putrescine, spermine, and cadaverine were detected in the growth medium inoculated with the commonly used inoculant A. brasilense Az39, which is widely applied in the cultivation of wheat, rice, and maize in Argentina [50,58]. It has also been postulated that Azospirillum inoculation in soil leads to cadaverine production and plays a role in plant development [12].

3.3.3. Phosphate Solubilization

Phosphorus is an essential macronutrient for plants and plays a crucial role in enhancing crop yield. It is involved in several fundamental biological processes, including respiration, signal transduction, energy transfer, cell division, photosynthesis, and macromolecule biosynthesis [59]. Plants primarily absorb phosphorus in the form of insoluble orthophosphate molecules. Compared with other macronutrients, phosphorus has low mobility in the soil because it tends to remain in a precipitated form as orthophosphate.

Phosphate-solubilizing microorganisms (PSM) play a vital role in converting insoluble phosphorus into bioavailable forms in plants [60]. These soil microbes release organic acids that solubilize phosphorus while simultaneously reducing soil pH. Additionally, PSM produces enzymes such as phosphatase and phytase, which facilitate the mineralization of phosphate, allowing plants to absorb it more efficiently [61,62]. Phosphate-solubilizing bacteria (PSB) not only improve phosphorus availability but also produce plant hormones, vitamins, and other growth-promoting compounds. Furthermore, these microorganisms enhance trace mineral availability and produce antibiotics that protect plants from disease [63,64].

Plants struggle to accumulate phosphorus because approximately 99% of the available phosphorus in the soil is either precipitated or immobilized, rendering it insoluble in water. PSM serves as a partial substitute for chemical phosphate fertilizers and contributes to the regulation of biogeochemical phosphorus cycles in both agricultural and natural environments. The most effective PSM functions as a biofertilizer, enhancing the use of rock phosphate for crop growth. These microbes convert insoluble phosphorus into soluble forms primarily HPO₄²⁻ and H₂PO₄ through mechanisms such as acidification, chelation, and ion exchange reactions. When applied to plants, soil, or rock phosphate fertilizers, PSM releases phosphorus, promotes plant growth, improves soil quality, and protects crops from diseases without causing environmental pollution. Therefore, the use of PSM alongside rock phosphate fertilizers represents an economically viable and environmentally friendly approach to sustainable agriculture [65,66].

However, the addition of phosphorus to commercial fertilizers is expensive. Moreover, excessive fertilizer use leads to soil and water contamination, negatively affecting both terrestrial and aquatic ecosystems [67]. Azospirillum species metabolize sugars to produce organic acids, which aid in phosphate solubilization [68]. However, not all Azospirillum strains possess this ability. Some strains have been found to solubilize phosphate even in the absence of root exudates, whereas other microorganisms utilize different mechanisms for phosphate solubilization [69].

4. Genetics of Nitrogen Fixation by Azospirillum

Azospirillum species can fix atmospheric nitrogen under low-oxygen conditions when ammonia is unavailable (Figure 4a). This process is mediated by the nitrogenase enzyme complex, which catalyses the conversion of atmospheric nitrogen (N₂) into ammonia. BNF is a critical process for plant growth and soil fertility. Ammonium is then further converted into nitrate (NO₃⁻) by nitrifying bacteria and transported through the xylem to the shoot as nitrate and nitrogenous organic compounds. Denitrifying bacteria can return nitrogen to the atmosphere [70].

The nitrogenase enzyme complex consists of two main components: dinitrogenase, a molybdenum-iron (MoFe) protein encoded by the nifDK gene that contains the active site responsible for nitrogen reduction, and dinitrogenase reductase, an iron (Fe) protein encoded by the nifH gene that provides electrons for nitrogenase activity [71] (Figure 4b). Electrons are transferred from reduced ferredoxin or flavodoxin through azoferredoxin to molybdoferredoxin. The BNF process requires 16 ATP molecules for every molecule of nitrogen reduced, with the hydrolysis of ATP facilitated by the NifH protein. The resulting ammonia (NH₃) is assimilated into organic forms, primarily glutamine and glutamate, which serve as key components in nitrogen metabolism [27].

Nitrogen fixation is an energy-intensive process, requiring at least 16 ATP molecules for the reduction of each N₂ molecule [72]. The synthesis and activity of nitrogenase are tightly regulated in all diazotrophic bacteria, including Azospirillum. Structural genes responsible for nitrogen fixation (nifHDK) in Azospirillum brasilense and Azospirillum lipoferum have been identified based on their homology with nifHDK genes in Klebsiella pneumoniae [73,74,75]. The A. brasilense genome contains additional genes (nifE, nifP, nifUS, nifV, and fixABC) that are clustered together and play essential roles in nitrogenase function and electron transport [76,77,78,79].

Multiple studies have demonstrated that nifHDK genes are exclusively expressed under nitrogen-limiting and microaerobic conditions [80,81,82,83,84]. In Azospirillum, transcription of nifHDK requires the alternative sigma factor σ⁵⁴ (RpoN or NtrA) and the transcriptional activator NifA (Table 2) [84,85,86,87]. Mutants with inactivated ntrA genes exhibit a nif- phenotype, confirming that both σ⁵⁴ and NifA are essential for activating nitrogenase genes [78,85]. The A. brasilense nifHDK operon is regulated by a σ⁵⁴-dependent promoter (-24/-12) with upstream activator sequences (UAS) required for NifA-mediated activation [81,88].

The nifA gene was identified through heterologous hybridization with the nifA gene from Bradyrhizobium japonicum and was located separately from the main nifHDK gene cluster [85,89]. The A. brasilense NifA protein shares conserved features with other NifA proteins, including a central regulatory domain and helix-turn-helix DNA-binding motif in the C-terminal region. Additionally, the conserved cysteine residues in NifA may play a role in oxygen sensitivity [90,91]. Although nifA transcription is constitutive, its expression levels vary depending on oxygen and nitrogen availability. Under nitrogen-fixing conditions, nifA transcription was maximized. However, in the presence of ammonia (NH₃) and low oxygen levels, transcription decreases to 60% of its derepressed state and further drops to 30% when both oxygen and ammonia are present [82,85].

Unlike Klebsiella pneumoniae, where the general nitrogen regulatory proteins NtrB and NtrC are required for nifA transcription [92], A. brasilense does not require ntrBC genes for nitrogen fixation [93]. A. brasilense mutants lacking ntrBC do not exhibit a nif- phenotype, although NtrC is necessary for optimal nitrogen fixation and maximal nifA expression under nitrogen-fixing conditions. However, NtrC does not directly activate the nifA promoter because no NtrC binding sites have been found upstream of nifA [85].

The constitutive expression of nifA leads to the production of inactive NifA, which is activated only under nitrogen-fixing conditions. This activation process involves glnB, which encodes the PII protein. A. brasilense glnB mutants exhibit a complete nif- phenotype, where nifA transcription remains unaffected, but nifH expression is abolished [94]. In A. brasilense, glnB and glnA are closely associated with the chromosome and play crucial roles in nitrogen fixation regulation.

Figure 4. Azospirillum facilitates biological nitrogen fixation in plants. (a) Schematic representation of biological nitrogen fixation in plants facilitated by the nitrogen-fixing bacterium Azospirillum. Atmospheric nitrogen (N₂) is converted into ammonium (NH₄⁺) or ammonia (NH₃) through the activity of the nif gene in root-associated bacteria. Ammonium is then further converted into nitrate (NO₃⁻) by nitrifying bacteria and transported through the xylem to the shoot as nitrate and nitrogenous organic compounds. Denitrifying bacteria can return nitrogen to the atmosphere. (b) Illustration of the nitrogenase enzyme complex and its role in nitrogen metabolism. Electrons are transferred from reduced ferredoxin or flavodoxin through azoferredoxin to molybdoferredoxin. The nitrogen fixation process requires 16 ATP molecules for every molecule of nitrogen reduced, with the hydrolysis of ATP facilitated by the NifH protein. The resulting ammonia (NH₃) is assimilated into organic forms, primarily glutamine and glutamate, which serve as key components in nitrogen metabolism. NifJ: Pyruvate flavodoxin/ferredoxin oxidoreductase. NifF: Flavodoxin/Ferredoxin [95].

Figure 4. Azospirillum facilitates biological nitrogen fixation in plants. (a) Schematic representation of biological nitrogen fixation in plants facilitated by the nitrogen-fixing bacterium Azospirillum. Atmospheric nitrogen (N₂) is converted into ammonium (NH₄⁺) or ammonia (NH₃) through the activity of the nif gene in root-associated bacteria. Ammonium is then further converted into nitrate (NO₃⁻) by nitrifying bacteria and transported through the xylem to the shoot as nitrate and nitrogenous organic compounds. Denitrifying bacteria can return nitrogen to the atmosphere. (b) Illustration of the nitrogenase enzyme complex and its role in nitrogen metabolism. Electrons are transferred from reduced ferredoxin or flavodoxin through azoferredoxin to molybdoferredoxin. The nitrogen fixation process requires 16 ATP molecules for every molecule of nitrogen reduced, with the hydrolysis of ATP facilitated by the NifH protein. The resulting ammonia (NH₃) is assimilated into organic forms, primarily glutamine and glutamate, which serve as key components in nitrogen metabolism. NifJ: Pyruvate flavodoxin/ferredoxin oxidoreductase. NifF: Flavodoxin/Ferredoxin [95].

Table 2. Bacterial genes involved in the interaction of Azospirillum with the plant root.

Gene	Function	References
nifH	Dinitrogenase reductase.	[21]
nifD	Subunit of dinitrogenase, FeMo-co biosynthesis,	[96]
nifK	Subunit of dinitrogenase	[96]
nifA	Nitrogen fixation (nif) genes transcriptional activator (Regulatory element)	[21]
nifN	Synthesis of FeMo-co	[96]
nifX	Involved in FeMo cosynthesis	[96]
nifB	Required for Fe-Mo cosynthesis	[96]
nifU	Participates in the mobilization of iron for the production and repair of Fe-S clusters	[96]
nifS	Participates in the mobilization of S for the production and repair of Fe-S clusters	[96]
glnA	Glutamine synthetase structural gene	[21]
amtB	Transporter of the structural gene ammonium	[21]
glnZ	PII homologue	[21]
glnD	The enzyme uridylyl-removing/uridylyl-transferase (UTase/UR)	[21]
ntrB	The two-component regulatory system sensor protein involved in overall nitrogen regulation	[21]
ntrC	The two-component regulatory system regulator protein involved in overall nitrogen regulation	[21]
ntrA, rpoN	A different sigma factor	[21]
draT	Reduced dinitrogenase ADP ribosyl-transferase	[21]
draG	Glycohydrolase activation by dinitrogenase reductase	[21]

Table 2. Bacterial genes involved in the interaction of Azospirillum with the plant root.

Gene	Function	References
nifH	Dinitrogenase reductase.	[21]
nifD	Subunit of dinitrogenase, FeMo-co biosynthesis,	[96]
nifK	Subunit of dinitrogenase	[96]
nifA	Nitrogen fixation (nif) genes transcriptional activator (Regulatory element)	[21]
nifN	Synthesis of FeMo-co	[96]
nifX	Involved in FeMo cosynthesis	[96]
nifB	Required for Fe-Mo cosynthesis	[96]
nifU	Participates in the mobilization of iron for the production and repair of Fe-S clusters	[96]
nifS	Participates in the mobilization of S for the production and repair of Fe-S clusters	[96]
glnA	Glutamine synthetase structural gene	[21]
amtB	Transporter of the structural gene ammonium	[21]
glnZ	PII homologue	[21]
glnD	The enzyme uridylyl-removing/uridylyl-transferase (UTase/UR)	[21]
ntrB	The two-component regulatory system sensor protein involved in overall nitrogen regulation	[21]
ntrC	The two-component regulatory system regulator protein involved in overall nitrogen regulation	[21]
ntrA, rpoN	A different sigma factor	[21]
draT	Reduced dinitrogenase ADP ribosyl-transferase	[21]
draG	Glycohydrolase activation by dinitrogenase reductase	[21]

5. Application of Azospirillum spp. In Stress Management, Phytoremediation, Biofortification, and Biocontrol in Modern Agriculture

Azospirillum spp. improve plant growth and yield under suboptimal conditions by enhancing the chlorophyll of 48.57 SPAD (soil plant analysis development) with nitrogen at 100 ppm, increasing potassium (K), calcium (Ca), soluble saccharides, and protein content [97]. Additionally, they help mitigate stresses, such as drought and high osmotic (NaCl) stress. Some strains can thrive even in mine tailings or contaminated soils. Under limited nutrient conditions, A. lipoferum produces catechol-type siderophores with antibacterial activities against various bacterial and fungal isolates.

Azospirillum has been widely used as a primary inoculant for commercial crop production worldwide. It colonizes vegetables, cereals, and various other plants, significantly promoting plant growth, making it a valuable bacterium for biofertilizer formulation. Unlike rhizobia, Azospirillum forms an associative relationship with non-leguminous plants without inducing the formation of new organs such as nodules.

Beyond agriculture, Azospirillum contributes to environmental sustainability by aiding soil stabilization, particularly in desert regions, where vegetation regeneration is challenging. Promoting plant growth in degraded soils helps to combat soil erosion and supports phytoremediation strategies. Through interactions with soil microbes, Azospirillum can assist in detoxifying contaminated environments, thereby fostering healthier ecosystems [98].

The bacterium benefits plants through associative nitrogen fixation, supplying between 20 and 40 kg N/ha in the rhizosphere. Additionally, it plays a role in hormone synthesis and ethylene precursor regulation, which helps adjust plant hormonal balance, further enhancing growth and stress resilience [99]. Azospirillum is also used as a biocontrol agent to improve the nutritional quality of food crops and to assist in soil decontamination. Notably, when introduced into maize plants, Azospirillum has been observed to induce substantial quantitative and qualitative changes in secondary metabolites, particularly benzoxazinoids, which may play a role in plant defence mechanisms [100].

Its diverse applications in biofertilization, biocontrol, and environmental restoration underscore Azospirillum’s importance as a sustainable agricultural tool with potential benefits for global food security and ecosystem health.

Several countries are currently concerned about the pollution of large portions of agricultural soils by heavy metals and agrochemicals. Phytoremediation, a remediation technique that uses soil microorganisms, has been found to reduce the harmful effects of these pollutants effectively.

In addition to its known agricultural benefits, Azospirillum possesses characteristics that can be advantageously applied to agroecosystems [12,101]. They play a crucial role in phytoremediation by colonizing the plant roots and enhancing hydrocarbon degradation in the rhizosphere. Studies have shown that Azospirillum can efficiently degrade polycyclic aromatic hydrocarbons (PAHs) in contaminated soil [102].

Through phytoremediation, A. brasilense has been found to stimulate the growth of the shrub Atriplex lentiformis (Quailbush) and alter the microbial population in the rhizosphere [103]. The strain A. brasilense SR80 is particularly effective in degrading crude oil and other petroleum hydrocarbons, contributing to bioremediation efforts [104].

Additionally, biofilms capable of hydrocarbon degradation have been found to contain Azospirillum species [105,106]. As part of the maize rhizosphere microbial community, Azospirillum contributes to the degradation of crude oil in contaminated soils [107]. Some studies have suggested that this bacterium forms biofilms enriched with nitrogenous compounds, which enhances microbial hydrocarbon degradation [105].

Furthermore, two Azospirillum species, A. rugosum [108] and A. oleiclasticum [11], were isolated from oil-bearing samples, and their ability to degrade oil in contaminated water was demonstrated. The metabolic capabilities of Azospirillum allow it to break down hydrocarbons, aid in soil bioremediation, and reduce the detrimental effects of pollutants on plant health.

Currently, increasing crop output and yield have become the primary focus of conventional agriculture, often at the expense of nutritional quality. Enhancing the nutritional value of food crops through various biotechnological methods, traditional plant breeding, and agronomic approaches is known as biofortification or biological fortification [109]. Biofortified foods are gaining popularity as an effective strategy for addressing nutritional deficiencies and providing healthier dietary alternatives. To promote environmentally friendly methods and improve the nutritional quality of crops, three primary approaches are used: transgenic (biotechnology), conventional (crop breeding), and agronomic (fertilization techniques). Studies have shown that inoculating crops such as sorghum, canola, and maize with Azospirillum spp. can enhance their nutritional quality [110]. Some studies have also suggested that Azospirillum may increase the levels of bioactive compounds, such as antioxidants, flavonoids, and carotenoids in plants, which offer significant health benefits to humans.

Although Azospirillum has long been reported to exhibit antipathogenic activity, its significance as a biocontrol agent has been overlooked. Azospirillum is believed to act as a biocontrol agent through several mechanisms, including competition for environmental resources, inhibition of parasitic weed germination, suppression of fungal growth through the production of toxic compounds, and promotion of plant growth to enhance pathogen resistance [29].

The combined use of mycorrhizal fungi, phosphate-solubilizing bacteria, and Azospirillum has been shown to reduce populations of the nematode Pratylenchus zeae and increase maize cob yield [111]. Additionally, Azospirillum spp. inhibit the germination of the parasitic plant witchweed (Striga hermonthica) in sorghum crops [112].

Although research on this topic remains limited, certain Azospirillum strains can produce bioactive compounds that are toxic to, or directly inhibit, phytopathogens. Many of these compounds are siderophores and small molecules that are capable of reversibly chelating iron. By sequestering iron, Azospirillum spp. gain a competitive advantage over microbes that do not produce siderophores, particularly in fungi. This mechanism restricts fungal growth by reducing the available iron in the rhizosphere while simultaneously enhancing iron uptake by the plant, thereby supporting plant health [113].

Plants possess specialized defence mechanisms to protect themselves against pathogenic attacks. Abiotic stresses, such as extreme temperatures, flooding, drought, salinity, and metal or nutrient imbalances, can negatively affect plant growth [114]. In response, certain plant growth-promoting bacteria (PGPB) can induce plant defence mechanisms, a process known as Induced Systemic Resistance (ISR), which enhances resistance against harmful bacteria, viruses, and fungi [115].

Plant systemic resistance is categorized into two types: systemic acquired resistance (SAR), triggered by pathogenic microbes, and Induced Systemic Resistance (ISR), triggered by non-pathogenic microbes such as PGPB [116,117].

When PGPB are introduced into the soil, plants exhibit four key responses: interaction of microbial antagonists within the rhizosphere, promotion of plant growth, reduction in symptom expression, and induction of structural and biochemical defense responses, including cell wall reinforcement, synthesis of phytoalexins, production of pathogenesis-related (PR) proteins, and activation of systemic resistance [118].

Non-pathogenic microorganisms trigger ISR, which originates in the initially affected tissues and spreads systemically throughout the plant. This enhances the ability of neighboring tissues to resist pathogenic infections, enabling a faster and more efficient defence response [119,120].

Broek and Vanderleyden [87] reviewed advancements in Azospirillum–plant root association, focusing on phytohormone biosynthesis, nitrogen fixation, root attachment, and genetic analysis of Azospirillum brasilense megaplasmids. Azospirillum fixes nitrogen under low oxygen- and nitrogen-deficient conditions, with nitrogenase activity regulated at the transcriptional and posttranslational levels. Root attachment occurs in two phases: an initial adsorption phase mediated by the polar flagellum and a subsequent anchoring phase facilitated by an unidentified calcofluor-binding surface polysaccharide. Genetic analysis of the p90 megaplasmid in A. brasilense revealed multiple genes involved in flagellation that are crucial for motility and attachment [87].

Although Azospirillum brasilense is the most extensively studied and widely applied species in agricultural bioinoculant development, other members of the genus, such as A. lipoferum, A. amazonense, A. halopraeferens, A. thiophilum, and A. irakense, also exhibit plant growth-promoting capabilities. The species has shown effective siderophore production, nitrogen fixation, and root colonization in cereal crops [121]. Gureeva et al. [122] reported that Azospirillum thiophilum, A. brasilense, A. picis, A. irakense, A. halopraeferens, A. lipoferum, and A. baldaniorum are also capable of reducing oxidative stress in Triticum aestivum L. (wheat) when exposed to heavy metals, such as Cu, Ni, and Pb. Sun et al. [123] stated that A. lipoferum CRT1 can trigger pre-germination or defense-related responses, enhance bacterial colonization on plant surfaces, reduce levels of energy-rich primary metabolites, and promote both root surface development and photosynthetic efficiency in three-leaf-stage plantlets. Despite their potential, these species remain underrepresented in the literature compared with A. brasilense, which has benefited from early discovery, ease of cultivation, and consistent results across diverse plant hosts. This historical research bias, combined with regulatory familiarity, has contributed to the dominance of A. brasilense in commercial and academic contexts. Nevertheless, expanding the exploration of other Azospirillum species could reveal novel interactions and broader applications, particularly in region-specific or stress-prone agricultural systems. Therefore, further comparative studies are warranted.

Azospirillum spp. are effective plant growth-promoting rhizobacteria (PGPR) that enhance crop tolerance to various abiotic stresses, including drought, salinity, and heavy metal contamination. Arzanesh et al. [124] demonstrated that Azospirillum lipoferum strains (B1, B2, and B3) significantly improved wheat (Triticum aestivum L.) growth under drought stress. Strain B3, which exhibited high nitrogen fixation, auxin production, phosphate solubilization, and ACC deaminase activity, increased wheat yield by 43% under moderate (S1) and 109% under severe (S2) drought stress, compared to uninoculated controls. Strain B2, which showed siderophore production, showed the highest drought resistance. da Cunha et al. [125] reported that salt (NaCl > 0.3 mol L⁻¹) and thermal (>35 °C) stress reduced Azospirillum brasilense growth, but salt-stressed inoculants improved viability on maize roots, indicating osmoadaptation. Gureeva et al. [122] found that A. picis B-2897T mitigated heavy metal toxicity (Ni and Pb), reducing their uptake in wheat seedlings and enhancing the expression of detoxification genes (NDOR and GST). These studies validated Azospirillum as a versatile agent for stress mitigation. The Azospirillum genus can activate plant defence mechanisms and enhance crop yields under stressful conditions after inoculation into the rhizosphere (Figure 5). They are also resistant to various biotic and abiotic stressors [114]. They can tolerate certain metals and toxic compounds, including hydrocarbons, pesticides, and other environmental pollutants.

6. Molecular Tools Techniques to Study Azospirillum Species

Multiomics approaches have emerged as powerful tools in biotechnology, enabling a comprehensive understanding of complex biological systems to unravel molecular mechanisms and optimize applications in agriculture and environmental sustainability [126,127]. Ferrarezi et al. [128] used a combination of microbiomics (species-specific qPCR, 16S rRNA metataxonomics, and metagenomics) and plant phenomics to study the performance of Azospirillum brasilense Ab-V5 in maize grown in irradiated and native soils with varying microbial compositions. Their findings demonstrated that the efficiency of Azospirillum depends on its persistence and functional niche occupation within the holobiont, with enhanced plant growth observed in diluted native soils and under irradiated conditions. Genes involved in ribose metabolism, amino acid pathways, and cresol degradation were notably enriched in favorable treatments, suggesting that Azospirillum recruits beneficial microbial functions. Similarly, Coniglio et al. [129] employed 16S rRNA sequencing to reveal that Azospirillum argentinense Az39 colonized maize rhizospheres and significantly altered the microbial community structure, increasing the abundance of beneficial genera, such as Burkholderia, Massilia, and Sphingobium. Co-occurrence network analysis further highlighted the positive interactions with Pseudomonas and functional shifts linked to nitrogen cycling and chemoheterotrophy. Bigatton et al. [130] and Omotayo et al. [131] also emphasized microbial gene profiling using metagenomics, confirming that PGPBs influence the abundance of nitrogen-related genes and stress-resistance functions in the rhizosphere. Collectively, these studies illustrate that integrating multiomics technologies provides a deeper understanding of how Azospirillum-based biofertilizers function, offering new strategies for optimizing microbial products in sustainable agriculture.

Azospirillum and other related PGPRs have been studied using molecular approaches, including gene functional analysis, gene knockout, genetically modified strain creation, and gene expression research. While more stable, wide-range vectors or unstable suicide plasmids have been utilized to effect chromosomal alterations, conjugation has also been used for genetic transformation. In this process, cell-to-cell contact occurs, and mobilizable genetic elements are transferred by a conjugative pilus. Azospirillum has been subjected to genetic transformation to employ constitutively expressed reporter genes for genetic labelling, promoterless reporter gene translation cassettes for gene expression studies, genetic engineering through the introduction of new genes, and random and site-directed transposon-induced mutagenesis for gene functional analysis. Plasmids were present in all the strains of A. lipoferum and A. brasilense. Up to six plasmids, ranging in size from four MDa to more than 300 MDa, are present in some strains [132,133]. A. brasilense Sp7 has two plasmids with molecular weights greater than 300 MDa, and three other large plasmids, including p115. The bacterial RNA polymerase sigma factor component is responsible for promoter specificity [134]. Plasmid vectors can be used to transform DNA into recipient cells. For example, the cosmid pLAFR3 was used to build Azospirillum genomic libraries [135]. As suicide vectors enable DNA mobilization into Azospirillum without stable integration of the entire vector, they are useful for chromosomal exchange, transposon mutagenesis, and gene deletion. Rather, vector-borne DNA is substituted for the host DNA by double recombination processes. Biparental conjugation is used for genetic modification of Azospirillum. In this process, both donor and recipient strains are necessary for transformation. The donor strain, which is frequently E. coli, must incorporate the transfer functions of the broad-host-range Inc P-type plasmid RP4 into its genome to mobilize plasmids that are not self-transmissible. Two strains of plasmid-mobilizing E. coli donors, Azospirillum SM10 and Azospirillum S17-1, can use a variety of gram-negative bacteria as recipients [136]. When plasmid DNA from another strain of E. coli that does not have RP4 transfer elements is recruited into Azospirillum, the third parent strain acts as a helper to deliver the transfer gene in trans. This process is known as triparental conjugation. A common helper strain for E. coli HB101 is pRK2013 (tra+), which is a helper plasmid [137].

The discovery of denitrification genes in mobile genetic elements in Azospirillum sp. TSH58 suggests that horizontal gene transfer is a mechanism by which this strain acquires nitrogen-cycling capabilities [138]. Transcriptomic investigations of Azospirillum brasilense Sp245 have revealed short RNAs that are responsive to nitrogen deficiency, suggesting that these RNAs may play a role in controlling interactions between microbes and plants [139].

7. Genetic Engineering in Azospirillum and Its Benefits

The non-pathogenic soil bacteria Azospirillum have been studied to encourage the growth of a variety of plants, making them a desirable model to comprehend beneficial, non-symbiotic plant-bacteria relationships. Identification of specific genes in these bacteria that promote plant growth is crucial for enhancing productivity and improving crop quality through genetic engineering, allelic exchange mutagenesis, transgene expression, and fusion of reporter genes [140]. Azospirillum contains a nitrogenase enzyme complex in its genome that supplies fixed nitrogen to the global nitrogen cycle. Because nitrogen fixation is thought to be the primary method by which Azospirillum promotes plant development, genetic studies initially concentrated on this process. Later, it was discovered that Azospirillum spp., pathogens, symbionts, and plant growth-promoting rhizobacteria were identical in terms of host plant recognition and affinity phenomena, hormone synthesis, and root morphological changes [141]. One strategy for identifying the genes of Azospirillum involved in interactions with plants is to compare the Azospirillum genome with the genomes of other plant-associated bacteria. The p90-encoded genes of A. brasilense Sp7 play a crucial role in its interaction with plant roots. Sequencing, annotation, and in silico studies of this 90-MDa plasmid have revealed the presence of a substantial number of genes encoding enzymes involved in the production of surface polysaccharides [142]. A. lipoferum’s 300-MDa plasmid encodes melanin, which is created through Tn5 mutagenesis. [143]. Transposon mutagenesis revealed that the 150-MDa plasmid of A. lipoferum [144] and p85 of A. brasilense Sp245 [145] encode enzymes producing anthranilic acid, a precursor of tryptophan (Trp). The identification and characterization of these genes, as well as additional genes, will aid in understanding the mechanisms by which this bacterium induces plant growth and development. Furthermore, enhancing the evolutionary stability of Azospirillum sp. bioengineered systems for ammonia production, utilizing metabolic engineering during biomanufacturing, shifting control between growth and production, optimizing pathways of essential gene production, employing cell-sorting techniques to homogenize a heterogeneous population at the early inoculum stage, reducing mutation rates by deleting host genes implicated in genome plasticity, and using CRISPR interference against mobile elements are valuable approaches [146]. Along with nitrogen-fixing genes, Azospirillum sp. possesses genes for phytohormone synthesis. Inoculating wheat seedlings with the A. brasilense NifP IAAc morphotype improved the number and size of lateral roots compared with the wild type. However, inoculation with NifP, a low IAA producer, resulted in no reaction from the plant [147]. Azospirillum inoculation can mimic the effects of IAA on wheat root elongation and branching [148]. However, synthetic hormones do not always replicate the effects of Azospirillum spp. [149]. It has been discovered that tryptophan-2-monooxygenase (TMO) activity exists in A. brasilense, indicating the presence of the IAM route [150,151]. The iaaM gene, which encodes TMO in Pseudomonas syringae, shows partial similarity to genomic DNA from A. brasilense. In nutrient-rich medium, the A. brasilense ipdC gene was highly expressed, whereas in minimal nutrient medium, IAA synthesis was high, and ipdC expression was low. These findings imply that distinct IAA production pathways are activated depending on the growth medium used [152]. Modification of the genes responsible for the synthesis of plant hormones can be useful for ensuring consistent hormone production and increasing plant yield. Root exudates may play a crucial role in the bacterial colonization of cereal roots and nitrogen fixation. Azospirillum obtains carbon from organic acids. By identifying, cloning, and sequencing a gene that promotes the production of carbohydrates in A. brasilense, Chattopadhyay et al. [153] used a 2.8-kb genomic region of A. brasilense RG to complement a pleiotropic carbohydrate mutant (car mutant) of the same strain that overlapped the fru operon and was incapable of producing carbohydrates. Three ORFs were identified by DNA sequence analysis: carR and carS for ORFs 2 and 3, respectively, which are responsible for utilizing carbohydrates to create carbon. Azospirillum may benefit from improved carbohydrate metabolism if these two genes are genetically modified to consume more carbohydrates, which would encourage plant growth. More research should be conducted on the manipulation, insertion, and modification of genes found in Azospirillum sp. to create more productive strains of the bacterium that generate exopolysaccharides (EPS), have the ability to form biofilms, and improve colonization and root adhesion. Thus, through genetic engineering, Azospirillum can be developed as a powerful tool for sustainable agriculture to address global concerns, including soil erosion, climate change, and food security.

Gene knockout and CRISPR/Cas9 technologies have significantly advanced the field of genetic engineering by enabling precise and targeted genome modifications. However, they have certain limitations. A primary concern with CRISPR/Cas9 is the risk of off-target effects, in which unintended mutations occur in genomic regions with sequence similarity to the target site, potentially leading to undesirable or harmful phenotypic outcomes. In the case of gene knockouts, the complete disruption of gene function may trigger compensatory mechanisms or pleiotropic effects, making it difficult to accurately interpret gene function. Moreover, the regulatory landscape for genetically modified organisms (GMOs) remains complex and highly restrictive in many regions, particularly in terms of agricultural and environmental applications. Approval processes can be lengthy and costly, and public resistance to GMOs further complicates their adoption [154]. These challenges highlight the necessity of developing more precise genome editing tools and fostering regulatory frameworks that balance innovation with biosafety and ethical considerations.

8. Formulation of Inocula and Industrial Production of Azospirillum Species

The formulation of bioinoculants is a multi-step process aimed at ensuring microbial survival, stability, and functionality for sustainable agriculture. It starts with selecting microbial strains that exhibit plant growth-promoting (PGP) traits such as nitrogen fixation, nutrient solubilization, siderophore and antibiotic production, and phytohormone synthesis. These strains must demonstrate genetic stability, adaptability to environmental conditions, and efficient plant colonization. Carrier materials play a critical role in transporting these beneficial microbes from the laboratory to the field. Common carriers include peat, talc, vermiculite, alginate, and charcoal, whose properties—moisture retention, pH, C: N ratio, and biodegradability—affect microbial viability and shelf life. Sterilization of these carriers through autoclaving or gamma irradiation helps prevent contamination during formulation. For instance, a peat and sugar-based carrier for Azospirillum brasilense significantly improved wheat growth [155], while a CMC (Carboxymethyl Cellulose) or Cellulose Gum is a modified starch or additive carrier which have been combined with MgO to enhance the shelf life and root colonization of Azospirillum amazonense in sugarcane [156].

Desiccation tolerance of microbial strains is tested using techniques like freeze-drying or spray-drying, often with protectants like trehalose or stress priming to enhance resilience. Storage stability is evaluated by monitoring colony-forming units (CFU), with a minimum threshold of ≥10⁸ CFU/g required for effective formulations. Microencapsulation technologies use alginate or synthetic polymers to provide controlled release and protection against environmental stress. Liquid formulations incorporating stabilizers like (Polyvinylpyrrolidone) PVP, trehalose, and glycerol further improve shelf life and application stability. Validation through field trials, registration, and commercial testing is essential for deployment. These strategies collectively improve soil fertility, reduce chemical input, and promote sustainable agriculture [157,158]. Without a suitable carrier, microbial suspensions of Azospirillum spp. lose viability rapidly when applied to soil or seed surfaces (Figure 6) [159]. Historically, bacterial suspensions have been applied directly to seeds or seedlings. In South America, Azospirillum-based inoculants are widely used for crop enhancement [160], although soil inoculation remains less effective compared to rhizobia. Seed coating with biofertilizer formulations enhances early colonization and symbiotic interactions, ensuring direct root contact [161]. Soil applications, in liquid or granular form, increase microbial diversity and promote rhizospheric activity. A minimum of 10⁶ to 10⁷ Azospirillum brasilense cells per plant is required for efficacy [162]. Such inoculants enhance nutrient cycling, soil quality, and are suitable for various crops and soil types [163].

Effective inoculants are essential for successful biofertilizer application and must satisfy three critical criteria. First, they should promote robust microbial growth, ensuring the beneficial microbes can establish themselves and proliferate effectively. Second, they must sustain a viable microbial population under optimal conditions, including proper storage (temperature 4–25 °C, low humidity, darkness), environmental stability (temperature, humidity, light, pH, oxygen levels), and an appropriate method of application (seed coating or seed treatment, soil application, foliar spray, root dipping), which collectively preserve their efficacy over time [4,158]. Third, at the time of use, the inoculant should deliver an adequate number of live, active microbes to the plant rhizosphere to stimulate a measurable plant growth response, nutrient uptake, or other intended agricultural benefits. In essence, the formulation phase is pivotal, as it ensures the successful transfer of living microbes from production to application. Despite minor advances, traditional seed coating technology has remained largely unchanged. Typically, seeds are coated with a peat-based inoculant using water or adhesives, followed by drying. Small seeds are also treated with finely crushed limestone and adhesives like jaggery or gum acacia to retain inoculants during seeding. Fluidized bed techniques and mechanical mixers such as tumblers and spinning drums are also used. Larger farms often utilize automated seeders equipped with inoculant tanks and application chambers. Adhesives prevent inoculant loss during handling and seeding, particularly for powdered inoculants applied with air seeders [163]. Liquid inoculants may be sprayed directly onto seeds with or without dissolved adhesives before drying. However, seed inoculation has inherent drawbacks: (a) limited inoculant per seed, especially small ones, which may fall below the Azospirillum efficacy threshold [162]; (b) poor adhesive binding may cause detachment during sowing; (c) seedling exposure to environmental stress may lead to microbial desiccation and death; (d) some seeds release antimicrobial compounds that inhibit inoculants; and (e) pesticides or fungicides on seeds may harm microbial viability [12,29].

Field application of Azospirillum-based biofertilizers remains challenging. Although controlled studies show promise, inconsistent performance in the field due to varying soil types, climates, and crop species hampers scalability. Storage and shelf-life issues also persist, with some formulations requiring refrigeration or degrading under fluctuating temperatures. Efficacy also varies with application methods such as seed coating, soil drenching, or foliar spraying. Adoption among farmers is further limited by a lack of awareness, skepticism, and inadequate extension support. For broader implementation, formulations must be cost-effective, user-friendly, and supported by effective outreach. While success stories like improved maize yields in Brazilian fields show great promise, real-world adoption hinges on overcoming these practical and socioeconomic barriers [164].

Recently, techniques for immobilizing live cells have gained widespread use in agriculture. Azospirillum immobilization restricts the mobility of microorganisms in solid or semi-solid media, thereby extending their beneficial effects. Immobilized bacterial cells retain high catalytic activity, which enhances their adaptation and survival in harsh environments [165]. Microbial cell encapsulation simplifies soil application while protecting bacteria from environmental stress. This approach provides a high cell-loading capacity, maintains cell viability, and improves microbial product efficiency [166].

The selection of carriers for immobilization is primarily based on factors such as a large surface area, mechanical stability, regeneration capacity, and non-toxicity toward immobilized organisms. In addition, carriers should be cost-effective and offer maximum permeability [167]. For example, ionic gelation has been used to create 2% alginate beads containing immobilized Bradyrhizobium sp. SEMIA6144 or Azospirillum brasilense Az39 cells were stored at 4 °C for up to one year. The alginate matrix improved bacterial survivability and interactions with Arachis hypogaea, ensuring the continuous release of cells [23].

Other immobilization materials for Azospirillum species include agar, carrageenan, polyacrylamide, chitosan, and silica-based compounds. The plant growth-promoting rhizobacterium A. brasilense strain Az has been studied for its potential role in enhancing tomato seedling growth, as well as its production of hydrolytic enzymes and nitrogen fixation [168]. Both free and calcium alginate-encapsulated cells were used in inoculation studies, and all fresh and dry macrobeads showed high encapsulation capacity (EC%) for the inoculant [168].

The immobilization of microorganisms, particularly Azospirillum spp., has become a powerful biotechnological tool in agriculture, offering a long-term strategy for improving plant growth and soil health. This technique ensures the sustained activity and survival of beneficial bacteria while enabling their controlled release and protection from environmental stressors. Advances in carrier materials, such as alginate beads, have further enhanced the encapsulation process by improving mechanical stability, permeability, and microbial interactions with plant systems. The ability of immobilized Azospirillum to produce key enzymes and promote plant development highlights its potential for eco-friendly agricultural applications, paving the way for innovative approaches for sustainable farming and environmental management. Various immobilization methods have been developed to improve bacterial stability, activity, and longevity under diverse environmental conditions [168].

9. Field Applications, Commercial Products, and Barriers to Adoption

The global Azospirillum bacterial fertilizer market reached US$620 million in 2022 and is projected to grow to US$923.02 million by 2030 at a CAGR of 5.1% during the forecast period of 2023–2030 [169]. This market expansion reflects an increasing emphasis on sustainable agricultural practices, soil health awareness, and organic farming. According to the FiBL 2021 survey, organic agriculture is practiced in 187 countries, with Australia leading with 35.69 million hectares of organic farmland. India, in particular, is home to 30% of all global organic producers and has 2.30 million hectares under organic cultivation. This surge in organic farming has been further supported by government initiatives. For example, the Indian Union Cabinet approved an Rs 1.08 lakh crore fertilizer subsidy in May 2023 to ensure the affordability and availability of fertilizers during the 2023–2024 Kharif season. These incentives, alongside growing consumer demand for eco-friendly farming solutions, have significantly encouraged the adoption of biofertilizers, such as Azospirillum [169]. Azospirillum functions as an effective biological soil conditioner by improving soil physical, chemical, and biological properties. As a nitrogen-fixing, plant growth-promoting rhizobacterium (PGPR), it colonizes the rhizosphere and enhances root proliferation through phytohormone (e.g., IAA, gibberellins) production [158,170]. This increased root biomass releases organic exudates that help bind soil particles, improving soil aggregation and structure. Azospirillum also produces exopolysaccharides (EPS), which enhance soil moisture retention, aeration, and reduce erosion and compaction risks. Its presence boosts microbial activity and organic matter stabilization, contributing to better nutrient cycling and soil fertility. Moreover, it supports the formation of stable microaggregates that increase water infiltration and root penetration [171,172]. Soil inoculation is effective in a wide range of crops, including grains, cereals, pulses, oilseeds, vegetables, and commercial crops. The Asia-Pacific region holds one-third of the Azospirillum bacteria fertilizer market, with countries such as India, China, and Vietnam leading the adoption owing to strong agricultural bases and government support. For instance, Japan’s Green Food System Strategy targets a 30% reduction in chemical fertilizer use by 2050, promoting the integration of biofertilizers, such as Azospirillum [169]. Despite the promising outlook, barriers to widespread adoption remain. One significant challenge is the limited awareness among farmers regarding the benefits and application of Azospirillum fertilizers. Traditional farming practices often dominate, and the perceived risks associated with new biofertilizer technologies discourage experimentation. Additionally, challenges related to storage stability, strain-specific efficacy, and inconsistent field performance have made some farmers reluctant to adopt these products. The COVID-19 pandemic and geopolitical issues, such as the Russia Ukraine war, also impacted production and supply chains, leading to disruptions in fertilizer availability and increasing prices. Reduced production capacity and logistical constraints from key producers such as Russia and Belarus further affected the global fertilizer industry. Nevertheless, ongoing research and development are aimed at overcoming these challenges. For example, Verdesian Life Sciences launched Accolade in January 2023, a liquid biological product containing Azospirillum brasilense to promote early plant growth. Similarly, Embrapa Soja (PR) developed a microbial combination of Azospirillum brasilense and Pseudomonas fluorescens that enhanced Brachiaria pasture productivity by 22% [169].

The Azospirillum bacteria fertilizer market spans several global regions, including North American countries such as Canada, the United States, and Mexico, and in Europe, the market encompasses Germany, Italy, Poland, France, Spain, the Netherlands, Sweden, Denmark, Belgium, Switzerland, the United Kingdom, Russia, Turkey, and other parts of Europe. In the Asia-Pacific region, significant contributors include India, China, Japan, Vietnam, South Korea, Indonesia, Australia, Thailand, Philippines, New Zealand, Singapore, Malaysia, and the rest of the APAC. South America includes Brazil, Argentina, and other countries within the continent. The Middle East and Africa region comprises markets in the UAE, Saudi Arabia, South Africa, Oman, Qatar, Kuwait, and other MEA nations [173]. Among these, Asia-Pacific and African countries are the major consumers of fertilizers because of their rapidly growing populations, which directly drives the demand for increased agricultural productivity. This has led to an upsurge in fertilizer usage, particularly in Asia. However, the adverse effects of chemical fertilizers, such as soil degradation and health hazards, have prompted governments in these regions to promote eco-friendly alternatives, such as organic manure and biofertilizers, including Azospirillum-based products, to mitigate environmental damage. Each country within the market is analyzed for its specific regulatory environment, domestic consumption patterns, production capacity, and import-export dynamics. Important analytical variables include raw-material costs, price trends, production sites, and supply chain logistics. These factors are critical for understanding current market behavior and future trends. Additionally, the presence of international companies was examined in relation to the challenges posed by local competition, domestic tariffs, and trade regulations. Key players shaping the Azospirillum bacteria fertilizer market include The Mosaic Company, Gujarat State Fertilizers & Chemicals Limited (GSFC), National Fertilizers Limited, Agrium Inc., Novozymes A/S, and Chr. Hansen Holding A/S, Rashtriya Chemicals and Fertilizers Limited, Madras Fertilizers Limited, Lallemand Inc., Jaipur Bio Fertilizers, Mapleton Agri Biotec Pvt. Ltd., International Pannacea Limited, Manidharma Biotech Private Limited, Kan Biosystems, Nutramax Laboratories Consumer Care, Inc., Kiwa Bio-Tech Products Group Corporation, Biomax, Vegalab SA, and Biomaxnaturals [173]. A wide range of commercial Azospirillum-based biofertilizers is now available, offering diverse formulations for different agricultural needs. These include Azospirillum (Progress-AS) by Harmony Ecotech Pvt. Ltd., Grow Complex Azospirillum by Growtech Agri Science & Research Pvt. Ltd., Azospirillum Bio Fertilizer by Apm Crops, Bio Promotor Azospirillum by Manidharma Biotech Pvt. Ltd., Power King Azospirillum Liquid by Hindustan Bec Tech India Pvt. Ltd., Navashakti Azospirillum by Navashakti Bio Crop Care Science Pvt. Ltd., Nitro Samrudhhi Azospirillum by Altret Bio Tech Ltd., K-Azospir Power by Khandelwal Bio Fertilizer, Azofix by Amruth Organic Fertilizers, Prabhaazos Azospirillum by Prabhat Fertilizer & Chemical Works, Quick Dry Sun Agro Azos by Laksitha Agro Biotech Pvt. Ltd., AZOSFER by Tari Bio-Tech, AZOLAM by Peak Chemical Industries Ltd., Bio Azospirillum by Special Biochem Pvt. Ltd., Azospirillum by Nova Agri Tech Ltd., Grotop Azospirillum Biofertilizer by Md Biocoals (P) Ltd., Azospirillum Bio Fertilizer by Criyagen Agri and Biotech Pvt. Ltd., Grow Agro Azospirillum Powder by Bhola Agro Industry, and BIO ‘N’ RICH Azospirillum by Sks Bioproducts Pvt. Ltd. Despite commercial success, practical challenges hinder broader adoption. These include farmers’ limited awareness of Azospirillum benefits, hesitancy to shift from conventional practices, concerns over storage stability, inconsistent field performance, and the need for crop-specific formulations. Bridging the knowledge gap through training, policy support, and product refinement is essential for advancing the role of Azospirillum in sustainable agriculture.

10. Novel Nitrogen Fixing Azospirillum Species and Their Efficiency

Azospirillum irakense and Azospirillum amazonense were recently reclassified as Niveispirillum irakense and Nitrospirillum amazonense, respectively [174], based on their phylogenetic, physiological, chemotaxonomic, and phenotypic characteristics. A newly identified nitrogen-fixing species, Azospirillum agricola sp., was isolated from Taiwanese agricultural soil and designated strain CC-HIH038T [175]. Nitrospirillum viridazoti sp., another efficient nitrogen-fixing species, was identified in strain BR 11140T, isolated from grasses and classified under the genus Nitrospirillum [176].

The rhizobacterium Azospirillum sp. strain Sp245T, initially thought to belong to Azospirillum brasilense, has been recognized for its plant growth-promoting properties, particularly its ability to fix atmospheric nitrogen and produce beneficial compounds. In Brazil, Azospirillum sp. Sp245T and related bacteria have been isolated from various plant root surfaces [177].

A newly discovered nitrogen-fixing and heavy oil-degrading bacterium, Azospirillum oleiclasticum, was found in an oil production mixture in the Yumen Oilfield in China. Two strains, RWY-5-1-1T and ROY-1-1-2, which exhibit both nitrogen fixation and heavy oil degradation capabilities, were identified from this mixture [11].

In Russia’s Tver region, a methane oxidation enrichment study conducted in a Sphagnum-dominated elevated peatland led to the identification of a unique nitrogen-fixing bacterium, Azospirillum palustre sp., known as strain B2T. Morphological, chemotaxonomic, and genetic characteristics were analyzed [178].

Additionally, Azospirillum sp. strain CC-LY788T of the Azospirillum species was isolated from cultivated soil in Taiwan [179].

Table 3. Impact of various Azospirillum sp. on the growth and yield of different crops.

Species	Applied Crops	Improved Growth/Yield	Reference
A. lipoferum	Maize	Height 35.33–43.89%	[180]
A. brasilense	Pak choi	Biomass 26–255%	[181]
A. lipoferum	Barley	Root elongation 12.5%, Root biomass 22.22%	[182]
A. brasilense	Tomato	Root biomass 118%	[183]
A. lipoferum	Wheat	Wheat yields up to 109%	[184]
Azospirillum sp. TS13	Komatsuna	Dry weight 40–51%	[185]
A. brasilense	Cucumber	Root length 73.65% Root weight 55.32%, root tips 35.85%	[186]
A. brasilense and Azospirillum sp. BNM-65	Cherry tomato	Dry weight 81–107%, leaves 32–43%, Shoot root dry weight 37–80%, Height 12–143%	[187]
A. lipoferum	Green gram	10.26% of shoot length, 18.28% of fresh weight, 18.45% of dry weight	[188]

summarizes the impact of the application of different Azospirillum sp. for sustainable agriculture.

11. Quorum Sensing (AHLs Pathway) in Azospirillum

Quorum sensing (QS) regulates gene expression in response to fluctuations in cell density. Quorum-sensing bacteria produce and release chemical signaling molecules known as autoinducers, which increase in concentration with increasing cell density [189,190,191]. These bacteria regulate the expression of specific genes through autoinducers, particularly N-acyl-homoserine lactones (AHLs). AHLs control many cellular functions that are essential for plant–bacteria interactions.

QS-mediated regulation is crucial for modulating physiological and metabolic activities in Azospirillum, including nitrogen fixation, biofilm formation, motility, and exopolysaccharide synthesis [192]. As a member of Alphaproteobacteria, Azospirillum promotes plant growth primarily by producing phytohormones, with auxin (indole-3-acetic acid, IAA) being the most abundant. IAA enhances the development of lateral roots and root hairs, leading to improved nutrient and water uptake by inoculated plants.

Despite the agricultural importance of Azospirillum and the availability of sequenced genomes, including Azospirillum sp. B510, A. lipoferum 4 B, A. brasilense Sp245, CBG497, and Az39; there are limited studies on the ability of these bacteria to produce AHL-like molecules or engage in quorum-sensing mechanisms [17,20,193]. Interestingly, Azospirillum sp. Az39 has been found to degrade AHLs and a wide range of other signaling molecules [194].

Owing to the agricultural and economic significance of Azospirillum, interactions between Azospirillum and other Azospirillum, Azospirillum and bacteria, and Azospirillum and plants mediated by quorum sensing remain poorly understood. This finding highlights the need for further genomic and functional studies to elucidate the mechanisms underlying these interactions.

12. Effects of Azospirillum-Based Nano-Biofertilizers on Crop Growth

In recent years, nanotechnology has emerged as a promising tool for revolutionizing sustainable agriculture by enhancing nutrient use efficiency and crop productivity (

Table 4. Synergistic Effects of Nanoparticles and Biofertilizers on Crop Growth, Physiology, and Stress Tolerance in Plants.

Crop	Nanoparticles Used	Biofertilizer Used	Irrigation Level	Growth Parameters Improved	Effects	Notable Outcomes	References
Not specified	Azospirillum-capped ZnO NPs	Azospirillum strains	Not specified	Seed germination (95%), LAI (45.6%), root/shoot length, biomass	Eco-friendly, increased chlorophyll and carotenoids	Potential for sustainable agriculture	[192]
Wheat	Titanium dioxide (nTiO2)	Azospirillum brasilense	Field trial (normal irrigation)	LAI, photosynthesis, nutrient uptake, and antioxidant enzymes	nTiO2 > 40 mg/L harmful; mitigated by A. brasilense	Best combo: 30 mg/L nTiO2 + A. brasilense	[193]
Cotton (Giza 96)	Zinc oxide (ZnO-NPs)	Azospirillum sp.	15, 30, 45-day intervals	Plant height, dry weight, leaf area, chlorophyll, seed yield	CAT, POD, PPO, proline increased under drought	Combined treatment, most effective in both seasons	[194]
Triticale	Nano Fe-Si oxide	A. lipoferum, P. putida	Full, moderate, severe (booting/head)	Chlorophyll index (50.23%), RWC (43.97%), stomatal conductance	Reduced F0, electrolyte leakage; improved FV, yield	Improved physiology and yield under drought	[195]
Wheat	Nano Zn, Fe, Zn-Fe oxide	Azotobacter, Azospirillum, Pseudomonas	Normal, moderate, severe	Yield (88%), photosynthetic pigments, PSII efficiency, RWC	Proline, sugars, CAT, POD, and PPO increased under drought	Zn-Fe oxide + Azotobacter best under stress	[196]

) [171]. Among various innovations, Azospirillum-based nanofertilizers have gained considerable attention because of their dual role as biofertilizers and nanomaterials. Azospirillum, a well-known plant growth-promoting rhizobacterium, facilitates nitrogen fixation and hormone production and improves root development. When combined with nanoparticles such as zinc oxide or titanium dioxide, these bio-nanofertilizers demonstrate enhanced effectiveness in nutrient delivery, stress tolerance, and physiological improvement of crops [2,170]. This synergy not only boosts growth and yield but also reduces the environmental risks associated with conventional chemical fertilizers. The integration of Azospirillum with nanotechnology offers a sustainable approach to address the challenges of food security and climate resilience in modern agriculture.

13. Ecological Effects of the Application of Azospirillum spp.

Although Azospirillum spp. are widely recognized for their plant growth-promoting effects, their introduction into soil ecosystems is risky. One major concern is the disruption of native microbial communities. Inoculation can shift the balance of soil microbiota, potentially altering nutrient cycling, microbial decomposition, and overall functionality of the soil ecosystem [197]. This may result in reduced microbial diversity and increased competition for beneficial indigenous organisms. Moreover, Azospirillum introduction, particularly of genetically modified strains, can have unintended consequences on the broader soil food web, including mesofauna and macrofauna, as demonstrated by litter bag studies assessing decomposition dynamics and mesofaunal populations [198]. Changes in plant composition due to Azospirillum inoculation may indirectly affect detritivores and their predators, such as earthworms and arthropods, ultimately affecting soil health. Another crucial risk is horizontal gene transfer (HGT) which is a natural mechanism among microbes that allows genetic material including antibiotic resistance genes, to move across species via conjugation or plasmid integration [98,199]. Genetically engineered Azospirillum strains developed using HGT may unintentionally transfer genes to nontarget soil microbes, raising ecological and regulatory concerns. To mitigate these risks, long-term monitoring strategies, including assessments of microbial shifts, faunal populations, and soil function using standardized tools, such as litter bags, should be implemented alongside molecular tracking of introduced strains. These biosafety measures are vital for ensuring the use of microbial inoculants under increasing global scrutiny.

14. Future Prospects

Azospirillum holds immense promise for sustainable agriculture because of its multifunctional role in promoting plant growth through nitrogen fixation, hormone production, phosphate solubilization, and biocontrol. However, realizing their full potential requires addressing several research gaps and practical challenges. Future studies should focus on developing crop-specific Azospirillum strains with enhanced stress tolerance and rhizospheric competitiveness. Targeting key nif genes through genetic engineering could improve the nitrogen fixation efficiency under diverse environmental conditions. Research questions should explore how Azospirillum interacts with the native soil microbiota and how these interactions influence root colonization and growth-promotion efficacy. The use of advanced “omics” tools such as genomics, transcriptomics, and metabolomics can help unravel the molecular mechanisms underpinning Azospirillum-plant interactions. Region-specific field trials are essential for evaluating strain performance under varying soil types and climatic conditions. Challenges such as inoculant viability, large-scale production, and field-level applications must also be addressed through improved formulation technologies and delivery systems. Ultimately, integrating Azospirillum into modern agricultural practices will require interdisciplinary strategies involving microbiology, agronomy, and biotechnology, along with farmer education and policy support, to promote widespread and effective adoption. Given these challenges, research on Azospirillum remains a dynamic field, with ongoing efforts to address environmental limitations and microbial interactions. Future advancements in biotechnological applications, improved inoculant formulations, and tailored agricultural strategies are critical for maximizing the potential for sustainable farming. The research gaps and prospects for Azospirillum spp. are summarized in

Table 5. Gaps and Research Innovations.

Molecular Engineering	Despite the nitrogen-fixing capabilities of Azospirillum, the efficiency of this process can vary under different environmental conditions. Engineering strains with enhanced nitrogenase activity or greater tolerance to environmental stressors (e.g., heat, oxygen, nutrient limitations) could significantly improve their performance in agricultural settings.
Co-expression of Nitrogen Fixation Pathways	A lot of Azospirillum strains depend on the nif gene cluster to fix nitrogen. However, more studies could be conducted on co-expressing other nitrogenase systems, like the vnf or anf clusters, to make them work better in places with few nutrients.
Oxygen Sensitivity and Adaptation	Azospirillum’s nitrogenase is highly sensitive to oxygen, making its activity difficult to maintain under oxygen-rich conditions. Innovative strategies to improve the bacterium’s tolerance to oxidative stress, such as the development of oxygen-scavenging systems or genetic modifications to enhance its ability to withstand aerobic conditions, could lead to more effective applications in diverse environments.
Abiotic Stress Resistance	Improving the tolerance of Azospirillum to abiotic stresses (such as salinity, drought, or extreme temperatures) would make the bacteria more versatile and beneficial for plants growing in challenging environments.
Symbiotic Relationships with Plants	Research into the specific signaling mechanisms between Azospirillum and host plants can help optimize their symbiotic interactions. Developing customised inoculants that are more efficient for crops may result from a thorough understanding of the molecular interactions of Azospirillum with various plant species.
Plant Growth-Promotion Mechanisms	Azospirillum is known for fixing nitrogen, but it also produces siderophores, plant hormones, and other secondary metabolites that aid in plant growth. Researchers may be able to increase the positive effects of these extra processes on plant development and stress resistance by recognising and comprehending them.
Nitrogenase Pathways	Alternative nitrogenases (V- and Fe-dependent) could be more efficient than Mo-dependent pathways.
Metagenomics of Soil and Rhizosphere Communities	Studying the complex microbiomes in the rhizosphere and soil can provide insights into how Azospirillum interacts with other microbes. By analyzing metagenomic data from natural environments, researchers can uncover new, more effective strains or microbial consortia that enhance nitrogen fixation or have synergistic effects on plant growth.
Crop Specific Nitrogen Fixation	There is a limitation in transferring the nif genes into cereals like rice and wheat. Need crop-specific engineering to optimize nitrogenase activity under different conditions.
Gene Editing	Optimised Azospirillum strains may be produced by selectively altering genes related to nitrogen fixation, stress tolerance, or plant signalling using CRISPR/Cas9 or other genome-editing methods. This would make it possible to adjust gene expression and produce strains that are suited to particular crops or environmental circumstances.
Cross-species Genetic Exchange	Research into the possibility of transferring beneficial genes from other nitrogen-fixing organisms (like Rhizobium or other Diazotrophs) into Azospirillum could create super-efficient strains capable of better adaptation to different environmental conditions.
Efficient Mass Production	To make Azospirillum inoculants commercially viable, efficient large-scale production methods are to be developed. Optimising culture media, fermentation procedures, and downstream processing are all part of this strategy to increase the production and activity of Azospirillum strains.
Formulation of Bioinoculants	It is important to make stable Azospirillum mixtures that are easy to use on plants or soil while still keeping the bacteria’s effectiveness and viability.
Long-term Effectiveness	Further research is necessary to determine the long-term efficacy of Azospirillum in various soil types and habitats. For sustainable agricultural methods, it is crucial to pursue further research on the persistence of these bacteria in the soil and their potential for either long-term advantages or detrimental effects on soil health.
Biosafety Considerations	The possible environmental impact of releasing transgenic Azospirillum strains must be carefully evaluated. Further research is needed to assess the biosafety of these strains, including the potential for horizontal gene transfer to nontarget organisms.
Carbon–Nitrogen Interaction	Understanding the reduced impacts of rising CO2 levels and improving nitrogen fixation by Azospirillum in the development of plants is an interesting topic of study now. This in-depth understanding could help to trap carbon in soil and lessen the impact of chemical fertilisers on the environment.
Adapting to Changing Environments	The development of Azospirillum strains capable of efficiently fixing nitrogen in higher temperatures, altering precipitation patterns, and elevating salinity will be essential in sustaining agricultural productivity under altered climate conditions.

.

15. Conclusions

Azospirillum sp. have emerged as a valuable tool for promoting sustainable agricultural practices. Its genetic diversity and adaptability demonstrate its evolutionary resilience and ability to thrive in diverse environments, making it a promising candidate for bioinoculant development. Advances in molecular research have provided insights into the various pathways and regulatory mechanisms through which Azospirillum enhances plant growth and yield. This review highlights the significance of Azospirillum spp. in achieving sustainable agricultural goals by improving soil health, increasing crop productivity, and mitigating adverse environmental effects. Further research on the immobilization of Azospirillum could enhance its applicability to challenging agricultural conditions, ensuring its effective use in diverse farming systems.