African Journal of
Agricultural Research

  • Abbreviation: Afr. J. Agric. Res.
  • Language: English
  • ISSN: 1991-637X
  • DOI: 10.5897/AJAR
  • Start Year: 2006
  • Published Articles: 6754

Full Length Research Paper

Agronomic efficiency of inoculant based on Azospirillum brasilense associated with nitrogen fertilization at maize

Vandeir Francisco Guimaraes
  • Vandeir Francisco Guimaraes
  • Universidade Estadual do Oeste do Paranaacute, Street Pernambuco deg 1777, Marechal Cacircndido Rondon, Center, 85960-000, PO Box: 91, Brazil.
  • Google Scholar
Marcelo Andreotti
  • Marcelo Andreotti
  • Bolsista de Produtividade CNPq, Departamento de Fitossanidade, Engenharia Rural e Solos, Universidade Estadual Paulista – UNESP, Ilha Solteira, SP, Brazil.
  • Google Scholar
Jeferson Klein
  • Jeferson Klein
  • University of Anhanguera-Uniderp. Av. Ceará, 333 - Vila Antonio Vendas, Campo Grande - MS, Zip code: 79003-010, Brazil.
  • Google Scholar

  •  Received: 22 November 2016
  •  Accepted: 14 February 2017
  •  Published: 01 June 2017


The objective was to evaluate the agronomic efficiency in the field of the inoculant Fluid and Turfoso containing the bacterium Azospirillum brasiliense, strains AbV5 and AbV6 applied by treatment of seeds in the corn crop associated with nitrogen fertilization in different localities. Four experiments were carried out in different sites involving two in Paraná and one each from Mato Grosso do sul and Santa Catarina. The design was randomized blocks with 6 treatments: T1- 0 kg ha-1  of nitrogen (N), without inoculation (control); T2 - 80 kg ha-1  of N, without inoculation; T3 - 160 kg ha-1  of N, without inoculation; T4 - 80 kg ha-1  of N + seed inoculation AzoTotal® 'Liquid'; T5 - 80 kg ha-1  of N + seed inoculation Nitro 1000 Gramines 'Liquid'; T6 - 80 kg ha-1  of N + seed inoculation Nitro 1000 'Peat' grasses. The evaluations were composed of nutrient content in leaves and grains, and at the end of the cycle length and ear diameter, number of rows of grains and grains per row, mass of one thousand grains and productivity. The results showed that the inoculation of the seeds, regardless of the physical nature of the inoculant, was efficient for the maize crop, reducing the use of mineral nitrogen in all evaluated sites. It is concluded that the use of inoculation with A. brasilense regardless of the physical nature of the inoculant reduces the need for nitrogen fertilization of the corn crop by 50%, without reducing the final yield of the crop.


Key words: Zea mays, biological nitrogen fixation, productivity, Azospirillum brasilense, sustainable agriculture.


The corn crop (Zea mays L.) is the most cultivated cereal in Brazil and in the world. National production in the 2015/2016 harvest was 83.33 million tons of grain (Conab, 2016; USDA, 2016). In the world scenario it has great social, economic and cultural relevance, being relevant for human  consumption  and  mainly  for  animal  consumption.
In relation to mineral nutrition corn is one of the most demanding into fertilizer, mainly the nitrogenous one (Carvalho et al., 2013), since this material needs high amout of nitrogen (Moda et al., 2014). In this study, the use of nitrogen fertilizers was considered to be an important determinant of the nutrient availability of the nutrient in the crop (Ferreira et al., 2007). Therefore, nitrogen fertilization is necessary because of the insufficient amount that the soil provides for adequate plant growth. This situation is particularly important for the maize crop, since, among the nutrients that influence its productivity, the N is one of the most absorbed during the development cycle of the plants.
The use of nitrogen fertilizers in maize crop presents high costs and high environmental impact (Garcia et al., 2013). In addition, there is a low efficiency of the current available sources, which are based on the 50% mark (Costa et al., 2003). The quest for maximizing the use of N is a challenge. One of the ways to increase yield and minimize the production costs is linked to plants that have a more developed root system (Costa et al., 2015), able to better exploit the soil and increase nutrient and water absorption. In this way, better development conditions are provided to the plant, resulting in increased productivity (Bassoi et al., 1994).
An alternative to reduce the use of mineral fertilizers is the use of seed inoculation by diazotrophic bacteria (Novakowiski et al., 2011; Quadros et al., 2014; Inagaki et al., 2015), widely used in leguminous crops.
Among the diazotic bacteria, the Azospirillum genus can colonize plant roots and shoots (Pedrinho et al., 2010). This is a promising alternative, since these microorganisms increase the availability of N to the plant, by breaking the triple bond of the atmospheric N2 and making it available to the plants. This practice can provide up to 50% of the total N to the plant (Piccinin et al., 2013). In addition to the above, this specie is capable of producing plant growth promoting substances (Santi et al., 2013), with the most important being indole acetic acid, an auxin (Radwan et al., 2005; Perrig et al. 2007), resulting in a higher root development (Rodrigues et al., 2014) and thus increasing the area of root system exploration and nutrient absorption (Ferreira et al., 2013).
The use of diazotrophic bacteria as an alternative to increase the availability of nitrogen to crops may be a less costly and ecologically viable option, since it contributes to the reduction of atmospheric CO2 up to 0.309 Mg CO2-eq ha-1 demonstrated in Brachiaria (Hungria et al., 2016). In light of this finding, several studies have been conducted to verify the potential of Azospirillum spp. (Dartora et al., 2013; Repke et al., 2013; Guimarães et al., 2014; Quadros et al., 2014).
In this line, inoculation of the seeds with A. brasilense increases foliar area and shoot dry matter by 12% on the foliar area and dry issue of aerial part (Marini et al., 2015). Costa et al. (2015), showed  an  increase  in  plant height, dry stem and root mass, chlorophyll content, a thousand-grain mass, and final crop yield of the second crop. This increase was 29%.
The objective of the present work was to evaluate the agronomic efficiency in the field of   Fluid and Turfoso inoculant containing the bacterium Azospirillum brasiliense, strains AbV5 and AbV6 applied by treatment of seeds in maize crop associated with nitrogen fertilization in different localities.


The study was carried out by conducting experiments in four sites with different edaphoclimatic conditions in the 2013/2014 harvest. Four experiments were carried out in different sites involving two in Paraná and one each from Mato Grosso do sul and Santa Catarina., all in Brazil (Table 1).
The areas where the experiments were developed were being cultivated with annual and perennial crops under no-tillage system for at least five years. Therefore, in order to characterize it initially, that is to say, before sowing of the maize, samples were taken in twenty profiles of tradition for the collection of the soil with deformed structure, realized with a screw thread in the depth of 0 to 0.20 m, whose chemical and physical characteristics are presented in Table 2. 
The count of diazotrophic microorganisms to determine the population of bacteria in cell numbers per mL was performed by estimating the Most Probable Number (MPN) using the MacCrady table in NFB (Azospirillum spp.) where the Semi-solid medium was used for bacterial growth according to methodology (Döbereiner et al., 1995). The results of the counts of diazotrophic microorganisms by the estimation of MPN in soils of the experimental areas were as follows: Site 1: 2x106 g-1 soil; site 2: 1.1x107 g-1 soil; site 3: 1.8x106 g-1 soil and site 4: 4.5 x106 g-1 soil.
The inoculant 'Nitro 1000 Gramines liquid' has the following characteristics: 2.0x108 CFU mL-1 (Colony Forming Units) of A. brasilense strains AbV5 and AbV6; Physical nature: Fluid; Density: 1.0 g / mL; Target Culture: Maize (Z. mays L.); Dosage tested 100 mL for 25 kg of seed: Lot: 001/2013; Manufacture: 10/31/13. The inoculant 'Nitro 1000 Gramines Peaty' has the following characteristics: Guarantee: 2.0x108 CFU g-1 of A. brasilense strains AbV5 and AbV6; Physical nature: Solid; Density: 1.0 g / mL; Target Culture: Maize (Zea mays L.); Dosage tested: 100 g for 25 kg of seed; Lot: 001/2013; Manufacture: 10/31/13.
The AzoTotal® inoculant was used as reference (standard inoculant) in the four experiments. The inoculant presents 2.0x108 CFU mL-1 of A. brasilense, strains AbV5 and AbV6; Physical nature: Liquid; Density: 1.0 g / mL. The dosage used in the test 100 mL for 25 kg of seed: Lot: 1101213; Manufactured: 09/13/13.
All the inoculants used were submitted to laboratory tests of concentration, purity and characterization. The analyzes followed official methods, according to Normative Instruction number 30, dated November 12, 2010 (MAPA). AzoTotal® Inoculant presented 2.15x108 CFU mL-1; Nitro 1000 Grasses 'Liquid' presented 2.33x108 CFU mL-1 and Nitro 1000 Grasses 'Turfa' presented 2.12x108 CFU g-1.
The four experiments used Piooner® 30F53 YH hydrid simple and were conducted in a randomized block design with six treatments and four replicates. The treatments were: T1- 0 kg ha-1  of nitrogen (N), without inoculation (control); T2 - 80 kg ha-1  of N, without inoculation; T3 - 160 kg ha-1  of N, without inoculation; T4 - 80 kg ha-1  of N + seed inoculation with standard AzoTotal® 'Liquid' inoculant at the dose of 100 mL 25 kg-1  of seeds; T5 - 80 kg ha-1  of N + seed inoculation with inoculant Nitro 1000 Gramines 'Liquid' at the dose of 100 mL 25 kg-1  of seeds;  T6 - 80 kg ha-1  of  N  +  seed inoculation with inoculant Nitro 1000 'Peat' grasses at the dose of 100 g 25 kg-1  seeds.
Nitrogen fertilization was performed in two applications. At the time of sowing, 30 kg ha-1 of N was applied in all treatments, with the exception of T1 (control). In the V6 stage of maize 50 kg ha-1 of N were applied in treatments with 80 kg ha-1 of N (T2, T4, T5 and T6). For T3 treatment, 80 kg ha-1 of N at the V4 stage and 50 kg ha-1 of N at the V8 stage were applied. Urea nitrogen (45% N) was used as the source of both the sowing and coverage.
Prior to corn sowing, the areas were desiccated with glyphosate herbicide at a dose of 4 L of p.c. ha-1. At this moment the cultural remains were collected for analysis of N, P and K in the samples (Table 3).
The experiments were implanted with the following sowing dates: Site 1 (10/10/2013); Site 2 (11/05/2013); Site 3 (10/25/2013) and Site 4 (12/9/2013). The seed were treated with (trichloromethylthio) cyclohex-4-ene-1,2-dicarboxy fungicide at a dose of  0.2 kg  100 kg-1 of seeds, as well as with insecticide imidacloprid + Thiodicarb at the dose of 0.2 L 100 kg-1 of seeds.
Inoculation of the seeds was realized in high density plastic bags, where the inoculants were directly deposited according to the treatments. Then they were agitated for approximately two minutes to standardize the distribution of the inoculant in the seeds. Sixty minutes after inoculation, the sowing was done, being the standard procedure for the four experiments.
Phosphorus fertilization (80 kg ha-1 of P2O5) and potassium (60 kg ha-1 of K2O) were applied to the sowing furrow using the mechanized fertilizer sowing machine (Embrapa, 2012).
Sowing of the experiments was carried out with the aid of a manual seeder (matracas), with five seeds per meter being distributed in the sowing furrow, reaching a final population of 70000 ha-1 plants. Each experimental plot consisted of 6 lines of 0.70 m spacing, 6 m long and 4.2 m wide, totaling 25.2 m2 per plot, spaced apart by 1 m, and total area of 604.80 m2. To obtain the useful area of the plots the outer lateral lines and 1.0 m of the ends of the lines of each plot were disregarded.
During the conduction of the experiments, the control of weeds, pests and diseases were carried out according to the needs of the crop (Embrapa, 2012).
When the plants were in the full flowering stage, they were collected the middle third of 10 leaves opposite and immediately below the main spike, per plot. After harvesting the cobs and threshing of the grains, samples of grains corresponding to each experimental plot were taken. These were dried at 65°C in a forced air circulation oven until mass reached constant. Afterwards, they were ground and analyzed for N, P and K content (Malavolta et al., 1997).Foliar and grain samples  were  ground  and  subjected  to  sulfur digestion according to the methodology of Embrapa (2009).
The experiments harvest was performed on the following dates: Site 1 (05/02/2014); Site 2 (3/26/2014); Site 3 (03/24/2014) and Site 4 (04/09/2014). The components of the production were determined by sampling ten ears per useful plot. The evaluations were length of cobs (CE, expressed in cm), ear diameter (DE, in mm), number of rows per cobs (NFGE), number of grains per row in the cobs (NGF) and mass of thousand grains (MMG, in grams).
For the determination of grain yield, all the cobs of the useful plot were milled and the grains weighed. The results were expressed in kg ha-1, correcting the values for 13% moisture in the wet basis.
The data, after tabulation, were submitted to analysis of variance by the Fisher-Snedecor test (test F) and the means of the treatments were compared by Duncan's test (P ≤ 0.05). The analyses were carried out using the GENES computer program of the Federal University of Viçosa (UFV) (Cruz, 2013).


The results obtained Site 1 showed that nutrient contents in foliar tissue and maize grains were significantly influenced by treatments with the exception of foliar K content and P content in the grains. The N and P contents in the foliar and K tissues in the grains were superior in the treatment with 160 kg ha-1 N, without inoculation; however, they presented significant difference only of the control, without addition of N and inoculation. For the N content in the grains, the highest levels were obtained in plants whose seeds were treated with turfous inoculant; however, this treatment only differed statistically from the control (Table 4).
For the production variables, there was no significant effect on the length of spikes (CE), number of rows per spike (NFGE) and number of grains per cobs (NGF) (Table 5). Regarding the cob diameter (DE) and mass of a thousand grains (MMG), the treatment was 160 kg ha-1 of N, which was statistically higher to the control. When the productivity was observed,  the  agronomic  efficiency of A. brasilense was verified through seed inoculation, since the treatments that received inoculation with A. brasilense + 80 kg ha-1 N, were equal to the treatment with 160 kg ha-1 N (Table 5).
For Site 2, there were no significant effects of treatments on P and K accumulation in foliar tissue and P in corn grains. For the N contents in the foliar tissue and N and K in the grains, a significant effect of the treatments was observed. The foliar N content was higher when 160 kg ha-1 N was used, however, it did not differ statistically from the treatments that received inoculation with A. brasilense. When the N and K contents were observed, the treatments 160 kg ha-1 N and turfous inoculant were superior to the control (Table 6).
Regarding the production components for Site 2, no significant differences were observed in the DE and NFGE. For the CE, NGF and MMG the lowest averages were provided by the control, treatments with inoculation of the seeds with A. brasilense showed the highest averages, except for the standard inoculant, which promoted intermediate results (Table 7).
Productivity was significantly influenced. Seed inoculation treatment with A. brasilense promoted higher means, and fertilization with 160 kg ha-1 N was used. Seed inoculations with standard, liquid and turfous inoculant exceeded the treatment with 80 kg ha-1 N promoting increments of 15.65; 23.16 and 26.22%, respectively (Table 7).
In the experiment conducted at Site 3 no effect of treatments on N accumulation in foliar tissue as well as N, P and K on the grains was observed. For the accumulation of foliar P the control obtained higher average values, while for the accumulation of K the highest average was observed with the fertilization of 160 kg ha-1 N, however differentiating only from the control (Table 8).
At Site 3 all the productive variables were significantly influenced by the treatments with nitrogen fertilization and inoculation of seeds with A. brasilense. The variables CE, DE and NGF were superior to the other treatments when the fertilization with 160 kg ha-1 N.
When evaluating MMG and productivity seed inoculations promoted averages similar to fertilization with 160 kg ha-1 N, which was the highest average. Seed inoculations with standard, liquid and turfous inoculant exceeded the treatment with 80 kg ha-1 N promoting increases of 22.05, 20.19 and 27.80%, respectively (Table 9).
For the experiment conducted at Site 4, N, P and K foliar contents as well as N content in maize grains were not influenced by the treatments used (Table 10).
Unlike the previous site, at Site 4, the variables CE, DE, NFGE, NGF and MMG were not influenced by the treatments used. Productivity was  statistically  influenced by the treatments employed, and the highest average was provided by fertilization with 80 kg ha-1 N, which differed from the control and fertilization treatments with 160 kg ha-1 N (Table 11).


Results of the interaction between diazotrophic bacteria and maize in terms of agronomic potential, nitrogen fixation or growth promotion, depends on many biotic and environmental factors such as plant genotype, soil microbiological community and nitrogen availability (Roesch et al., 2006).
It is important to note that the effect of inoculation with Azospirillum in the experiments conducted can not be correlated only with the increase of N, but also with other nutrients (Holguin and Bashan, 1996; Ferreira et al., 2013). The results obtained in the present research for potassium in Site 3 and phosphorus at Sites 1 and 3.
Thus, based on research data with field inoculation experiments (Okon and Vanderleyden, 1997), the genus Azospirillum spp. promotes gains in productivity of important crops in the most varied conditions of climate and soil. However, they point out that the gain with inoculation goes beyond simply aiding in the biological fixation of N2, also helping in the increase of the absorption surface of the roots of the plant and, consequently, in the increase of the volume of the explored soil, being able to increase the absorption of other nutrients.
According to the authors Okon and Vanderleyden (1997), this finding is justified by the fact that the inoculation modifies the morphology of  the  root  system, increasing not only the number of radicels but also the diameter of the lateral and adventitious roots.
The modifications of the root system is related that Azospirillum spp. In plants, produce and stimulate the production of growth promoting substances, among them auxins, gibberilins and cytokinins, and not only the biological fixation of nitrogen.
According to Cantarella (2007), the N foliar sufficient level is 27.5 to 32.5 g kg-1 N, our result showed below the critical level for the maize crop. For the contents of the other nutrients, the values are within the appropriate, regardless of the treatments. When considering the critical levels established P and K sufficiency range in foliar tissue is, 2.5 to 4.0, and 17.0 to 22.5 g kg-1, respectively (Cruz et al., 2008). In this way, the results of P and K in all studied sites are presented in the range of sufficiency,  except  for  the  K  content   obtained   in  the control treatment in Site 3, which was below the critical level.
The results demonstrated for the foliar N content evidenced that the effect of plant growth promotion by the action of A. brasilense is not restricted to biological fixation of nitrogen, although it contributes in part to the supply of N to the corn plant. About 50% as demonstrated in the present study. However, part of this 50% may have been supplied via the promotion of root growth, due to the induction of plant hormone synthesis, such as auxins (Radwan et al., 2004; Kuss et al., 2007), increasing the nutrient absorption capacity from the decomposition of the pre-existing straw.
Results similar to the present study are demonstrated (Salomone and Dobereiner, 1996) which verified increases in productivity with the inoculation of Azospirillum spp. In different crop conditions. In this same sense Cavallet et al. (2000), verified higher yield indexes in the corn crop, as a consequence of inoculation A. brasilense.
Okon and Labandera-Gonzalez (1994), when evaluating twenty years of studies with the inoculation of Azopsirillum sp. it was found that 60 to 70% of the experiments had positive results. In this sense, the beneficial effects of inoculation with A. brasilense have already been reported in several studies in the literature (Dartora et al., 2013; Repke et al., 2013; Quadros et al., 2014; Costa et al., 2015; Marini et al., 2015; Morais et al., 2015), in addition to the increase in productivity, the characteristics that these bacteria have in synthesizing growth-related phyton- mones are shown in Figure, such as auxins, gibber- linins and cytokinins (Kuss et al., 2007; Perrig et al., 2007) and the availability of N2 present in the soil in absorbable forms for plants.
For the experiment conducted at Site 4, it is worth highlighting that after flowering, the high incidence of foliar diseases in maize was observed, which may have impaired the photosynthetic process and the transsite of assimilates to the grains, explaining in part the average productivity of grains.
In general, the results observed in the present work, involving four sites, developed in three distinct regions, show the agronomic efficiency of A. brasilense (strains AbV5 and AbV6) in promoting plant growth, contributing to good maize of the hybrid Piooner® 30F53 YH, which received inoculation via seed, with half of the nitrogen dose recommended.


The inoculation of the seeds of corn with 100 mL to 25 kg of seed with the liquid and turfous inoculants, based on the bacterium Azospirillum brasilense, presents agronomic efficiency.
Inoculation with Azospirillum brasilense, regardless of the physical nature of the inoculant (liquid or turfous), allowed to reduce nitrogen fertilization in the corn crop by 50% without compromising final crop yield.


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