**2. Materials and methods**

**1. Introduction**

100 Wheat Improvement, Management and Utilization

in the country [12].

by Bashan et al. [16].

N2

Wheat (*Triticum aestivum* L.) is an annual cycle plant, considered among the cool season cereal,

The final crop yield is defined according to the cultivar used, the amount of agricultural supplies, and management techniques employed. The increasing use of high-yield potential wheat has implicated in more frequent use of agricultural supplies, among which nitrogen fertilization shown to be important in defining the grain yield [2]. Therefore, there is a need to study wheat cultivars verifying their response to the uptake and utilization of nutrients in the

Nitrogen fertilization is one of the highest costs of the production process of nonleguminous crops [4]. Wheat, corn, and rice crops utilize approximately 60% of the N fertilizer produced in the world [5]. The use of N fertilizer must be carefully controlled to ensure good yield and

Several authors reported a positive response of nitrogen fertilization on grain yield of wheat [3, 7–11]. Due to the high cost of fertilizers and awareness in support of sustainable agriculture and less polluting, in which the research is growing, one possibility would be to use inoculants containing bacteria that promote growth and increase the productivity of plants. Studies on biological nitrogen fixation (BNF) by *Azospirillum* in grass have been carried out in Brazil. Until recently, no commercial inoculants with these bacteria are available

Although the plant genotype performs an essential role in the colonization of bacteria, cultivars with high and low potential of association exist [13]. Several studies have been published confirming that *Azospirillum* produces phytohormones that stimulate root growth in many plant species. The components released by *Azospirillum brasilense* responsible for stimulating root growth are indoleacetic acid (IAA), gibberellins, and cytokinins [14]. Inoculation with *Azospirillum* can improve the leaf photosynthetic parameters, including chlorophyll content and stomata conductance, greater proline content in shoots and roots, improvement in water potential, an increase in water content in the apoplast, more elasticity of the cell wall, more biomass production, and greater plant size which were reported by Barassi et al. [15]. Increases in photosynthetic pigments such as chlorophyll a and b and auxiliary photoprotective pigments, such as violaxantine, zeaxantin, ateroxantine, lutein, neoxanthin, and beta-carotene, which result in greener plants without water-related stress, were verified

In addition, the increase in root development caused by inoculation with *Azospirillum* is involved with several other effects. Increases in water and mineral uptake have been reported, as well as greater tolerance to stresses, such as salinity and drought, resulting in a more vigorous and productive plant [17, 18]. According to Dobbelaere et al. [19], positive responses to inoculation with *A*. *brasilense* are obtained even when the crops are grown in soils with high N content available, which indicates that the plant responses occur not only due to the

fixed but also mainly depending on the production of phytohormone growth promoters

one that has greater economic importance, with large grain yield capacity [1].

soil and their performance and cultural practices in different environments [3].

manage N in the soil; N fertilizer increases production costs for farmers [6].

The wheat experiment was conducted in 2014, in an experimental area that belongs to the UNESP Engineering Faculty, located in Selvíria—MS/Brazil—with the following geographical coordinates 20°22′S and 51°22′W and an altitude of 335 m. Soil in this experimental area was classified as Distroferric Red Oxisol with clay texture (with values of particle size of 420, 50 kg−1, and 530 g of sand, silt, and clay, respectively), according to Embrapa [22], which has been cultivated with annual cultures over 27 years and the last 11 years with no-tillage system. The area was under corn cultivation before sowing wheat. The annual average temperature was 23.5°C, annual average pluvial precipitation was 1370 mm, and annual average relative air humidity was between 70 and 80%.

Glyphosate [1800 g ha−1 of active ingredient (a.i.) and 2,4-D (670 g ha−1 of a.i.)] herbicides were used for desiccation and applied in 2 weeks prior to sowing wheat. Chemical attributes of the soil in the tillable layer were determined before the wheat experiment began. The methods proposed by van Raij et al. [23] showed the following results: 13 mg dm−3 of P (resin), 6 mg dm−3 of S═SO<sup>4</sup> , 23 g dm−3 of organic matter (OM), pH (CaCl<sup>2</sup> ) of 4.8, 2.6 mmol<sup>c</sup>  dm−3 of K+ , 13.0 mmol<sup>c</sup> dm−3 of Ca2+, 8.0 mmol<sup>c</sup> dm−3 of Mg2+, 42.0 mmol<sup>c</sup> dm−3 of H + Al, 5.9 mg dm−3 of Cu, 30.0 mg dm−3 of Fe, 93.9 mg dm−3 of Mn, 1.0 mg dm−3 of Zn (DTPA), 0.24 mg dm−3 of B (hot water), and 36% base saturation. After soil chemical analysis, 2.5 t ha−1 of dolomitic limestone (with 88% relative total neutralizing power) was directly applied as topdressing 80 days before the wheat was sown in 2014 in order to elevate base saturation to 70%, as recommended by Cantarella et al. [24].

The experimental design was a randomized block with four replications, in a factorial scheme 5 × 2, with five N rates (0, 50, 100, 150, and 200 kg ha−1, as urea) applied as topdressing at the growth stage 3.2 on Zadok's scale [25], with and without seed inoculation with *A*. *brasilense*. Wheat seeds were inoculated with 300 mL ha−1 of inoculant liquid of *A*. *brasilense* bacteria AbV5 and AbV6 strains (guaranteed minimum analysis of 2 × 10<sup>8</sup> UFC mL−1). The inoculant was mixed with the seeds using a cement mixer, 1 h before planting and after the seed treatments with carbendazim + thiram fungicides (45 + 105 g a.i. per 100 kg of seed) and thiodicarb + imidacloprid insecticides (45 + 135 g a.i. per 100 kg of seed). Each plot consisted of 6 m in length with 12 lines and an inter-row spacing of 0.17 m. The usable area of the plot was eight center lines, excluding 0.5 m extremities. The plot size was 10.20 m<sup>2</sup> .

Were applied 350 kg ha−1 of the 08-28-16 formulation in the forms of urea, triple superphosphate, and potassium chloride, respectively, at wheat sowing was applied. The experiments were conducted in a no-tillage system. The area in both crops was irrigated by a central pivot sprinkler system. The water coverage was 14 mm over a period of around 72 h. The cultivar used was the CD 116, and sowing was done with an experimental machine on 05/16/14, with 80 seeds being sown per meter. Metsulfuron-methyl (3.0 g a.i. ha−1), a postemergence herbicide, was applied 20 days after emergence (DAE) to control weeds, like *Ipomoea grandifolia*, *Tridax procumbens*, and *Spermacoce latifolia*. The seedling emergence was 6 days after sowing. Topdressing with nitrogen fertilization was performed at 35 DAE, manually distributing the fertilizer on the soil surface (no incorporation) beside and approximately 8 cm of sowing lines in order to avoid the contact of the fertilizer with the plants. After this topdressing, the area was irrigated by sprinkling (depth 14 mm) at night to minimize N losses by volatilization of ammonia, a procedure common in the irrigated wheat crop. The plants were harvested 110 days after wheat emergence.

Concentrations of N, P, K, Ca, Mg, S, Cu, Fe, Mn, and Zn were measured in the grain and straw (above the soil) of wheat at harvest occasion (the end of the crop cycle), in 10 plants per useful area of plot. The determination of nutrients was carried out as described by Malavolta et al. [26]. The wheat was harvested from the plants in the useful area of each plot, and grain yield was calculated after mechanical threshing. Data were transformed into kg ha−1 and corrected for 13% moisture (wet basis). The agronomic efficiency of the treatments was determined:

<sup>A</sup><sup>E</sup> <sup>=</sup> grain yield with fertilizer <sup>−</sup> grain yield without fertilizer \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ amount of <sup>N</sup> applied . (1)

The results were subjected to analysis of variance and Tukey's test at 5% probability to compare the averages of plants that had been inoculated with *A*. *brasilense* with those that had not been inoculated. Regression equations were fitted for the effect of N rates using the Sisvar program [27]. For the Pearson correlation analysis, separated from inoculated and non-inoculated treatments, we used SAS program [28].
