Azospirillum Genus

The Azospirillum genus of bacteria was discovered by J. Dobereiner and became important in the 1970s for its ability to fix atmospheric nitrogen, hence the genus name Azospirillum¹¹. The genus has a broad geographical distribution, and can be found in temperate and tropical regions¹.

Azospirillum-genus bacteria are Gram-negative and free-living. They are cane-shaped, have active movement, measure 0.8 to 2 μm in diameter and 2 to 4 μm in length, and have intracellular polyhydroxybutyrate granules¹². Optimum development temperature range for these bacteria is 28 to 41 °C. These bacteria are strictly aerobic when in a N₂-free environment and supplied with nitrogen or microaerophilic sources, which requires them to fix nitrogen¹¹. However, to promote a microaerophilic environment when in a semi-solid medium these bacteria produce a thin veil-like film containing the oxygen concentration essential for nitrogen fixation and growth initiation.

Azospirillum sp. have a flexible carbon and nitrogen metabolism which augments their ability to compete for rhizosphere colonization¹². When present in the rhizosphere they colonize both the mucigel layer around the roots (external colonization) and root intercellular spaces (internal colonization)⁸. Colonization occurs mainly in the areas of elongation and root hairs¹¹. They are highly competitive when colonizing the rhizosphere, and to maintain metabolism make use of different nitrogen sources such as ammonia, nitrite, nitrate, molecular nitrogen and amino acids, as well as carbon sources such as organic acids (malate, pyruvate, succinate and fructose)¹⁰.

Fifteen Azospirillum species have been identified to date: A. lipoferum, A. brasilense, A. amazonense, A. halopraeferens, A. irakense, A. largimobile, A. doebereinereae, A. oryzae, A. melinis, A. canadiana, A. zeae, A. rugosum, A. palatum, A. picis and A. thiophilum. Of these the most widely researched are A. lipoferum and A. brasilense, both of which are common in tropical grasses and forages¹. Not all Azospirillum species can be found colonizing plants in various regions; A. amazonense, for instance, has only been isolated in Brazil¹². Azospirillum brasilense, however, is widely distributed in tropical and subtropical soils, a characteristic that contributed to its ample study¹³. Experiments have shown that A. brasilense promotes plant growth and consequently increases productivity¹⁴. It exhibits satisfactory results when associated with Poaceae family plants such as corn, oats, wheat and rice¹⁵.

Inoculum for plants

Widely known as biofertilizer, inoculants intended to promote plant growth contain live microorganisms that implement various mechanisms. Commercial production of biofertilizers in Latin America began in 1898¹⁶, and Brazil is one of the largest current markets in the region¹⁷. Inoculants can improve competitiveness in the agricultural sector and is a promising and sustainable technology capable of partially replacing nitrogen fertilization¹⁸.

Brazil’s Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento da Brasil - MAPAB)¹⁹, requires that commercial inoculants contain at least 10⁹ cells per gram or milliliter of product at their expiry date, that they must be prepared with a sterile vehicle and be free of unspecified microorganisms up to the 1x10^-5 dilution factor. Traits decisive for inoculant efficacy in promoting plant growth include the strain, its competitiveness, and the number of necessary or viable cells for rapid colonization of the rhizosphere and plant tissue²⁰. They need to be produced using non-toxic vehicles, and be water-soluble and associated with competitive lines²¹.

Inoculation success depends on selection of efficient, stress-resistant strains, since diazotrophic bacteria are found in various soil types and their persistence is determined by soil salinity, pH and moisture²². Azospirillum genus bacteria promote plant growth through mechanisms such as greater tolerance to environmental stressors, phytohormone production and nitrogen fixation²³.

A major initiative to develop Azospirillum inoculates in Brazil began in 1996 when the Department of Biochemistry and Molecular Biology of the Federal University of Paraná partnered with the Brazilian Agricultural Research Company (Empresa Brasileira de Pesquisa Agropecuária - Embrapa Soja) to do field research on Azospirillum efficiency. Years of research produced Azospirillum lines that survived longer in soils and produced more plant growth¹⁶. A total of eighteen trials were run which were divided into two sets of nine trials each. The first set evaluated peat inoculants containing a single A. brasilense or A. lipoferum strain in corn using five trials with three harvests and in wheat using four trials with two harvests. The second set evaluated a mixture of two A. brasilense strains (Ab-V5 and Ab-V6) in peat or liquid inoculants in four corn trials and four wheat trials. This research contributed to the release of four A. brasilense strains (Ab-V4, Ab-V5, Ab-V6 and Ab-V7) for commercial inoculant production. Only Ab-V5 and Ab-V6 are widely used as inoculants because they provide the most satisfactory results in corn and wheat¹⁶. Due to their ease of application liquid inoculants were chosen over peat inoculants for marketing. In 2010, a liquid inoculant containing Azospirillum strains and protective molecules for tropical conditions was developed in an association between Embrapa and the private sector.

Azospirillum strains are sold worldwide in countries including Argentina, Mexico, Italy, France, Australia, Pakistan, Germany, the United States, Africa, Belgium, India and Uruguay²⁴. The success of Azospirillum inoculants is the result of their beneficial effects in agricultural crops; for instance, they have a 60 to 70 % success rate and provide 5 to 30 % productivity gains in various cereal crops²⁵.

Action mode of Azospirillum bacteria

Azospirillum spp. bacteria supply nitrogen to plants via different mechanisms (Table 1).

Production of growth regulators is one of the main mechanisms by which these bacteria affect plant growth since they influence growth in plants, modify root morphology and maximize use of soil resources, which in turn augment BFN and reduction of the assimilable nitrate available in soils²⁴.

Azospirillum bacteria also reduce nitrate (NO₃ ^-) in the roots, promoting plant growth due to consequent lower energy expenditure required from the plant to reduce nitrate to ammonia; this energy is allocated to other vital processes. These bacteria can influence glutamine synthetase activity in the roots of corn plants²³. Glutamine synthetase is extremely important in the nitrogen incorporation process and essential for plants to express their full productive potential²⁶. Other indirect mechanisms through which Azospirillum bacteria can promote plant growth is by reducing the appearance of fungi or soil pathogens via production of siderophores, chitinases, glucanases and antibiosis.

Growth of the plant root system, which can occur in response to growth-promoting substances²⁵, can result in greater mineral and water absorption. Auxin, specifically indole-3-acetic acid (IAA), is the principal hormone promoting root growth¹⁵. Azospirillum lineages produce IAA in addition to other indole compounds such as cytokinins and gibberillins⁸. At least three biosynthetic pathways exist for IAA production in Azospirillum, two of which (indole-3-acetamide and indole-3-pyruvate) depend on tryptophan²⁷. Indole-3-acetic acid is the main indole secreted by Azospirillum²⁴, although indole-3-butyric acid (IBA) has also been reported²³. This indole is an important reserve source in the Azospirillum genus²⁸.

Production of IAA by Azospirillum very probably affects root growth. For example, inoculation of tomato and red pepper seeds with A. brasilense, which is known to produce IAA, increased average root length from 7.21 (control) to 9.68 cm in tomatoes and from 6.20 (control) to 6.76 cm in red pepper²⁹. The aerial portion of the plants in this study also grew more in the treatments than in the controls, highlighting the growth promoting ability of A. brasilense. Phytohormone synthesis in A. brasilense can differ between strains. In one study the 42 M line produced higher IAA levels than the sp7, Cd, Az39, 40 and 42 strains⁶.

In Azospirillum strains IAA may be a signal needed to maintain a symbiotic plant-Azospirillum interaction³⁰, since this signaling function is shaped by coevolutionary processes between bacteria and their host plant³¹. This phytohormone also affects photosynthesis, and biosynthesis of some metabolites and other phytohormones such as cytokinins and gibberellins³². In plants cytokinins regulate cell division and new tissue formation both in the aerial part and roots³³. Gibberellins regulate plant growth, and promote division and elongation of primary roots³⁰.

Azospirillum strains can secrete abscisic acid (ABA) which forms part of the water stress defense mechanism in plants³⁴. This phytohormone induces plant responses to water, environmental and saline stress³⁵. For example, inoculation of A. lipoferum into corn plants resulted in high ABA levels and increased plant tolerance to drought probably related to ABA as well as prolines and polyamines³⁴. Polyamines (e.g. cadaverine, spermine and spermidine) are organic polymers associated with root growth and stress suppression in plants³³. In a study of rice seedlings inoculated with A. brasilense cadaverine production was associated with increased root growth and reduced osmotic stress³⁶. Corn plants inoculated with A. brasilense also are reported to exhibit greater drought resistance and higher biomass production³⁷, possibly due to the presence of cadaverine, among other phytohormones.

Growth-promoting bacteria such as Azospirillum can also act as biological control agents³⁵, which can occur through mechanisms such as parasitism, production of antibiotics, toxins and/or enzymes, interference in the plant-host recognition process and resistance induction³³. Facultative bacteria such as those in the genera Pseudomonas, Burkholderia, Azospirillum and Bacillus, produce and secrete secondary substances that can act as antibiotics, fungicides, antivirals and immunosuppressive agents³¹. In plants endophytic bacteria of this type provide the best biological control of pathogens via natural colonization of the rhizosphere and invasion of internal tissues, both of which are essential means for successful treatment of diseases affecting subterranean plant tissues.

The Azospirillum genus is not strictly considered to exercise biocontrol but Azospirillum strains do produce chemical compounds that modify plant metabolism and defense activity. For instance, A. brasilense has been shown to effectively control Agrobacterium tumefaciens and phytopathogenic fungi²⁵. Although the inhibition mode was not well defined, phenylacetic acid, from the auxin group, was identified and found to have antimicrobial activity. Phenylacetic acid is used in the defense mechanism and bacterial competition in the host²⁵. Biological defense in plants follows two metabolic pathways involving jasmonic acid and salicylic acid¹⁷, while bacteria-induced systemic resistance is activated by the jasmonic acid and ethylene signaling pathway¹⁵.

Co-inoculation with rhizobia

Co-inoculation with bacteria that may or may not be symbiotic is common in legume crops³⁸. Different microorganisms are used that produce a synergistic effect that surpasses production results obtained when strains are used separately³⁹. In vitro assays indicate that some bacteria mixtures provide a synergistic interaction that can improve nutrient supply, remove inhibiting products and/or stimulate physical or biochemical mechanisms³⁵. For example, when associated with rhizobia in legumes A. brasilense produces a beneficial effect caused by production of phytohormones that promote root system development, thus allowing more effective exploitation of available soil volume³⁸.

In future co-inoculation between Azospirillum and other microorganisms may become a major research focus³⁵. Co-inoculation produces higher success rates in plants since it results in increased productivity due to balanced nutrition and improved absorption of nitrogen, phosphorus and other minerals⁵. It also stimulates root nodule function and increases nodule numbers and weight. Co-inoculation of Bradyrhizobium japonicum and A. brasilense in soybeans provided better yields, even under water and nutrient deficit conditions³⁹. In a study of B. japonicum/A. brasilense co-inoculation with and without water deficit, co-inoculation increased nodule weight under water deficit conditions and produced better yields and a greater dry matter accumulation under both conditions⁴⁰. Studies of root growth and nodulation in soybean co-inoculated with B. japonicum/A. brasilense found that addition of A. brasilense helped to stimulate root growth and improved nodule onset and development through excretion of metabolic products, especially IAA³⁶. The co-inoculated plants also exhibited a greater number of nodules and a higher percentage of nodulated plants.

Azospirillum in forage grasses

Grassland degradation is a major problem in livestock systems. For example, an estimated 80 % of grasslands in Brazil exhibit some level of degradation⁴¹. Of the factors that can lead to grassland degradation low nutrient supply for plants is the principal cause since it results in low grass quality and productivity⁴². Among soil nutrients, N is the main limiting factor for grass growth and development in the tropics⁴³. Mineral fertilization is widely used to ensure proper grass growth, but this is expensive and environmentally damaging since around 50 % of applied N is lost due to volatilization or leaching¹⁴.

This situation highlights the potential benefits of BFN in tropical grasses as a means to restoring forage productivity and quality. Diazotrophic bacteria (a.k.a. PGPB) are the main force behind BFN. The process itself involves conversion of N₂ into other nitrogenous substances which are then assimilated by plants through synthesis of protein and nucleic acids¹. The nitrogen cycle in grassland ecosystems strongly depends on BFN and diazotrophic bacteria to supply N to plants. Inoculation of PGPB is therefore a promising technique for promoting growth and nutrition in forage plants. It is a viable alternative for totally or partially replacing N-fertilizer and thus contributing to natural resources conservation⁴⁴.

The benefits of PGPB in crops such as corn, wheat and rice are clear, but knowledge of the mechanisms involved requires extensive further research. Studies evaluating the effects of PGPB in establishing and maintaining pastures are fundamental to attaining cost-effective and sustainable grassland systems. Using BFN supports sustainability since it can totally or partially replace nitrogen fertilizers, which are produced from non-renewable fossil fuels⁴⁵. Any lowering of N fertilizer use will also mitigate environmental pollution derived from its production and use, with the added dividend of reducing greenhouse gas emissions⁸.

A metanalysis of 22 yr of data on in-field PGPB inoculation concluded that Azospirillum spp. promotes growth in several grass species under different environmental conditions⁵. These gains went beyond BFN to include modification of root production and a consequent broadening of absorption area and the volume of exploited soil. Most of the research supporting this conclusion has involved inoculation of Azospirillum in forage grasses.

Azospirillum brasilense was reported to increase dry matter production and foliar growth in pot-grown millet Setaria italica⁴⁶. In a study of forage grasses native to the Pantanal Matogrossense in South America, inoculation with diazotrophic bacteria had noticeable effects at 60 and 90 d of culture⁴⁷. Combined inoculation of an A. brasilense/A. lipoferum association in the same study resulted in greater dry matter production in the aerial portion and roots, as well as N accumulation. Higher dry matter production than in a control was also observed with inoculation of A. brasilense in the grasses in a natural pasture⁴⁸.

Inoculation is also reported to favor the simultaneous production of dry matter in the aerial portion and roots, and high levels of phosphorus (P) and N accumulation due to more efficient use of these nutrients¹⁴. This higher dry matter accumulation rate may be related to an increase in photosynthetic enzyme activity and N assimilation⁸. Similar increases in dry matter production have been observed with inoculation of Marandú grass, indicating this technique is a sustainable alternative for increasing forage production⁴⁹. Azospirillum inoculation has also increased productivity in other grasses⁵⁰, which may be due to excretion of plant hormones that enhance macro- and micronutrient absorption³⁵.

Use of PGPB is reported to raise nitrogen accumulation and overall yields. In a study using Pennisetum and Panicum species grasses inoculation with A. brasilense increased N content by approximately 40 kg ha^-1 year^-1 via BFN²⁵. In another study BFN was found to raise N accumulation 7 to 10 kg ha^-1 mo^-1 during the summer, which varied by grass genotype; this represents 39% of the N necessary for plant development and productivity⁵¹. Dry matter accumulation rate in the same study was 15 kg ha^-1 d^-1 in the inoculated treatments, almost four-fold higher than the 4 kg ha^-1 d^-1 in a control without inoculation and fertilization.

Inoculation of A. brasilense in Brachiaria brizantha Staf cv. Marandu, produced better yield, greater tiller generation and a longer grazing period than uninoculated and unfertilized plants⁴⁹. In this same system root dry matter production increased with A. brasilense inoculation³³. Using the same grass cultivar, another study found that A. brasilense inoculation produced 9 % more leaves and 12 % more tillers than a control, although no differences in plant height were observed between inoculation and N fertilization treatments⁵².

Application of A. brasilense in forage grass production contributes to increasing dry matter content, N content and plant height, making PGPB inoculation a viable alternative for partial replacement of N fertilizer. It can be a vital element in efforts to combine animal production with environmental conservation, which will become more feasible as soil biotechnology is broadly employed to promote soil conservation and fertility and improve plant nutrition. However, more research is needed on the mechanisms and effects of PGPB inoculation on dry matter content, N content, plant height, leaf area and crude protein content so recommendations can be made regarding its combination with N fertilizer¹. In addition, there is still no conclusive data indicating whether the effects of inoculation are due to BFN or hormonal action.

Azospirillum in corn

Yield in corn responds to a combination of genetic potential, environmental conditions and soil fertility. However, attaining maximum yields requires application of large amounts of fertilizers, particularly nitrogen. One option for reducing use of N fertilizers with corn is application of Azospirillum spp. strains, since it can reduce their use by 30 to 50 kg ha^-17.

More specifically, inoculation with A. brasilense increases photosynthesis and dry matter accumulation rates⁸. When associated with unimproved and cultured genotypes with low N availability, Azospirillum inoculation leads to greater dry matter production, grain yield and N accumulation in corn⁵. It can also increase overall grain production by 9 %⁵³, and productivity by 30 %⁸. Some authors report better results when inoculating Azospirillum in corn seeds, with average increases of 70 % in dry root mass and 43.5 % in aerial dry mass⁵⁴.

Inoculation process

Inoculation is a simple procedure in which seeds are mixed with an inoculant. It is preferably done in the shade in the morning to prevent direct sunlight and excessive heat from killing the bacteria. Mixing is done in a rotating drum, cement mixer, seed treatment machine or other machine. It is important to uniformly distribute the inoculant on the seeds and verify that each seed is covered by inoculant before sowing. For this technique to be effective seeds must be of good quality and the proper dose of inoculant/kg seed must be applied. After inoculant application the seeds are dried for about 30 min before sowing, all the while protected from sunlight and heat. For peat mixture inoculants a sugar solution or other adhesive substance (gum arabic, arrowroot flour, cassava flour, wheat flour and/or various types of cells and polymers) is recommended to moisten the seeds. In this case, a 10 % sugar solution (100 g sugar/L water) is prepared, the seeds mixed into it at a proportion of 250 to 300 ml sugar solution per 50 kg seeds, and finally the peat mixed in at the proportion indicated by the manufacturer⁸. In either process, the seeds have a thin layer of inoculant on their surface when it is complete. It is extremely important to verify that all seeds have been covered with inoculant since this will ensure nitrogen fixation and expression of their full genetic potential.

Inoculated seeds should be sown the same day they are inoculated to ensure the highest number of viable microorganisms. If this is not feasible, the seeds can be stored in ventilated sheds by spreading them out on a smooth surface in layers less than 30 cm thick¹². The inoculant contains live microorganisms which are sensitive to hot environments¹⁶, and seeds should therefore be sown a maximum of 24 h post- inoculation. If this period is exceeded, the seeds must be re-inoculated to improve efficacy. During sowing, the seed container on sowing machines can become quite hot (35+ °C), which can kill the bacteria; should this occur sowing should be stopped and the container cooled. Direct inoculation in the seed container is not recommended because inoculant distribution is not uniform, which reduces inoculation efficiency¹⁶.

Inoculation of small lots of seeds can be done with clean plastic bags, basins or buckets previously sterilized with 70% alcohol, as long as all seeds are covered with a thin layer of inoculant. Other aspects of the process follow the same criteria as when using a rotating drum or concrete mixer, namely that it be done in the shade, that inoculated seeds be allowed to dry for 30 min before sowing, and that doses be calculated following manufacturer instructions to ensure maximum efficiency¹².

For beans planted in small areas direct inoculation in the furrow is an alternative technique. Application can be done with a manual or motorized sprayer directly in the furrow. If using this technique, the inoculant must be mixed with water following manufacturer instructions and the sprayer to be used cannot have residues of agrochemicals such as pesticides or herbicides as these can decrease bacteria viability and lower efficiency. Even though the inoculant consists of soil bacteria, the operator must wear personal protection gear.

Final considerations

Use of Azospirillum spp. in grasses can span the gap between productivity and sustainability. Inoculants based on these microorganisms can potentially reduce nitrogen fertilizer use without affecting productivity, resulting in savings and greater profitability. Grasses such as sugarcane, corn and forages are known to respond well to Azospirillum sp. inoculation, but further study is needed under different conditions to support its broader adoption among producers. This technique can increase competitiveness for some agricultural products vis-à-vis conventionally-grown crops. Grassland inoculation in livestock production can increase forage mass production, mitigate degradation risks and improve production rates.