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Azospirillum lipoferum

Table of contents

Other Names

Spirillum lipoferum

Synopsis

Azospirillum lipoferum

1. Identity: Taxonomy, Natural Source, and Common Forms

Taxonomic Classification

Azospirillum lipoferum is a species of microaerophilic, gram-negative, rod-shaped, nitrogen-fixing bacteria. Its full taxonomic classification places it in the Domain Bacteria; Phylum Proteobacteria; Class Alphaproteobacteria; Order Rhodospirillales; Family Rhodospirillaceae; Genus Azospirillum; Species Azospirillum lipoferum.

The organism was first described by Martinus Beijerinck, who named it Spirillum lipoferum in 1925. The classification was later revised by Tarrand et al., who renamed it Azospirillum lipoferum, a name meaning "small, fat bearing, spiral." Specifically, Spirillum lipoferum was reclassified in 1978 as Azospirillum lipoferum by Jeffery Tarrand, Noel Krieg, and Döbereiner, who also added Azospirillum brasilense to the genus. The prefix "Azo-" comes from the French word "azote," which means nitrogen.

Morphology

Cells are gram-negative, vibroid to straight rods, sometimes curved, highly pleomorphic, with an abundant cytoplasmic accumulation of poly β-hydroxybutyrate. They are motile, inclined to salts and organic acids as carbon sources, and oxidase-positive. When grown in broth, cells have a single polar flagellum, but when grown on agar at 30°C, lateral flagella of shorter wavelength are also formed. Strains belonging to group II (which includes A. lipoferum) are distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium.

Natural Source and Ecological Niche

Bacteria of the genus Azospirillum are non-nodule forming microorganisms commonly found in the soils of tropical, subtropical, and temperate ecosystems. They colonize the surface and the interior of roots, both in wild and agricultural plants — a relationship termed a rhizosphere association. In Brazil, during the 1970s, similar strains of this species were found associated with the roots of grain plants by scientists led by Dr. Johanna Döbereiner, whose group discovered that these bacteria had the ability to fix nitrogen.

Almost all known members of the family Rhodospirillaceae are found in aquatic environments, suggesting that Azospirillum represents a lineage that may have transitioned to terrestrial environments much later than the Precambrian split of "hydrobacteria" and "terrabacteria."

Azospirillum lipoferum is currently most notable for its ability to enhance the success of certain agricultural plant products such as maize, rice, and wheat. While the genus contains several species, the worldwide distribution and advantages of inoculation were primarily illustrated with A. brasilense and A. lipoferum.

Common Preparations and Formulations

Azospirillum strains are marketed as biofertilizers in various countries, including Africa, Argentina, Australia, Belgium, Brazil, Germany, France, Italy, Mexico, Pakistan, Uruguay, and the USA. Commercial biofertilizer products based on A. lipoferum are prepared in several carrier formats. Azospirillum lipoferum CRT1 is a natural PGPR isolate that is commercially used on maize to stimulate plant growth and field yield, with a peat-based seed-coating formulation providing 105–6 CFU of bacteria per seed.

Biofertilizers manufactured in several countries are generally carrier-based and may suffer from short shelf life, poor quality, high contamination, and low and unpredictable field performance. Common carrier systems include peat (solid), talc-based powders, liquid broth, and granular formulations applied to seeds or soil. Several strategies exist for applying plant growth-promoting microorganisms in agriculture, including inoculating seeds, soil, or plant roots with beneficial microorganisms; seed treatment involves coating seeds with microbial inoculants before planting.

2. Historical and Traditional Use

Azospirillum lipoferum is a microorganism with no documented history in traditional herbal medicine or pre-scientific ethnobotanical use — it was not consciously employed as a remedy or agricultural amendment by any named culture prior to its microbiological discovery. Its "traditional" use is therefore scientific-agronomic in nature and dates from the early twentieth century.

The genus Azospirillum celebrates 100 years since its discovery in 1925 by Martinus Willem Beijerinck, who worked with Spirillum lipoferum as a starting species. Döbereiner and Day (1976) reported the nitrogen-fixing potential of some grasses attributable to the activity of S. lipoferum in their roots. This discovery re-framed the organism as a biologically and agronomically important entity, directly motivating the taxonomic revision in 1978.

Brazil has a long tradition in research on nitrogen fixation in Azospirillum-grass associations. Following this tradition, A. brasilense and A. lipoferum were the two species first formally described, and A. argentinense became an inoculant recommended in Argentina for more than 30 years. The organism's practical use as a deliberate soil amendment arose from mid-twentieth-century agronomic science in Latin America, eventually spreading to commercial biofertilizer markets worldwide.

The application of Azospirillum spp. represents an exceptional case of translational biology, in which the use of a microorganism first showed promising potential in the field to boost crop growth and productivity, and over time, the different facets of this beneficial behavior were scrutinized.

3. Key Constituents and Active Compounds

Azospirillum lipoferum is a living microorganism rather than a plant-derived extract, so its "active constituents" are functional biochemicals that it synthesizes and secretes. The following categories of biologically active compounds and capacities have been characterized in the peer-reviewed literature.

3.1 Indole-3-Acetic Acid (IAA) and Related Auxins

One of the main mechanisms of action is the ability to produce a large amount of the phytohormone indole-3-acetic acid (IAA). IAA is the first auxin group of growth stimulants identified to play a central role in plant growth. It functions as a biological process regulator, supporting cell elongation by altering conditions such as membrane permeability, increasing water permeability, reducing cell wall pressure, and increasing cell synthesis.

The majority (90%) of IAA produced by azospirilla is produced via the tryptophan-dependent pathway, with the indole-3-pyruvate decarboxylase (IpdC) being the rate-limiting enzyme. This pathway involves the transamination of tryptophan to yield indole-3-pyruvate (IPyA), followed by a decarboxylation reaction to form indole-3-acetaldehyde (IAAld), and the subsequent oxidation reaction results in IAA formation.

3.2 Gibberellins

Gibberellins A1, A3, and iso-A3 have been identified in cultures of A. lipoferum. Gibberellin production by bacteria is involved in plant growth promotion and yield increase. Gibberellins contribute to stem elongation and seed germination.

3.3 Cytokinins

The hormonal repertoire of Azospirillum includes the production and regulation of cytokinins, gibberellins, salicylic acid, IAA, and ethylene. These substances stimulate root elongation and enhance nutrient and water uptake. Cytokinins promote cell division and delay leaf senescence, thereby supporting plant growth.

3.4 ACC Deaminase

The bacterium expresses ACC deaminase, which hydrolyzes the ethylene precursor 1-aminocyclopropane-1-carboxylate, reducing plant ethylene levels and promoting growth. Elevated ethylene is associated with plant stress responses; its reduction via ACC deaminase enables continued root and shoot development under adverse conditions.

3.5 Abscisic Acid (ABA)

Several PGPR species are known to induce drought stress tolerance through the ABA pathway; endophytic Azospirillum lipoferum has been shown to enhance ABA levels, which alleviated drought stress effects in inoculated maize plants. Bacterial inoculation enhanced two-fold the ABA content in host plant seedlings, which accounts for the performance of inoculated plants under adverse environmental conditions such as salt or drought. These findings help to understand the contribution of Azospirillum to protect plants from abiotic stress through bacterial ABA production and inducing ABA signaling in plant hosts.

3.6 Siderophores

Under limited nutrient conditions, A. lipoferum produces catechol-type siderophores with antibacterial activities against various bacterial and fungal isolates. Several isolates have the genetic ability to chelate ferric iron with siderophores. Siderophores chelate ferric iron (Fe³⁺) in the rhizosphere, improving iron availability to plants.

3.7 Nitrogenase Enzyme Complex

Nitrogenase activity in A. lipoferum is regulated according to the intracellular nitrogen and O₂ level. Posttranslational control of nitrogenase, in response to ammonium and anaerobiosis, involves ADP-ribosylation of the nitrogenase iron protein, mediated by the enzymes DraT and DraG.

3.8 Poly-β-Hydroxybutyrate (PHB)

These bacteria are spiral or slightly curved rod-shaped nonspore-forming cells with polyhydroxybutyrate (PHB) granules. PHB serves as an intracellular carbon and energy storage compound and is implicated in the bacterium's survival and stress resistance.

4. Mechanisms of Action

During more than 35 years of study of the Azospirillum–plant interaction, over 20 proposals were suggested for the mechanism of action. These include single phytohormone activity, multiple phytohormones, nitrogen fixation, assortments of small-sized molecules and enzymes, enhanced membrane activity, proliferation of the root system, enhanced water and mineral uptake, mobilization of minerals, mitigation of environmental stressors of plants, and direct and indirect biological control of numerous phytopathogens. By volume, the largest number of published findings involves hormonal activities, nitrogen fixation, and root proliferation.

Researchers have proposed the "Multiple Mechanisms Theory," based on the assumption that there is no single mechanism involved in the promotion of plant growth by Azospirillum, but rather a combination of a few or many mechanisms in each case of inoculation. These may vary according to the plant species, the Azospirillum strain, and environmental conditions when the interaction occurred.

4.1 Biological Nitrogen Fixation

Azospirillum sp. is able to fix 20–40 kg N ha⁻¹, and due to its inoculation, yields increase on average by 5–10%. It was reported that Azospirillum benefits the plant directly via associative nitrogen fixation (in the range of 20–40 kg N/ha in the rhizosphere), synthesis of hormones, and modulation of plant hormonal balance by deamination of the ethylene precursor.

Nitrogen fixation is mediated by the nitrogenase complex and governed by the nif gene system. Although initial efforts were aimed at clarifying the importance of biological nitrogen fixation in plant growth in the face of root colonization with Azospirillum, recent advances show that these bacteria also activate the mechanisms of acquisition of phosphorus and iron, two essential nutrients for fulfilling the plant's life cycle. Importantly, the main obstacle for a significant contribution of nitrogen fixation by Azospirillum is the lack of direct transfer of fixed nitrogen to the plant, a key limitation acknowledged in the scientific literature.

4.2 Phytohormone-Mediated Root Morphology Changes

All analyzed Azospirillum strains have the potential to alter the hormonal balance of their host by secreting plant hormones such as auxins and cytokinins and by expressing enzymes capable of metabolizing the precursor of ethylene. Maize plantlets artificially inoculated with A. lipoferum display an altered root system phenotype with more numerous, longer lateral roots under field and greenhouse conditions, allowing roots to explore a larger volume of soil in search of nutrients and water.

4.3 Photosynthesis and Carbon Metabolism

Inoculation of maize seeds with A. lipoferum leads to a reduction of ascending sap glucose content, lifting its feedback down-regulatory inhibition of leaf photosynthetic potential. Both this activity in A. lipoferum-inoculated maize and the accumulation of higher quantities of photosynthetic pigments in A. brasilense-inoculated wheat foster carbon fixation to sustain increased plant growth.

4.4 Phosphate Solubilization

A. lipoferum demonstrates phosphate solubilization ability, though weaker than that of specialized phosphate-solubilizing bacteria. The mechanism involves organic acid production (primarily acetic acid) that reduces medium pH and releases soluble phosphate from calcium phosphate complexes.

4.5 Root Colonization and Chemotaxis

Whole-genome expression array analyses with RNA samples obtained from planktonic cells, sessile cells, and root-adhering cells have shown that root-associated Azospirillum cells grow in an active sessile-like state and gene expression is tightly adjusted to the host plant. Adaptation to rice seems to involve genes related to reactive oxygen species (ROS) detoxification and multidrug efflux, as well as complex regulatory networks.

Chemotaxis and motility are primordial for the initiation of root colonization in a wide range of rhizobacteria. The chemotaxis system integrates environmental signals into an appropriate bacterial response by using a dedicated signal transduction pathway.

5. Scientific Evidence by Area of Use

5.1 Cereal Crop Yield Enhancement (Wheat, Maize, Rice)

Overview and meta-analytic evidence: A meta-analysis of field trials conducted between 1981 and 2008 revealed average enhancements of grain and forage yields of 15% and 27%, respectively (n = 91), after wheat inoculation with Azospirillum in the absence of nitrogen fertilization.

Field variability: A review of field assays conducted worldwide found that all reports of assays conducted in Brazil and Mexico described positive yield-enhancements by Azospirillum. However, as few as 60% of field assays conducted in Egypt, India, and Uruguay concluded in yield-enhancements, while only 5 such cases were seen out of 6 years of experimentation in 12 locations in France. Positive effects on yield were even considered "erratic" in field assays conducted in the USA, where no correlation was found with host plant, soil type, and N fertilization.

Maize-specific field study (Scientific Reports, 2017): Correlations between modifications of yield and 6-leaf plantlet characteristics were estimated on maize in four fields with contrasting soil properties over two consecutive years using the commercial isolate A. lipoferum CRT1. In both years, plantlet metabolome, photosynthetic potential, and organ morphology were found to display field- and inoculation-specific signatures. Metabolomic analyses revealed that A. lipoferum CRT1 mostly affected sugar metabolism with no suggested impact on N and P assimilation. Mineral nitrogen feeding increased yield but did not affect yield enhancement by the bacterial partner. However, greater improvements of leaf photosynthetic potential correlated with yield diminutions, and larger plantlets in all of their proportions correlated with yield enhancements.

Brazil field trials (Plant and Soil, 2010): Selection and evaluation of Azospirillum strains for maize (Zea mays L.) and wheat (Triticum aestivum L.) crops was performed following protocols established by Brazilian legislature, requiring field experiments in at least two different localities representing the crop growing regions and for at least two seasons. In a first set of nine trials performed at Londrina and Ponta Grossa, southern Brazil, nine Azospirillum strains were evaluated after application to seeds as peat-based inoculants.

Maize nitrogen fertilizer reduction: It was concluded that it is more economical to utilize N-fertilizers at 100 kg/ha, and inoculating seeds of maize with Azospirillum lipoferum can lessen the N-fertilizer load up to 50% and enhance its grain production up to 12% compared to uninoculated plants receiving 200 kg/ha of fertilizer, and enhance grain production up to 36% compared to plants grown in the absence of A. lipoferum or fertilizers.

Evidence strength: The evidence base for cereal yield enhancement is extensive, drawn from hundreds of field trials across multiple continents. However, results are highly variable across geographic locations, soil types, and crop varieties. Meta-analyses show overall positive trends, but individual field outcomes are inconsistent, particularly outside tropical and subtropical environments.

5.2 Drought Stress Tolerance

Wheat greenhouse experiments: It was 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 drought stress and by 109% under severe drought stress, compared to uninoculated controls. Strain B2, which showed siderophore production, showed the highest drought resistance.

Wheat seedling water status: Inoculation of wheat seedlings with Azospirillum lipoferum strains reduced leaf water potential and increased leaf water content during drought stress, enhanced production of IAA, and improved root development and lateral root formation for water and nutrient uptake.

Wheat yield under water stress (field study): In the presence of water stress, A. lipoferum treatment showed 24.79% and 11.31% increases in straw fresh and dry weights per plant, 54.33% and 58.67% in spike fresh and dry weights per plant, 79.35% and 3.32% in grain yield per plant and 1000-kernel weight respectively, compared to non-inoculated control.

Maize ABA-mediated tolerance: Maize tolerance for a water deficit was enhanced with inoculation by Azospirillum lipoferum owing to enhanced ABA content and the shrinking of GA and ABA synthesis inhibitors. Similarly, Arabidopsis thaliana tolerance to drought stress was improved by Azospirillum brasilense because of raised ABA levels.

Evidence strength: Drought tolerance evidence is predominantly laboratory- and greenhouse-based, with multiple well-controlled experiments on wheat and maize demonstrating significant improvements. Field validation is more limited. Evidence is preliminary to moderate in strength; no randomized controlled trials in food systems or human-consumption contexts exist.

5.3 Salt and Abiotic Stress Tolerance

Inoculation of A. lipoferum strain JA4 showed improved plant growth (higher plant height and dry weight of root as well as shoots) under continuous irrigation with 160 mM NaCl when contrasted with uninoculated control plants. Azospirillum spp. are effective plant growth-promoting rhizobacteria (PGPR) that enhance crop tolerance to various abiotic stresses, including drought, salinity, and heavy metal contamination.

Evidence strength: Mostly greenhouse/laboratory studies. Promising, but evidence is preliminary and strain-dependent.

5.4 Non-Cereal Crops and Broader Agricultural Applications

The application of biofertilizers containing Azospirillum sp. strains is recommended for non-legume plants such as paddy, millets, oilseeds, sugarcane, banana, coconut, oil palm, cotton, chilli, lime, coffee, tea, spices, and herbs. It was estimated that up to 50% of the nitrogen content in sugarcane could be supplied by Azospirillum sp.

5.5 Transcriptomic and Genomic Evidence

Unique transcriptomic fingerprints were observed in two rice varieties (Cigalon and Nipponbare) after inoculation with A. lipoferum 4B or A. lipoferum B510. The authors observed altered expression of 1,243 and 2,141 genes in Cigalon and Nipponbare varieties, respectively, by strain 4B.

Many genes involved in colonization of plant roots, plant-growth promotion (such as those involved in phytohormone biosynthesis), and properties involved in rhizosphere adaptation (such as catabolism of phenolic compounds, uptake of iron) are restricted to a particular strain and/or species, strongly suggesting niche-specific adaptation.

Evidence strength: Genomic and transcriptomic evidence is robust and mechanistically informative, but is largely preclinical and does not translate directly to human health outcomes.

6. Body Systems and Health Areas Associated with Azospirillum lipoferum

It is important to note that Azospirillum lipoferum is currently classified and regulated as an agricultural biofertilizer, not as a human dietary supplement, probiotic, or pharmaceutical agent. Its documented associations with "health" are indirect and relate to agricultural systems, food security, and sustainable farming. No peer-reviewed clinical studies involving human subjects, animals as consumers, or direct human physiological endpoints have been identified in the literature.

  • Soil and Rhizosphere Health: Decades of work involving laboratory and field research endorse various beneficial properties, such as plant rooting, mineral nutrition, hormonal strengthening, and the activation of cellular and molecular responses, which lead to better growth, development, and productivity.
  • Food Security and Nutritional Quality: Although initial efforts were aimed at clarifying the importance of biological nitrogen fixation in plant growth, recent advances show that these bacteria also activate the mechanisms of acquisition of phosphorus and iron. A deeper understanding of the molecular basis of Azospirillum in biofertilizer and biostimulant development will contribute to sustainable agricultural practices and help to meet global food security demands.
  • Reduction of Synthetic Fertilizer Use: By the use of Azospirillum inoculants in agricultural production, up to 25–30% of the quantity of nitrogen fertilizer can be saved.
  • Plant Immune and Stress-Response Priming: Apart from nitrogen fixation, production of growth-promoting substances, disease resistance, and drought tolerance are additional benefits of Azospirillum inoculation.

7. Dosage Forms and Doses Reported in Studies

Azospirillum lipoferum inoculant doses in the scientific literature are expressed in colony-forming units (CFU) per seed, per gram of carrier, or per hectare of land. The following represent dose information as stated in peer-reviewed sources:

  • Generally, the number of inoculant cells necessary to induce plant stimulation should be in the order of 106 to 107 per seed. Azospirillum lipoferum CRT1 is commercially used on maize with a peat-based seed-coating formulation providing 105–6 CFU of bacteria per seed.
  • Phytostimulation can also be achieved at lower inoculation levels; positive effects of A. lipoferum CRT1 on maize physiology were evidenced with only 104–5 inoculant cells per seed.
  • The supplied biofertilizer to be used in fields should be of good quality and contain 107 viable cells per gram as an inoculum.
  • At field scale, Azospirillum fixes nitrogen from 10 to 40 kg per hectare.

Formulation types documented in studies include: peat-based carrier inoculants (seed coating), talc-based powders, and liquid broth inoculants applied to seeds or soil. No human oral dosage or dietary supplement dosage for A. lipoferum has been defined in any peer-reviewed source, as the organism is not used as a human supplement.

8. Safety Considerations

8.1 Pathogenicity Status

None of the species or strains of Azospirillum is reported as a human or plant pathogen. It is considered the safest bacterium that can be used as a biofertilizer at commercial level for several crops.

8.2 Non-Target Organism Effects

Inoculation effects on microbial functional groups in the rhizosphere are site-specific. Field research assessed the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the size and/or diversity of selected microbial functional groups important for plant growth, using quantitative PCR and Illumina MiSeq metabarcoding. The potential for impacts on native rhizosphere microbial community structure is an area of ongoing research.

8.3 Compatibility with Chemical Inputs

Physical or chemical incompatibilities may arise when A. lipoferum is co-applied with high concentrations of synthetic fertilizers or broad-spectrum biocides; sequential rather than simultaneous application is recommended to maintain cell viability.

8.4 Regulatory and Biosafety Classification

In many countries, biofertilizers must be registered with agricultural or environmental authorities before they can be marketed. This process often requires detailed documentation of the product's composition, efficacy, and safety. Regulatory agencies often set minimum standards for the presence of viable Azospirillum cells, as well as limits on contaminants such as pathogens and non-target microorganisms. Brazilian legislation, for example, outlines specific microbiological quality standards for inoculants, including acceptable levels of contamination.

Microbial strains selected for biofertilizer development should preferably belong to the low-risk group of non-pathogenic BSL-1 microorganisms. Major safety guidelines are essential for using PGPRs as biofertilizers.

8.5 Context-Specific Limitations

A number of obstacles remain for ascribing nitrogen fixation and auxin production a fundamental role in the plant response. Although Azospirillum is known to produce IAA in free-living cultures, it is still not clear to what extent this occurs in the rhizosphere and how this process would be regulated.

Various studies reported that PGPR inoculant functioning under field conditions is impacted by soil type, plant genotype, and the PGPR strains used. Unlike greenhouse conditions, field inoculation of phytobeneficial microorganisms is associated with variable levels of success.

Death of organisms in inoculated seeds is one of the important factors contributing to the failure of inoculation response in field conditions.

References

Health Conditions

Health conditions that Azospirillum lipoferum may help support.

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Body Systems

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