Azospirillum spp. potential for maize growth and yield

The importance of biotechnology involved in the availability of nutrients to plants in different production systems is well known. In the search for agricultural sustainability, biological nitrogen fixation process stands out, especially in tropical regions where soil organic matter can be rapidly mineralized. In this aspect, researches have pointed out the potentialities of the use of diazotrophic bacteria, as well as other growth-promoting bacteria in Poaceae. Maize crop, especially, stands out in the international scenario, requiring a deepening of the research aiming to raise the contribution potential of microorganisms including Azospirillum spp. in reducing the consumption of fertilizers from nonrenewable sources while promoting an increase in agricultural productivity and mitigating environmental impacts.


INTRODUCTION
Maize (Zea mays L.), one of the most important cereals grown throughout the world, is used as a source of food for humans, animal feed and as raw material for industries.The United States of America is the world's largest producer of this crop, followed by China and Brazil, respectively (USDA, 2018).The maize crop is generally influenced by environmental stress problems, among which are those related to low soil fertility, mainly in terms of nitrogen (N) availability (Novakowiski et al., 2011).In this way, nitrogen fertilizers are commonly used in maize crops in order to supply the deficiency of this nutrient in the soil, meet the physiological needs of the plant and provide high yields.However, due to the economic and environmental cost of industrial manufacturing processes for the increasing food demand, it is necessary to develop technological innovations and incorporate them into the agricultural activity aiming to rationalize the use of nitrogen fertilizers.Among the viable alternatives, the use of beneficials generated by the prokaryotic microorganisms is able to fix atmospheric nitrogen and make them available to the plants through association with the plant roots.The microorganisms with these features are representatives of several bacterial phylogenetic groups, called diazotrophic, capable of colonizing both the interior of the roots and the rhizosphere of plants.This type of symbiosis generates a number of benefits including the stimulus to root growth, making it more voluminous and so absorb larger quantities of water and nutrients (Martínez-Morales et al., 2003).Probably because of the higher root volume and better plant nutrition, there are also several reports of higher tolerance to plant pathogens (Correa et al., 2008).
Usually, inoculation with Azospirillum brasilense provides an increase of dry mass, N accumulation in plants and grain yield, especially if there is correlation between bacteria and unimproved genotypes under conditions of low N availability (Okon and Vanderleyden, 1997).In addition, plant nutritional status, exudate quality, competing microorganisms and strain choice are also factors that may influence the interaction between the maize plant and the bacterium as well as the efficiency of biological nitrogen fixation (BNF) (Quadros, 2009).Due to the incompatibility of A. brasilense with the chemical products used in seed treatment (Croes et al., 1993), there is a need to study alternative methods of seed inoculation.Thus, a method that has been developed is the inoculation of bacteria in the sowing furrow.According to Basi et al. (2011), inoculation with A. brasilense (Abv5/Abv6 strains) in the seeds or on the sowing furrow increased the yield of the maize crop independently of the N rate applied in topdressing (Pereira et al., 2015).
In addition to the increase in maize productivity, there are reports in the literature about nitrogen fertilizer economics when the crop is submitted to inoculation with A. brasilense (Cheng et al., 2011).The researches related to the efficiency of the use of inoculants based on A. brasilense were neglected for many years due to the inconsistency of the results, however, recently they have been more focused on the necessity for development of more sustainable agriculture.Gramineae have some advantages when compared to Leguminosae.They have a fasciculated root system, having advantages over the pivotal system of legumes to extract water and nutrients from the soil, which, together with other physiological factors, promotes greater photosynthetic activity.Therefore, the interest in the biological fixation in Gramineae is great.Not all of the nitrogen required in maize is provided by the bacteria, making the technique a form of N supplementation for the crop.However, this alternative may lead to a reduction in the use of nitrogenous fertilizers and this cost may be equal to or higher than legumes that may be self-sufficient for nitrogen (Döbereiner, 1992).In this way, several investigations involving the association of maize genotypes with A. brasilense found positive results, so that Hungria (2011) obtained yield increases of 30% in relation to the uninoculated control and without nitrogen topdressing fertilization when maize genotypes were inoculated with A. brasilense whereas Braccini et al. (2012) observed a 20% increase in grain yield under the same conditions.Müller et al. (2016) obtained an increase of up to 28% of productivity in maize, when plants were inoculated with A. brasilense at different doses of N. Portugal et al. (2016) verified higher yields in the four N doses tested (0, 30, 60 and 90 kg ha -1 ) by spraying A. brasilense on maize leaves.
However, the adoption of this technology in the agricultural systems is still incipient, because the results vary according to the cultivar, edaphoclimatic conditions, inoculation types and methodology of conduction of the research.Genotypes may interfere with nitrogen uptake, so the identification, selection, and use of more efficient maize genotypes in nitrogen uptake and assimilation is an important strategy, as they contribute to the development of the crop, as well as reduce the contamination of the environment by nitrogen residues (Reis Júnior et al., 2000).Thus, to find responsive maize genotypes to the inoculation with bacteria of the genus Azospirillum, associating them with genetic and breeding programs aiming to increase grain production are really important for the development of new cultivars.

MAIZE CROP
Maize (Zea mays L.) which belongs to the family of Gramineae (Poaceae), is a monoic, allogamous, annual, robust, erect and diploid (2n = 2x = 20) crop (Paterniani and Campos, 1999).It originates from the teosinte, a Mexican subspecies (Zea mays ssp.Mexicana (Schrader) Iltis), which has been cultivated in many parts of the world for more than 8,000 years (the United States of America, China, India, Brazil, France, Indonesia, South Africa and others).It is a polytypic species, being probably one of the greater genetic variability between the cultivated plants.There is genetic variability for practically all traits of the plant and the maintenance of this variability is due to the establishment of germplasm banks, which maintains the diversity of individualized types and under controlled conditions of races (Fornasieri Filho, 2007).
Botanically, maize belongs to the group of C4 plants, its root system is the fasciculate type reaching up to 3 m deep in the soil, while a great part of the roots are in the 0 to 30 cm layer.The stem is a full culm type, consisting of nodes and internodes, while its lanceolate leaves are inserted alternately in the stem, in addition to presenting male inflorescences, tassel, and female inflorescences and ear, and the fruit being classified as cariopse (Ritchie et al., 1993).The domestication of the maize crop occurred through visual selection in the field, considering important traits such as productivity, resistance to diseases and adaptability, among others, giving rise to varieties known nowadays.Thus, from a Gramineae with several stems, small spikelets and with few seeds, it became an erect plant, with a single culm, monoic, with larger ears containing higher seed quantity and quality.
Maize has a great adaptability, represented by varied genotypes, which allows the cultivation from Ecuador to the limit of temperate lands and from sea level to altitudes above 3,600 m, in tropical, subtropical and temperate climates (Barros and Calado, 2014).It is possible that the maize crop may be the one with the greatest genetic variability among the cultivated plants, since there are genetic differences for practically all the traits of plants, being the maintenance of this variability, kept in germplasm banks, which maintains the diversity of individualized types and under controlled conditions of races (Fornasieri Filho, 2007).
Commercially, there are varieties and maize hybrids on the market.Hybrids are suitable for production of systems that use high technology, being single hybrid, single modified hybrid, triple hybrid, triple modified hybrid and double hybrid.While the varieties are recommended for less technical plantings, being obtained by natural pollination through the selection of groups of plants with desirable characteristics, presenting some variability, but with common genetic characteristics (Cruz et al., 2010).
In socioeconomic terms, maize has an undisputed role in the world, due to its exceptional position among exploited agricultural species (Môro and Fritsche, 2015).According to projections, by 2022, maize grain for animal feed will be the most traded in international markets, accounting for 80% of world trade.This position is consolidated due to maize being the most produced cereal in the world, fundamental for animal and human feed, as well as indispensable and driving of several agroindustrial complexes in function of their productive potential, chemical composition, and nutritional value.Demand for the crop has been particularly high in China, driven mainly by animal growth and industrial processes, which account for over 90% of the country's maize imports (USDA, 2017).
Among the nutrients most required by the maize crop is the N, which is important at the initial stage of development of the plant (second week after emergence), when it is with four leaves fully expanded.It is at this stage that the developing root system already shows a considerable percentage of absorbent hairs and differentiated branches and the addition of N stimulates its proliferation with consequent development of the aerial part.Also at this stage the process of floral differentiation begins, which originates the beginnings of the panicle and ear, as well as, it defines the production potential.This implies the necessity of the availability of at least 30 kg ha -1 of N at this stage in order not to limit this physiological event (Fancelli, 1997).
Worldwide, it is estimated that maize will reach 1,038.80 million tons of grain produced in the 2017/18 season.The United States, China, and Brazil are the world's largest producers, respectively, totaling a combined production of 672 million tons (USDA, 2017).Over the last five years, the USA maize production has grown by more than 110 million tons at an average yield of 11,000 kg ha -1 (USDA, 2017), surpassing the total Brazilian production, which increased by 25 million tons since 2010 (CONAB, 2017), more than 30% of the total produced by the country (FAO, 2017).From the data presented, it can be inferred that even the crop presenting high productive potential is evidenced by grain yields of up to 16,000 kg ha -1 in some countries not only in experimental conditions and also by technified farmers (Cantarella, 1993).Their productivity is complex and depends on the interaction between genetic, environmental factors (Argenta et al., 2001) and welldefined management techniques.
The projections in the coming years are that, there will be a substantial increase in the use of fertilizers in Brazil for attending to the intensification of agriculture and the recovery of degraded areas.Considering Brazilian fertilizer production is insufficient to meet national demand, mostly imported from out of the country, as in the period from January to September 2017, 75% (19,182 tons) of fertilizers usage in Brazilian agriculture came from abroad, indicating an increase of 10.3% in relation to the same period of 2016.There were important nutritional growths in nitrogenous fertilizers of 9.2%, phosphates of 23.6% and potassium of 6.6% (ANDA, 2017).

Biological nitrogen fixation (BNF)
Nitrogen is essential for the proper functioning of plants, as it participates in the composition of the related amino acids, protein, chlorophyll, photosynthesis and many essential enzymes for cell maintenance and development as well as it is present in the processes of ionic absorption, respiration, multiplication and differentiation of cellular and genetic inheritance which are essential for the growth and development of the aerial part and the root system (Marschner, 1995;Epstein and Bloom, 2006;Malavolta, 2006;Grassi Filho, 2010;Taiz and Zeiger, 2013).It is known, for example, that N represents about 40% of the total cost of production of the maize crop (Barros Neto, 2008) and about 50% of this applied nitrogen undergoes the action of ammonia volatilization, denitrification, erosion, microbial immobilization and leaching (Reis Júnior et al., 2010) into the water until it reaches the water table, rivers and lagoons, consequently polluting the environments (Lewis et al., 1984).
BNF involves the transformation of atmospheric nitrogen (N 2 ) to forms assimilable by the plant: ammonium (NH 4+ ) or nitrate (NO 3-) through dinitrogenases enzymes existent in diazotrophic bacteria present in the soil (Novakowiski et al., 2011).This process provides nitrogen compounds directly to plants through associations, or when organisms die and release them into the environment, providing the necessary nitrogen for plant development (Lindemann and Glover, 2003).
According to Rudnik et al. (1997), BNF is a process related to the need of the environment and the fixing species, because of enzyme nitrogenase, which is responsible for the reduction of N 2 , is inactive in the presence of ammonia.BNF is a significant process in the agricultural sector, with the biological process contributing most of the fixed N. It is estimated that it provides about 175 million tons of N to the biosphere, or 65% of the total which makes it the second most important biological process on the planet after photosynthesis, along with organic decomposition (Moreira and Siqueira, 2006).However, only biological nitrogen fixation is not able to provide all the necessary N to the development of crops that demand a larger amount of this nutrient, so nutritional supplementation with nitrogen fertilizer formulations is necessary.In this way, it is important to know well the history of the planting area, as well as the predecessor crop, in order to define more accurately the N doses, sources and parceling to be applied (Portugal et al., 2017).
Environments of degraded areas, with poor soils, substrates devoid of organic matter, can be stimulated by BNF, when compared with areas with climax vegetation, provided with rich substrates in organic matter, because the cycling that occurs in these environments guarantees the preservation of metabolism and growth rate (Moreira et al., 2010).BNF is a process that depends on several factors.In order for the bacterium to establish a positive interaction with the plant, it is indispensable to use selected A. brasilense strains (Hungria, 2011) capable to compete with the microorganisms already present in the soil.Another factor to be taken into account is the choice of the genotype to be inoculated since the beneficial relationship between the hybrid and the bacterium is determined by the quality of the exudates released by the roots of the plant (Nehl et al., 1996).This phenomenon is known as chemotaxis, where each genotype releases a different amount of exudate with different chemical composition, which may or may not be attractive and serve as a carbon source (malate, pyruvate, succinate, and fructose) for the inoculated bacteria (Quadros, 2009).
As for the survival of this microorganism, it is known that A. brasilense has a low ability to survive for prolonged periods of time in most soils.The physicochemical conditions of the soil and the absence of the host plant can directly affect the population of the bacteria (Bashan et al., 1995).However, in unfavorable situations, these bacteria develop protection mechanisms such as cysts formation, poly-β-hydroxybutyrate, and melanin production, favoring their survival (Del Gallo and Fendirik, 1994).
The inoculation with diazotrophic bacteria can alter the root system morphology, a number of radicels and root diameter, probably due to the production of growth promoting substances (auxins, gibberellins, and cytokinins), and not only by BNF (Cavallet et al., 2000).The production of phytohormones helps the growth of plants, and can modify the morphology of the roots, which allows a greater volume of soil exploration and a higher nutrient uptake (Silva et al., 2004), greater tolerance to salinity, dryness (Bashan et al., 2004) and plant pathogens (Correa et al., 2008), resulting in more productive plants (Hungria, 2011).The inoculation with Azospirillum is carried out similarly to inoculate soybean seeds with Bradyrhizobium.The commercial product can be applied in solid form (as peat) or in liquid form.Also, it is necessary to be cautious of the temperature conditions, not leaving exposed to the sun and without joint application with agrochemicals, since they are living microorganisms (Hungria et al., 2010).
The most common inoculant application method is via seeds.In a study by Portugal et al. (2017) and according to Hungria (2011), seed inoculation associated with the addition of 24 kg ha -1 of N at sowing and 30 kg ha -1 of N at the flowering stage enable average yields around 7,000 kg ha -1 .However, seeding furrow inoculation has been studied as a way of avoiding toxicity of the products used in the treatment of seeds on the bacterium, since some chemicals can disorganize the flagellum used by A. brasilense in association with the plant (Croes et al., 1993).According to Basi et al. (2011), the application of A. brasilense (Abv5 and Abv6 strains) provided an increase in maize productivity, and the inoculation through the sowing groove did not differ from that in the seeds, showing an efficient application method.However, the selection of strains for inoculant manufacturing still needs a lot of research.There are currently technological packages using plant varieties and efficient bacterial strains, which can supply more than 50% of the N necessary to the plant (Bárbaro et al., 2008).

Plant growth-promoting bacteria: Azospirillum spp.
An alternative to achieve high yields of maize, with lower consumption of nitrogen fertilizers, is the inoculation of the crop with bacteria that have the capacity to supply nitrogen to the plants, known as plant growth-promoting bacteria (PGPB) which belong to the phylogenetic groups called diazotrophs (Moreira et al., 2010).PGPB are known as free-living bacteria in the soil, rhizosphere, rhizoplane, and phyllosphere that are beneficial to plants.PGPB endophytes residing within the plant have also been found.They directly affect plant growth by supplying substances that are generally scarce.PGPB may aid to uptake nitrogen nutrition of crops through various mechanisms.They are able to fix atmospheric nitrogen; solubilize phosphorus and iron, and produce plant hormones such as auxins, gibberellins, cytokinins, and ethylene.In addition, they promote higher plant tolerance to stresses, such as drought, high salinity, metal toxicity and pesticide loading (Bashan and De-Bashan, 2010).
Diazotrophs comprise a broad range of prokaryotic microorganisms, including representatives of archebacteria, cyanobacteria, gram-positive and gramnegative bacteria that exhibit great morphological, physiological, genetic and phylogenetic diversity.Such diversity guarantees not only the resilience of the processes that mediate in a given ecosystem but also the occurrence of this, in the most different terrestrial habitats (Moreira and Siqueira, 2006).This kind of bacteria can contribute to plant growth by the following characteristics: nitrogen supply, phytorium production, phosphate solubilization (Pedrinho, 2009), increase the activity of nitrate reductase when they grow endophytically in plants (Cassán et al., 2008), as well as acting as an agent for the biological control of pathogens (Correa et al., 2008).Chavarria and De Melo (2011) report that the use of microorganisms in agricultural practices has become increasing since nitrogen fertilization represents an important element in production costs.
The loss of diversity of soil microorganisms, especially diazotrophs, can alter the population structure of other organisms located along the trophic chain.Vital soil processes such as the decomposition of organic matter and the cycling of nutrients can suffer impacts taking the agricultural system to higher dependence on fertilizers.In this context, the knowledge of the phenotypic diversity and genetic structure of the populations present in the rhizosphere can help in the understanding of how the variations in the environment may be influencing the functionality of these populations.
The diazotrophic bacteria, the most studied PGPB, belonging to the Azospirillum genus do not form a symbiosis with the host plant (Bashan and Bashan, 2005), and Azospirillum spp. is among the most important bacteria involved in the fixation of N 2 in grasses (Cáceres, 1982).This bacterium is characterized by its rod shape, which are commonly uniflagellated, gramnegative, with characteristic vibratory movement and mixed flagellar pattern (Hall and Krieg, 1984).These microorganisms fit into the group of facultative endophytic diazotrophs, as they colonize both the interior of the roots, where their cells can penetrate into intercellular spaces and lodge, as well as in the external part of the roots, being found in the mucigel present in the rhizosphere of plants and occur frequently in tropical and subtropical soil (Bashan and Levanony, 1990;Baldani et al., 1997).
The Azospirillum genus, when inoculated, may not achieve the similar efficiency of the rhizobia-leguminous symbioses in the soil.The contribution of N fixed to Gramineae is around 25 to 50 kg N ha -1 year -1 , equivalent to the average supply of approximately 17% of crop demand (Moreira et al., 2010).Several studies have been carried out to identify microorganisms that have a symbiosis with Gramineae, as occurs in the soybean crop with the bacterium Bradyrhizobium japonicum.However, the bacterium A. brasilense has a great response potential in association with maize cultivation.The interest in the use of this development-promoting bacterium capable of contributing to plant nutrition has increased and tends to increase in the coming years, due to the high financial value invested annually with fertilizers and in relation to the search for sustainable agriculture (Hungria et al., 2010).
These bacteria have a wide ecological distribution, being found in association with monocotyledonous and dicotyledonous plants (Magalhães and Döbereiner, 1984;Döbereiner and Pedrosa, 1987;Lange and Moreira, 2002).It has been investigated that the effect of Azospirillum spp.not only on crop yield but also on the physiological causes the possibly of increase in yield (Bárbaro et al., 2008).It is possible to classify this bacteria as rhizocompetent bacteria, because the survival of this genus in the soil, in the absence of host plants, is related to different physiological mechanisms of protection (Bashan and Levanony, 1990;Del Gallo and Fendrik, 1994;Moreira et al., 2010), they are: melanin production, poly-β-hydroxybutyrate (PHB) and polysaccharides (Del Gallo and Fendrik, 1994), formation of cysts (cell aggregates) and change in cell shape.
The Azospirillum genus can act on the vegetative growth through the reduction of nitrate in the roots of the plants (Döbereiner et al., 1995;Cassán et al., 2008).Among the effects of the association between these bacteria and the plants are the biological nitrogen fixation capacity (Fukami et al., 2016), solubilization of inorganic phosphate, production of hormones such as auxins and cytokinins (Tien et al., 1979), gibberellins (Bottini et al., 1989), regulation of ethylene biosynthesis (Strzelczyk et al., 1994), as well as a variety of other bioactive molecules (Perrig et al., 2007); the solubilization of phosphates (Rodriguez et al., 2004); the biological control of pathogens (Correa et al., 2008); and the increase of plant resistance to different abiotic stresses (Yang et al., 2009).One of the most striking effects of inoculation with A. brasilense on root morphology is represented by the root hair proliferation, making them more voluminous, and consequently able to absorb larger amounts of water and nutrients (Saikia et al., 2012).The association of diazotrophic bacteria of the Azospirillum genus culminates with the increase of maize crop yield (Bashan and De-Bashan, 2010).
Other physiological responses caused by inoculation with Azospirillum include the improvement in photosynthetic parameters of leaves, including chlorophyll content and stomatal conductance, higher proline content in aerial part and roots, improvement in water potential, increase in water content of apoplast, higher cell wall elasticity, higher biomass production and higher plant height (Barassi et al., 2008).
Worldwide, the majority of inoculation evaluating experiments with Azospirillum spp. in the maize crop showed increases in grain yield (Kennedy et al., 2004;Kannan and Ponmurugan, 2010).In Brazil, Hungria et al. (2010) when inoculating selected species of A. brasilense and A. lipoferum in maize and wheat, found increases of 26 and 30% in grain yield of these crops, respectively, as well as increases in P and K uptake by plants.Increases in maize yield were also obtained by Cavallet et al. (2000), Novakowisk et al. (2011), Martins et al. (2012) and Araújo et al. (2014) with the inoculation of Azospirillum spp in the treatment of seeds, as in the sowing furrow or in foliar application.However, positive responses to increase in productivity are not always obtained with inoculation of the seeds with Azospirillum spp. as reported by Campos et al. (1999) in oat and wheat crops and by Müller et al. (2012) with the inoculation of A.brasilense in the sowing furrow and the treatment of seeds in the maize crop.Farinelli et al. (2012) evaluated the agronomic viability of the use of the inoculant (A.brasilense) in the treatment of seeds in the maize crop, associated to nitrogen topdressing (0, 90 and 120 kg ha -1 ).They verified that seed inoculation promoted improvements in the morphological and productive traits of maize and that the highest average of grain yield was achieved with the inoculant powder associated with the application of 120 kg ha -1 of N in topdressing.Vazquez et al. ( 2012) evaluated the effects of A. brasilense (liquid, peaty and control without inoculant) and the N rates in topdressing (0, 30, 60 and 120 kg ha -1 ) on the development of the plant and productivity of maize grains.The researchers found that the use of A. brasilense based liquid and peaty inoculant did not interfere with the agronomic traits and grain yield of maize and the fertilization with N applied in topdressing resulted in a linear increase in grain yield and the higher applied dose was not sufficient to obtain the maximum response.Duarte et al. (2012) evaluated the agronomic performance of two maize hybrids (DKB390YG and 30F35H) as a function of seed inoculation with Azospirillum spp.ABV 5 + ABV6 strains and N rates in topdressing (0, 30, 60, 90, 120 and 150 kg ha -1 ).They concluded that the effects of nitrogen fertilization and inoculation with Azospirillum on grain nutrition and grain yield of maize depended on the genetic material, with a positive response of DKB 390YG to the inoculation and a higher response of 30F35H to nitrogen fertilization compared to DKB 390YG.The researchers observed that inoculation increased leaf N concentration, but did not provide partial substitution of nitrogen fertilization in maize crop.Marini et al. (2015) evaluated the efficiency of inoculation of A. brasilense based commercial product via seed treatment (100 ml ha -1 of inoculant at a concentration of 2.0 × 10 8 UFC ml -1 ), in association with different levels of N topdressing fertilization (0, 40, 80, 120 and 160 kg ha -1 ) via urea, applied between the V 4 and V 6 stages, in two maize genotypes (30F53 and CD386).They verified that the inoculation provided increases of 11 and 12%, for leaf area and dry matter of maize aerial part, respectively.There was a differentiated response of maize hybrids to most of the analyzed variables.Grain yield data were adjusted to the cubic model, obtaining a higher value in the dose of 160 kg ha -1 of N by the 30F53 hybrid, with a linear effect increasing as a function of the N doses applied in the hybrid CD386, with an increase in yield of 14.6 kg ha -1 for each kg of N Revolti et al. 579 applied to the soil.
Laboratory results indicate that the beneficial effect of Azospirillum is probably due to obtaining plants with longer roots and larger seedlings, which present a faster initial growth.In field experiments, more roots (54%), higher dry matter in the aerial part (28%) and higher grain yield (7.1% -average of 221 places) were observed, mainly due to the higher number of grains, since there was no change in mean grain weight (Hungria, 2011).The results of studies with inoculation of this microorganism in maize are contradictory.Apparently, what is verified is that there are different ways to carry out the inoculation, with the main ones directly in the seeds, in the furrow of sowing or in the soil when the plant is already in development.Among the causes of variation of available results, it can be inferred that there may be an effect of the fungicides and insecticides applied to the commercial seeds on Azospirillum, the culture phase influences the response, or that there is a differential response of the genotypes used.
Considering the aforementioned, obtaining technical information on the application efficiency of inoculants based on A. brasilense via seed treatment, spraying in the interior of seeding furrow or foliar spraying (reaching the neck region), in the V 4 stage, can promote the reduction in the use of nitrogen fertilizers in maize crop, with increases in morphological, agronomic and grain yield components.
In Brazil, it is estimated that the use of inoculants containing selected strains of A. brasilense can result in an estimated saving of US$ 2 billion per year, considering fertilizer transport costs (Hungria et al., 2010).Consideration should also be given to the benefits of less environmental pollution resulting from the production and use of nitrogen fertilizers as well as the reduction in the emission of greenhouse gases.Thus, researches involving bacteria of the Azospirillum genus is developed by plant breeders, because these microorganisms can associate and provide benefits to crops of great economic importance, such as maize, sorghum, wheat, sugarcane, among others.However, the interaction of maize genotypes with the strains of bacteria of the Azospirillum genus is not yet fully elucidated, so research in this sense has been growing steadily in the world.

Genotype × inoculation interaction
Studies related to the interaction between genotypes × inoculation demonstrate that there is a differentiated response of the genotypes when they are inoculated with diazotrophic bacteria.Reis Júnior et al. (2000) pointed out that when BNF is related with non-leguminous species, the effect of plant genotype on N fixation is expressive.Thus, identifying, selecting and using less demanding genotypes for the N element are important tools (Revolti, 2014).
Usually, inoculation with A. brasilense provides an increase of dry mass, N accumulation in plants and grain yield, especially if the association is between bacteria and unimproved genotypes and under conditions of low N availability (Okon and Vanderleyden, 1997).In addition to these factors, the nutritional state of the plant, the quality of the exudates, the existence of competing microorganisms and the choice of the adapted strain to each region in terms of climate, management system and cultivars, are also factors that can influence the interaction between maize plant and bacterium and affect the efficiency of BNF (Quadros, 2009).According to Bartchechen et al. (2010), research involving Azospirillum in maize indicates that the interaction between the bacterium and the plant varies according to the cultivar, edaphoclimatic conditions, and methodologies of conduction of the trials.These methodologies mainly involve: a) forms of inoculation: seed coating, sowing furrow, application via leaf or plant spray; and b) experimental designs, control of pathogens and pests, and vegetative stage of the plant at the time of inoculation.
However, these bacteria naturally exist in most soils and present wide genetic diversity (Ardakani et al., 2011), making it necessary to use efficient strains in BNF and in the production of growth and development hormones capable to compete with native bacteria as well as to select maize genotypes responsive or suitable for this association (Basi, 2013).Thus, following the Brazilian legislation for inoculants, established by the Brazilian Ministry of Agriculture, Livestock and Supply, Embrapa Soybean researchers led by Hungria (2011) found that Ab-V4, Ab-V5, Ab-V6, and Ab-V7 showed higher soil survival, higher growth promotion and adaptation to technologies used in maize and wheat crops.Following the same study, maize yield increases up to 30% in relation to the control not inoculated with the bacteria.This fact justifies the reason why inoculant manufacturers opt for the Ab-V5 and Ab-V6 strains in their products intended for maize and wheat crops.
In practical terms, there are studies in which 85% of the experiments involving maize and bacteria of the Azospirillum genus responded positively, with an average productivity increase of 472 kg ha -1 (Díaz- Zorita and Fernandez, 2008).Several experiments conducted in Latin America during the last decades, have indicated in the majority, plant growth and/or productivity of the crops studied (Cassán and García de Salamone, 2008).Costa et al. (2015) studied the inoculation with A. brasilense in seeds and nitrogen doses in maize crop in Cerrado region and verified that the use of the bacterium promoted higher plant height, culm diameter, leaf chlorophyll index, culm and root dry mass, ear insertion height, thousand grain weight, and grain yield.Similarly, Cunha et al. (2014) also found positive results when inoculating maize seeds with the bacterium, obtaining an increase in productivity and reduction of nitrogen topdressing application by 16%.Morais et al. (2016), when testing several doses of inoculant containing A. brasilense applied to maize sowing, verified that the dose of 200 ml ha -1 promoted an increase in grain yield.Verona et al. (2010) observed that the inoculation provided greater culm diameter and greater weight in relation to the aerial part dry mass even in water stress.It is known that in addition to the leaves, most of the reserves produced by the plant are stored in the stalks, making this ratio of greater aerial part mass and larger culm diameter produce better storage conditions and a possible higher final production, since these reserves are indispensable for the good development of the plant, mainly in the reproductive phase, to supply the drains represented by the ears.
The benefits of inoculation of A. brasilense can be verified in other cultures.Sala et al. (2008) inoculated wheat seeds (Triticum aestivum hard L. and Triticum durum L.), providing 0, 60 and 120 kg ha -1 of urea (70% at sowing and 30%, 30 days after sowing) and they could see that there is an interaction of the endophytic bacteria response with nitrogen fertilization.As a result of the research, the highest cumulative amount of N was obtained with the inoculation of the A. brasilense IAC-AT-8 strain and with the addition of 60 kg ha -1 of N when compared to the control.They were also able to prove that when the dose of N increased from 60 to 120 kg ha -1 , there was a linear decrease in the nutrient utilization efficiency index.
However, not all of the necessary N in the maize crop provided the bacteria.It is an alternative that allows the producer to reduce the use of nitrogenous fertilizers achieving an economy equal to or greater than that found in legumes, which can be self-sufficient in N (Döbereiner, 1992).García de Salomone and Döbereiner (1996) evaluated different maize genotypes inoculated with Azospirillum and obtained different responses regarding the inoculation under the yield in the production, noting that there are variations in the interactions between maize genotypes and diazotrophic bacteria.

Scientific advances regarding the use of Azospirillum sp. in maize crop
A summary of the main results obtained from inoculation with Azospirillum sp. in recent years is shown in Table 1.

FINAL CONSIDERATIONS
Although there are plans to set up new industries and open up new areas for mineral exploration, the situation over the next ten years is quite critical.Thus, the use of Plant Growth-Promoting Bacteria that assist in the biological fixation of nitrogen is of great value for maize and other grasses, as it is an excellent economic

Species / methodology adopted Inoculation effects References
Azospirillum lipoferum strain (Accession no.GQ255950) Mitigation the deleterious effects of drought on maize.Benefits in corn crop in normal as well as drought stress conditions.Bano et al., 2013.Seed inoculation with Ab-V5 and Ab-V6 strains of A. brasilense Increase in maize plant height, yield of maize grains and plant's dry matter when compared to control.Braccini et al., 2012 Liquid inoculant carryng A. brasilense Ab-V5 The grain production was increased by 29% in the treatment with A. brasilense and nitrogen compared to nitrogen fertilization alone.

Ferreira et al., 2013
Seed inoculation with A. brasilense and five rates of N.
Decreasing of Fe concentration in leaves and increase of corn grain yield.Galindo et al., 2016 Seed inoculation with diferente doses os A. brasilense strains Ab-V5 and Ab-V6.
The dose of 200 mL ha -1 Azospirillum was noteworthy for grain production.Inoculation with the active strain of Azospirillum brasilense (strain 65B) Inoculation was performed just before sowing: bacterium suspension was mixed with maize seeds and used for spraying the field.
Inoculation of maize with A. brasilense bacteria contributed to an increase of that plant vigour and yield.

Hungria et al., 2010
Many strains of A. brasilense and A. lipoferum have been used to inoculate cultivars of different cultivars of species of plants, in including maize, in more than ten countries.
The data indicates 60-70% occurrence of success with statistically significant increases in yield of the order of 5-30%.
The present study in pots was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes.
Two maize genotypes produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp.strains or fertilized with the equivalent of 100 kg N ha -1 .The two genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR-mutants generally caused plant responses similar to those of the parent strains.
De Salamone et al., 1996 Association of doses of nitrogen fertilization with and without inoculation with A. brasilense strains Ab-V5 and Ab-V6.
There was a significant increase in both number and mass of commercial corn cobs with A. brasilense inoculation as compared with treatment without inoculation.The association of inoculation with A. brasilense and nitrogen increase more than 30% the corn cobs production.

Araújo et al., 2014
Four methods of inoculation with A. brasilense (Ab-V5 and Ab-V6) were compared: (1) standard seed inoculation -control treatment; (2) inoculation in the planting furrow at sowing; (3) leaf spray inoculation at the V2.5 stage of the maize plant growth cycle or 3rd tiller for wheat ; and (4) spray inoculation on the soil surface at the V2.5 stage of the maize plant growth cycle or 3rd tiller for wheat.
All inoculation techniques increased the abundance of diazotrophic bacteria in plant tissues, and foliar spray improved colonization of leaves, while soil inoculations favored root and rhizosphere colonization.In field experiments, inoculation with A. brasilense allowed for a 25 % reduction in the need for N fertilizers.Fukami et al., 2016 opportunity to be able to increase the efficiency of nutrient absorption, in addition to providing environmental benefits associated with reduced fertilizer use.However, the efficiency in the use of diazotrophic bacteria in maize is related to the all management involved during the development of the plant, since there is a need to know the physiological characteristics and water needs, nutritional, pest control and crop diseases, so that the actual influence of the growth-promoting bacteria present in the rhizosphere on the plants can be obtained.In addition, the total nitrogen supply of the crop will not only be supplied by the microorganisms, it is necessary to stagger the topdressing fertilization at the recommended doses for the crop, as shown in several types of research.
On this way, considering the existence of a wide range of available genotypes of maize, either commercially or in research institutes, there is a great importance to know the interaction of the genotype under study with the inoculation form of diazotrophic bacteria, in order to identify, select and use less demanding genotypes for the N element.This happens because, as occurs with A. brasilense and several microorganisms, there are genetic variations within the same species, which demands the development of research aimed to evaluate and relate more closely maize genotypes under study with the degrees of association of the existing strains.So it will be possible to delineate genetic breeding programs in an efficient way in order to develop cultivars more responsive to the inoculation with diazotrophic bacteria.Thus, in addition to the low cost for farmers, the use of beneficial bacteria containing Azospirillum contributes to the environment and may be the subject of future negotiations on carbon credits trading.The prospects are also that, in the coming years, the agronomic efficiency of inoculation with Azospirillum can be confirmed with other Gramineae.

Table 1 .
Main recent results obtained from inoculation with Azospirillum spp. in the corn crop.