1. Introduction

The nitrogen concentration in corn kernels can reach 12 g kg^-1 (12 kg t^-1), and the amount of absorbed N can reach 20g m^-2 (20 kg ha^-1). The importance of N is acknowledged for its functions in plant metabolism and its presence as a constituent of proteins, coenzymes, nucleic acids, cytochrome, chlorophyll, and pigments (Li et al., 2024). Nitrate reductase (NR) is the key regulatory enzyme in N metabolism because the NO₃^- absorbed by roots must be reduced to ammonium (NH₄⁺) before being incorporated into organic compounds in the root system and/or shoots (Kant, 2018). The NR enzyme acts during the first step of NO₃^- reduction in higher plants (Ganeteg e outros., 2017). The NO₃^- assimilation pathway in higher plants involves two sequential stages: first, the conversion of NO₃^- to nitrite (NO₂^-), mediated by the NR enzyme and by the nitrite reductase enzyme, which converts NO₂^- to ammonia (NH₃); then, NH₃ assimilation in to the amino acids glutamine and glutamate, which are used for translocating organic N from its sources to its sinks. The main enzymes involved are glutamine synthetase (GS) and glutamate synthase (GOGAT) (de Bang et al., 2021).

Various environmental stresses (e.g., N deficiency) that affect plants are known for generating reactive oxygen species (ROS) and consequent oxidative stress. The activation of oxidative stress results from an imbalance between oxidative and antioxidant compounds, favoring the excessive generation of free radicals or reducing their removal rate. Plants contain a complex antioxidant system for eliminating ROS, including antioxidant enzymes such as superoxide dismutase (SOD) and guaiacol peroxidase (POD) (Hernández et al., 2001). The free radicals inactivate enzymes and damage important cellular components, causing the degradation of chlorophyll and membrane phospholipids as well as the degradation of polysaccharides, the denaturation of enzymes, and DNA breakage (Richards et al., 2015). The defense mechanisms against free radicals are important in limiting the action of oxidative stress and in the destruction of ROS, which depend on the duration of the stress and the ability of the plant to survive these stresses. The plant may react with an increased expression of genes encoding enzymes with antioxidant functions (Munné-Bosch et al., 2013).

The search for sustainability in agricultural production systems has been increasing. One alternative found for minimizing the application of N fertilizers is the use of microrganisms that perform biological nitrogen fixation (BNF), which can supplement N fertilizer use, nourishing the plant and increasing its ability to resist stress (Jensen et al., 2012). Bacteria from the genera Azospirillum (Baldani et al., 1986) and Herbaspirillum (Olivares et al., 1996), which both fix atmospheric nitrogen (N₂), have been isolated from the leaves as well as the stems and especially the roots of various grasses. The companies have chosen to produce inoculants of A. brasilense with the strains Ab-V5 + Ab-V6, because they are effective with the cultures of corn and wheat. Hungria et al. (2010) evaluated the use of A. brasilense, strains Ab-V5 + Ab-V6, on corn and found a 27% increase in productivity.

Gárcia de Salomone and Döbereiner (1996) observed significant differences in the NR activity of corn genotypes inoculated with a mixture of Azospirillum spp. strains. The use of the species A. brasilense they increase the activity of the antioxidant system of plants in stressful conditions (Vurukonda et al., 2016). In addition, Azospirillum produces phytohormones that stimulate the growth of the roots of different plant species (Fukami et al., 2018).

Few studies have been carried out to identify the mechanisms of interaction between these bacteria and enzymes of N metabolism in corn. Thus, the objective was to work to evaluate the enzymatic and morphological changes of maize hybrids submitted to inoculation with A. brasilense in seed treatment and leaf pathway.

2. Materials and Methods

2.1. Study site and seedling growth

The study was conducted using a greenhouse hydroponic system in the Plant Biotechnology Laboratory of the Biology Department, Santa Maria Federal University (Universidade Federal de Santa Maria), Santa Maria, Rio Grande do Sul State, Brazil. The seeds of six corn hybrids were sown on germitest substrate in 17 L plastic boxes and placed at 25 °C with 0.0015 L of distilled water available per day to allow germination to occur. After two weeks, the hybrids had similar growth and uniform seedlings (shoot length of approximately 0.007 m) were selected and transplanted to the hydroponic system, which used a nutrient solution with no N available. The nutrient solution contained 221.96 mg L^-1 of CaCl₂, 246.40 mg L^-1of MgSO₄, 544.40 mg L^-1 of KH₂PO₄, and 47.99 µM FeSO₄/Na-EDTA; the complete micronutrient solution contained 0.03mg L^-1 of Mo, 0.26 mg L^-1of B, 0.06 mg L^-1 of Cu, 0.50 mg L^-1 of Mn, and 0.22 mg L^-1 of Zn. The pH of the solution was kept at 5.8 adjusted with addition of HCl, and the solution was replaced every seven days.

2.2. Experimental design and description of the treatments

Six experiments were conducted, in a completely randomized design, with five replicates, composed of six corn hybrids inoculated with A. brasilense at a dose of 0.0025 L kg^-1 of seed in the seed treatment, and 0.3 L ha^-1 in the leaf application, which occurred at the V2 stage (Ritchie et al., 1993); the bacteria were absent from the control treatment; totaling 90 plots. The inoculation of the seeds occurred before the sown on the germitest substrate, for application in the leaf, a backpack sprayer containing a light jet spray tip, with a working pressure of 30 lb in^-2 was used, which provides a spray volume of 200 L ha^-1.

The hybrids used were AG9045 (Agroceres Seeds), AG8025 (Agroceres Seeds), AG2040 (Agroceres Seeds), Feroz (Agroceres Seeds), BG7051 (BioGene), and BG706 (BioGene). The commercial product inoculant used consisted of combined A. brasilense bacterial colonies from the AbV5 and AbV6 strains at a concentration of 2.0x10⁸ colony-forming units (CFU) mL^-1. This combination of strains (AbV5 and AbV6) was used in all treatments that contained the bacteria. Each plot consisted of four plants, grown until stage V3 (Ritchie et al., 1993).

2.3. Biochemical variables, pigments, and morphology of roots and shoots

The seedlings were harvested, and the leaf and the roots were separated for the analyzes. The seedling structures utilized in the biochemical analyses of lipid peroxidation and in determining the activity of the SOD, POD, and NR enzymes as well as the level of total chlorophyll and carotenoids were immediately frozen in liquid N after their harvest and kept in a freezer (-80 °C) until analysis, for these analyzes two plants per plot were used. The morphological measurements of leaf area (LA), shoot dry weight, and root length, projected area, surface area, diameter, volume, number of branches, and dry weight were taken shortly after the plants were harvested, in two plants per plot.

Lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was estimated in accordance with the method of El-Moshaty et al. (1993). Leaf (0.5 g) and root (1.5 g) samples were macerated in liquid N and homogenized in 4 mL of 0.2 M citrate buffer (pH 6.5) containing 0.5% Triton X-100. The homogenate was centrifuged at 20,000 x g for 15 min at 4 °C. A 1-mL aliquot of supernatant was added to 1 mL of 20% (w/v) trichloroacetic acid (TCA) containing 0.5% (w/v) thiobarbituric acid (TBA). The mixture was heated at 95 °C for 40 min, then cooled in an ice bath for 15 min and centrifuged at 10,000 x g for 15 min. The absorbance of the supernatant was read at 532 and 600 nm (to correct for nonspecific turbidity). Lipid peroxidation is expressed as nmolmalondialdehyde (MDA) mg^-1of protein.

Leaf and root samples macerated in liquid N were used for the enzymatic analyses. For this purpose, a 0.5-g sample was homogenized in 3 mL of 0.05 M sodium phosphate buffer (pH 7.8) containing 1 mM EDTA and 2% (w/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 13,000 x g for 20 min at 4°C, and the supernatant was used to determine the enzyme activity (Zhu et al., 2004) and protein concentration (Bradford, 1976). The activity of the POD enzyme was determined in accordance with Zeraik et al. (2008), using guaiacol as the substrate. The reaction mixture contained 1.0 mL of potassium phosphate buffer (100 mM, pH 6.5), 1.0 mL of guaiacol (15 mM), and 1.0 mL of H₂O₂ (3 mM). After homogenization, 50 μL of the plant extract was added to this mixture. The enzyme activity was measured through the oxidation of guaiacol to tetraguaiacol detected by an increase in absorbance at 470 nm. The results are expressed in units per mg of protein (U mg^-1 protein). For this calculation, a molar extinction coefficient of 26.6 mM^-1cm^-1 was used.

The SOD activity was determined in accordance with the spectrophotometric method described by Giannopolitis and Ries (1977). The reaction mixture contained 50 Mm potassium phosphate buffer (pH 7.8), 13 mM methionine, 2 μM riboflavin, 75 μM nitro blue tetrazolium (NBT), 0.1 mM EDTA, and 100 μL of enzyme extract. The photochemical production of blue formazan from NBT was monitored via an increase in absorbance at 560 nm. The reaction was performed in test tubes at 25 °C inside a reaction chamber equipped with a 15-W fluorescent lamp.

The reaction was started by turning on the light and was stopped after 15 min by turning off the light. As a control, additional tubes with the reaction mixture were kept in the dark. One unit of SOD was defined as the amount of enzyme that inhibits NBT photoreduction by 50% (Beauchamp and Fridovich, 1971). In the assay, photochemically excited riboflavin is reduced by methionine to semiquinone, which donates one electron to the oxygen, forming the superoxide radical, which in turn converts NBT into blue formazan.

The leaf photosynthetic pigments (chlorophylls and carotenoids) were extracted in accordance with the method of Hiscox and Israelstam (1979) and estimated using the equation of Lichtenthaler (1987). Fresh leaf samples (0.05 g) were incubated at 65 °C with dimethylsulfoxide (DMSO) until the pigments were completely removed. The absorbance of the solution was measured in a spectrophotometer (Celm E-205D – Companhia Equipadora de Laboratórios Modernos, Alphaville, Barueri, São Paulo (SP) State, Brazil) at 645 and 470 nm for total chlorophyll and carotenoids.

The NR activity (µM NO₂^- g^-1FW h^-1, with FW representing fresh weight) was analyzed using the method described by Jaworski (1971) with modifications, calculating the enzyme activity based on the amount of NO₂^- released by the plant tissues into the incubation solution and using a standard curve. Initially, 0.5 g of each sample was macerated in 5 mL of 0.1 mM phosphate buffer (KNH₂PO₄) (pH 7.5) containing 1% (v/v) isopropanol and KNO₃^- (50 mM). The extracts were transferred to test tubes and then placed in a water bath at 37 °C for 30 min in the dark. The reaction was stopped by adding 1 mL of 1% sulfanilamide and 1 mL of 0.02% α-naphthyl (n-naphthyl-ethylenediamine). The contents of the tubes were left to rest for 15 min and then read in a spectrophotometer at 540 nm.

An image analysis system (WinRHIZO Pro LA2400; Regent Instruments Canada Inc., Quebec, Canada) was used to scan and analyze the morphological characteristics of the root and leaf. The root system and leaves were scanned separately on the scanner (Epson Expression 10000XL; Seiko Epson Corporation, Suwa, Nagano, Japan), in a transparent plastic tray filled with water. LA and root length, projected area, surface area, diameter, volume and number of branches were determined.

2.4. Statistical analysis

The data were subjected to the analysis of variance F-test (p≤0.05). When the F-test was significant, the Scott-Knott test was applied to test the difference between treatment means. The data were analyzed using SISVAR statistical software (Ferreira 2011).

3. Results

3.1. Pigments and biochemical variables of shoots

A significant effect was observed in all of the hybrids for the LA variable (Figure 1A). The seed application and the leaf application in V2 were shown to be efficient for the Feroz hybrid. In the present study, the increase in the LA did not result in a corresponding significant difference in the dry biomass of the shoots. Except for the AG2040 and Feroz hybrids, the inoculant application during the V2 stage significantly reduced the LA.

The leaf pigments were analyzed by measuring the chlorophyll and carotenoid levels (Figure 1B and C). The highest chlorophyll level was observed in the AG9045 hybrid when applying A. brasilense during the V2 development stage. The variable carotenoids differed among the hybrids - a relatively high carotenoid level occurred following the application of A. brasilense at V2 in the AG9045 and AG2040 hybrids, as well as in the control for the AG2040 and BG7051 hybrids.

There was reduced SOD activity with the application of A. brasilense at V2 to the BG7051 and AG2040 hybrids, and the heightened SOD activity following the V2 treatment of the BG7060 and Feroz hybrids (Figure 2A). A similar trend was observed for the activity of the antioxidant enzyme POD: the leaf application at V2 increased the POD activity in the shoots of the AG8025, AG2040, Feroz, and BG7060 hybrids (Figure 2A).

In general, the NR enzyme activity was greater in the shoots (Figure 2C) than in the roots (Figure 2C). The NR enzyme of the shoots showed greater activity in the control compared with the bacterial treatments for the AG9045, AG8025, Feroz, and BG7060 hybrids, with the presence of the bacterium reducing the NR activity (Figure 2C). The concentration of MDA (TBARS) one of the products of lipid peroxidation - rose with the application of the seed inoculation (TS) treatment in the AG9045, AG2040, and BG7051 hybrids (Figure 2D).

3.2. Biochemical variables and morphology of roots

For the AG2040 hybrid, the parameters root length, projected area, and surface area showed a positive response to inoculation with A. brasilense, both in the seed treatment and in the V2 stage leaf application (Figure 3A, B, and C). Additionally, for the AG8025 hybrid, the seed treatment with A. brasilense led to a positive response in the root length, projected area, surface area, volume, and number of tips (Figure 3D and E). The bacterium promoted increases in the variable root dry weight for the BG7060 hybrid, while for the AG8025 and AG2040 hybrids, a decrease occurred with the bacterial treatment (Figure 3F).

For the AG2040 corn hybrid in the V2 treatment, a significant increase in the activity of the SOD enzyme was observed in the roots (Figure 4A). The AG9045, AG8025, and BG7051 hybrids showed greater SOD activity without the bacterium (control treatment). The POD activity varied significantly among the treatments in all of the hybrids evaluated and more frequently generated increased activity in the A. brasilense-bacterium-exposed plants, decreasing lipid peroxidation (Figure 4B).

The bacterial TS significantly increased the activity of the NR enzyme in the root of the AG8025 hybrid, but not in the root of the other hybrids (Figure 4C). For the Feroz hybrid, greater lipid damage was observed with the TS (Figure 4D). For the AG2040, BG7060, and BG7051 hybrids, the control treatment showed greater lipid peroxidation.

4. Discussion

The enzymatic activity of the corn plant submitted to treatment with A. brasilense helps to understand the inconsistency of results obtained in the field, so the work was carried out in the hydroponic system as it is less complex than the soil. In addition, this work can be used to help identify important biomarkers of plants induced or repressed by the tested bacterial strains that have positive responses in some corn plants and can be used for future experiments in field or greenhouse plants. The enzymatic activities of corn plants inoculated with A. brasilense are less explored in scientific studies, but they are important to understand the dynamics of the interaction between the plant and the bacteria.

The positive effect of the seed application and the leaf application in V2, for hybrids AG2040 and Feroz, may be related to the presence of the bacterium inside the root and/or in the plant, contributing to the BNF and phytohormone production, which increases the LA (Figure 1A). In wheat, a greater LA and shoot dry biomass were observed when the plants were inoculated with A. brasilense without the addition of N (El-Sayed and Althubiani, 2015).

The higher pigment concentration leads to a greater absorption of light energy as well as increases in photosynthesis and in the production potential and vigor of the plants (Bashan et al., 2006). The results show that for the AG2040 hybrid (Figure 1B), the presence of the bacterium reduced the level of chlorophyll; this result may be related to some unmeasured stress or to a lesser amount of N available to the plant, through the BNF, for the synthesis of chlorophyll. This chlorophyll loss may cause a progressive decline in the photosynthetic capacity of the plants. Chlorophylls play an important role in photosynthesis because they are responsible for the capture of light energy. Thus, a lower chlorophyll concentration may lead to a decrease in the photosynthetic efficiency and affect other cellular processes, such as cell division and expansion (Björn et al., 2009).

In general, a reduction in the carotenoid levels was observed with the seed application of inoculant (Figure 1C). In addition to acting as accessory photosynthetic pigments, carotenoids have an essential role in photoprotection, helping to eliminate the free radicals that damage cellular components such as lipids. Therefore, these pigments are important in preventing stress-induced oxidative damage. In this regard, hybrids showing higher amounts of these pigments have an enhanced capacity to eliminate free radicals, whereas a reduced carotenoid concentration may indicate greater damage to the photosynthetic membranes in the hybrids evaluated. Hybrids with higher levels of these pigments would be expected to have a greater ability to tolerate stress conditions due to the close relationship among the pigments, photosynthetic potential, and grain yield (O’Neill et al., 2006).

To minimize the effects of oxidative stress, plants have an antioxidant system composed of enzymes, such as POD and SOD, thatactively participate in the elimination and breakdown of ROS (Foyer and Noctor, 2003). The reduced SOD activity with the application of A. brasilense at V2 to the BG7051 and AG2040 hybrids indicates that other antioxidant enzymes besides those analyzed in the presen tstudy may be operating and reducing the damage to membrane lipids (Figure 2A). The heightened SOD activity following the V2 treatment of the BG7060 and Feroz hybrids may be related to the lower lipid peroxidation level in this treatment (Figure 2A). These results indicate that the bacterial application effectively increased the defense capacity of corn plants against the oxidative stress induced by the absence of N.

The seed treatment with A. brasilense lowered the SOD activity in the AG8025 and AG2040 hybrids but increased this activity in the Feroz hybrid, which prevented lipid peroxidation (Figure 2A). SOD is the most effective antioxidant enzyme in all aerobic organisms and in subcellular spaces prone to oxidative stress. That environmental stresses often lead to increased ROS generation is well described in the literature; therefore, SOD is important in the stress tolerance of plants, providing the first line of defense against the toxic effects of high ROS levels.

This result, of POD, may be related to the lower peroxidation of membrane lipids in this treatment (Figure 2A). POD is responsible for the removal of hydrogen peroxide (H₂O₂), which is an ROS (Hameed et al., 2011) that can act detrimentally, protectively, or as a signaler, depending on the balance between the production and the elimination of the ROS at a given location and time. Thus, the increased SOD and POD activity when A. brasilense was applied reduced the lipid peroxidation and increased, in most treatments, the LA of the hybrids evaluated.

This result of NR enzyme activity may have occurred because N assimilation requires plants to expend substantial energy (Figure 2C), explaining why this process occurs predominantly in the leaves, which is the center for ATP synthesis, reducing agents, and electron donors. The NR expression and activity are strongly regulated by several factors, such as light, NO₃^- concentration, carbohydrates, and the availability of metal cofactors. The equilibrium of the enzyme is determined by the rate of its degradation and its synthesis. The rate and the amount of N assimilation by plants during their cycle depend on the activity of the enzymes involved and the availability of the energy required for the assimilation processes (Bredemeier and Mundstock, 2000).

For the treatments with A. brasilense, a lower NR activity was observed, which may have occurred because the bacteria of the Azospirillum genus were able to fix N₂, transforming it into NH₃, an N form that can be used by the plants; therefore, no need for NO₃^- reduction existed. Additionally, bacteria from the Azospirillum genus can function in plant growth through the reduction of NO₃^- to NH₃ (Ferreira et al., 1987); therefore, plants would not expend energy to reduce NO₃^- to NH₃, and this energy could be allocated to other vital metabolic processes. According to Machado et al. (1998), such bacteria can exert some influence on the GS activity in corn plant roots; however, the activity of this enzyme was not evaluated in the present study. Reis Junior et al. (2008) observed that with the predominance of NO₃^-, the activity of the NR enzyme increased. Furthermore, genetic variability for NR activity has already been reported (Purcino et al., 1994).

The concentration of MDA indicating the existence of higher stress due to N deficiency, which was reflected in the lower level of chlorophylls and carotenoids (Figure 2D). The accumulation of ROS, as a result of various environmental stresses, is among the main causes for the loss of plant yield components (Gill and Tuteja, 2010), because these stresses affect cellular constituents, such as nucleic acids and proteins, resulting in lipid peroxidation. For the BG7060 hybrid, oxidative stress decreased with the presence of the bacterium. Additionally, the inoculation of A. brasilense in the seeds reduced lipid peroxidation in the AG9045, AG2040, and BG7060 hybrids (Figure 2D). This result may have occurred due to an increased activity of the antioxidant enzymes, which prevented plant stress.

The results, namely, larger positive root responses with inoculation via the seeds compared with the leaves, may be due to an association of the bacterium with the root from the start of development, leading to benefits for the plant (Figure 3). Thus, the plants that received a leaf application during V2 may not have benefited more significantly because the experiment only lasted until the V3 stage. The root increases were probably due to the ability of the bacterium to produce phytohormones and make them available to the plant. In studies involving the inoculation of millet with A. brasilense, modifications in the root morphology were observed, with a greater number of lateral roots and an increase in root hairs, similar to the changes caused by applying indoleacetic acid (IAA) (Perrig et al., 2007).

Observing that (Figure 3) shows a significant improvement of AG8025 and AG2040 treated with seeds for number of branches, surface, projected area. The effect of the A. brasilense inoculation on the root development of the hybrids is related to the potential area explored by the plants. Diazotrophic bacteria can influence crop N nutrition, an effect that is indirectly related to an increased root. In the first studies on the associations between plants and Azospirillum spp., researchers believed that the benefits promoted by the bacterium were derived only from the BNF. However, studies have shown that these positive microbial effects are mainly due to morphological and physiological changes in the roots of inoculated plants (Cassán et al., 2014).

Changes in the root system are related to the growth-promoting substances mainly IAA produced by Azospirillum spp., which play an important role in promoting plant growth in general. Accordingly, in studies with A. brasilense in a liquid medium, the production of IAA increased within 96 h. Additionally, small but biologically significant amounts of gibberellin and kinetin were detected in the liquid medium (El-Sayed and Althubiani, 2015).

This result, for the AG2040 hybrid, may relate to the lower lipid peroxidation in the root (Figure 4A), resulting in a larger root system. For the AG9045, AG8025, and BG7051 hybrids, N absence can increase ROS production, thus leading to the inhibition of protein synthesis and an increased activity of antioxidant enzymes (Figure 4A). For the POD results, the bacterium assisted in increasing the activity of the root antioxidant enzymes, thereby preventing lipid peroxidation (Figure 4B). The antioxidant enzymes participate in the direct elimination of H₂O₂, acting in the cytosol, vacuole, cell wall, and organelles where detoxification is necessary (Noctor and Foyer, 1998). The bacterium has a capacity promote an increase in morphological and biochemical characteristics, when interaction occurs between plant and bacteria, it is important to find the right conditions so that bacteria to be responsive in increasing the grain yield of the corn crop.

Unlike the response obtained for the NR activity in the shoots, the bacterial TS significantly increased the activity of the NR enzyme in the root of the AG8025 hybrid (Figure 4C), this result may be due to an association of the bacterium with the root of the plants that made more NO₃^- available to the roots, thereby increasing the enzyme activity. The NR activity decrease in the leaves, accompanied by an increase in yields, could be explained by an NO₃^- reduction in the roots due to the bacterial NR activity, as observed in wheat (Baldani et al., 1986).

N-deficient plants absorb NO₃^- faster than plants well supplied with N, indicating that the N demand of the plant also controls its rates of absorption. NR is the first enzyme in the N reduction chain within the N-uptake process of plants. Consequently, the NR activity may be indirectly related to crop yield. Plants with high NR activity have a greater ability to assimilate the available NO₃^- and thus a greater ability to respond to N fertilization (Purcino et al., 1994).

MDA is a product of lipid peroxidation. Thus, a higher level of this compound indicates oxidative stress. The higher POD activity in the A. brasilense treatments did not prevent damage, indicating that the competition for fixed N may have induced the production of the free radicals that oxidized the phospholipid components of the cell membranes, resulting in the increased MDA levels in the Feroz hybrid (Figure 4D). The ability of such competition to cause oxidative stress was recently observed, with the detection of H₂O₂ accumulation in the first leaf and root tissues (Afifi and Swanton, 2012).

When competition for N occurs, N availability to the crop declines, which causes stress and hinders plant physiological and biochemical processes. This condition of cellular stress can be evaluated using the lipid peroxidation levels (Huang et al., 2004). The greater lipid peroxidation for the AG2040, BG7060, and BG7051 hybrids, indicating that the bacterium assists the antioxidant enzymes, promoting a reduction in lipid peroxidation. Furthermore, the high POD activity may be related to the high H₂O₂ availability produced by the N-deficiency-induced stress. Thus, POD would be the main enzyme responsible for the breakdown of peroxide, given that POD is located in the cell wall. Consequently, the bacterium has the ability to promote an increase in morphological and biochemical characteristics when the interaction between plant and bacterium is suitable. However, determining the conditions required for the bacterium to promote an increased corn grain yield is important.

5. Conclusions

Different responses were observed in the interaction of maize hybrids with inoculation of A. brasilense via e seeds and leaf. The hybrids AG9045, AG8025 and AG2040 showed the highest activity of antioxidant enzymes when inoculated with A. brasilense in the seeds or in the leaf in V2. Inoculation via seeds treatment showed the best results, however the application in V2 can be an alternative for applications in the field. The bacterium positively affects the activity of antioxidant enzymes and increases the root system and leaf area of plants.

Acknowledgements

To the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Processes 312480/2020-2), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil - Finance code 001, and the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS – Processes 22/2551-0001644-8) for granting scholarships to the authors. To the scholarship students and volunteers for helping in data collection.

References

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