Nitrogen fertilization and leaf spraying with Azospirillum brasilense in wheat: effects on mineral nutrition and yield

The use of Azospirillum brasilense has the potential to improve plant nitrogen (N) use efficiency, while a better understanding of alternative management practices with inoculation is necessary. The aim was to examine the effects of the leaf application of A. brasilense in association with nitrogen fertilization on the wheat crop. The experiment was conducted in Lidianópolis, Paraná, Brazil, in a completely randomized block design with four replications. The treatments included four doses of A. brasilense for leaf application (0, 200, 400, and 600 ml ha) and four doses of N (0, 40, 80, and 120 kg N ha). The nutritional content, yield components, quality, and yield of the wheat crop were evaluated. There was no interaction among the factors, nor did the treatments have an isolated effect on spike length, the number of spikelets per spike, spikes per m, thousand grain weight, and test weight. However, doses of A. brasilense increased calcium and magnesium absorption at 283 and 380 ml ha, respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha, respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha, respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf. The results demonstrate that the inoculation of leaves with A. brasilense favored a higher absorption of divalent cationic macronutrients and that N was fundamental to increasing the yield, with the best responses observed between 47 and 61 kg ha.

calcium and magnesium absorption at 283 and 380 ml ha -1 , respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha -1 , respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha -1 , respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf. The results demonstrate that the inoculation of leaves with A. brasilense favored a higher absorption of divalent cationic macronutrients and that N was fundamental to increasing the yield, with the best responses observed between 47 and 61 kg ha -1 .

INTRODUCTION
Brazil sets itself apart from the world as one of the largest food producers. However, with regard to the wheat crop, national production has only supplied 50% of national demand (CEPEA 2018), which makes the country one of the largest importers of wheat (ABITRIGO 2017). In the 2017 harvest, the area designated for the wheat crop corresponded to approximately 2.0 million ha and resulted in a yield of 5.4 thousand tons, culminating in an average yield of 2225 kg ha -1 (CONAB 2018).
The state of Paraná in Brazil is the biggest cereal grain producer and has the largest cultivated area. Therefore, wheat is a very important winter crop for the South of the country and one of the few sources of income during the cold season (RIBEIRO et al. 2018). This is why it is necessary to develop techniques that allow for better use of the cultivated areas, particularly correct soil fertility management and mineral nutrition.
Nitrogen (N) is the most required nutrient for the wheat crop, and its extraction and exportation are in the order of 28 and 20 kg N for each ton of grain produced, respectively (SBCS/NEPAR 2017). According to MOREIRA & SIQUEIRA (2002), less than half of the N applied in the soil is absorbed by the plants, the rest is immobilized or lost through volatilization and/or leaching. In this sense, using biological resources for N management has the potential to increase N use efficiency (FERNANDES 2016).
The use of Azospirillum brasilense is an alternative for reducing the application of N fertilizer, either through biological N fixation or greater N use efficiency due to root system growth (BASHAN & de-BASHAN 2010). Moreover, bacteria of the genus Azospirillum have the potential to stimulate plant development through multiple mechanisms, including biosynthesis of phytohormones, better nitrogen nutrition, stress mitigation, and biological control of pathogenic microbiota (BASHAN & de-BASHAN 2010). The main hormone produced by strains of Azospirillum is the auxin, IAA, which is responsible for inducing elongation and cell division. Inoculation with Azospirillum is commonly applied to the seed, and the inoculant can be applied in either a liquid or solid form. However, the bacteria's contact with pesticides, such as insecticides and fungicides, that are used in seed treatments may compromise the inoculation technique (FUKAMI et al. 2016, MUNARETO et al. 2018, thereby requiring alternative methods to increase the bacteria's efficiency. In recent years, leaf application of Azospirillum has been the object of several studies (PEREIRA et al. 2017, RIBEIRO et al. 2018, CORREIA et al. 2020. In a study by OFFEMANN (2015), leaves were sprayed with A. brasilense, which promoted increases in the average internode length, average spike length, leaves, spikes, root dry mass, root volume, and the leaves' N content. MARKS et al. (2015) found that spraying leaves with Azospirillum resulted in significant increases in shoot dry mass compared to the controls without inoculation. RIBEIRO et al. (2018) showed that inoculation in wheat seeds and leaves with A. brasilense promoted plant height growth but did not influence yield components and grain yield. Additionally, according to the authors, there was no conformity in the inoculation techniques, presenting varying results in the two years of research.
Therefore, this study hypothesized that the leaf application of A. brasilense favors plant nutrition and has positive effects on crop yield components by reducing the dose of mineral N. The aim was to evaluate the effect of leaf inoculation with A. brasilense in association with N rates in the wheat crop.

MATERIAL AND METHODS
The experiment was conducted under field conditions in a no-till system in Lidianópolis, Paraná, Brazil, located at an altitude of 539 meters at the geographical coordinates 24° 05' 36'' S, 51° 41' 85'' W. The soil of the area was classified as Dystrophic Red Oxisol (SANTOS et al. 2013) with a composition of 83% clay, 7% sand, and 10% silt. The chemical characteristics prior to the experiment are presented in Table 1.
According to the Köppen-Geiger classification, the region has a humid subtropical climate (CFa), whose characteristics include average temperatures below 18 °C in the colder months and above 22 °C in the warmer months. Climate data on the precipitation and temperatures during the experiment were obtained from INMET (National Institute of Meteorology) and are shown in Figure 1. The sequential water balance was calculated using a program developed by ROLIM et al. (1998). Prior to the experiment, a wheat-soybean succession had been cultivated in the area in question in the last five years.    Fertilization was applied at sowing with 250 kg ha -1 of NPK 10-15-15. The treatments were applied to the total area in the tillering stage.
The doses of inoculant were diluted in water and applied to the leaves at night through a spray bar system with CO 2 pressurization, regulated to a flow of 100 L ha -1 . The Masterfix Gramíneas ® inoculant was used at a concentration of 100 million cells of A. brasilense per ml. Urea was used for nitrogen fertilization (45% N). The cultural trails followed technical recommendations. When 50% of the wheat plants were in the flowering stage, leaf tissue samples were taken, and fifty flag leaves per experimental unit were randomly collected. After taking the leaf samples, the leaves were dried to constant mass in a forced-air oven at 65 ºC. A Wiley mill was then used to grind the materials, which were subsequently weighed and digested with nitric-perchloric acid in order to determine elements of Ca, Mg, K, P, and S (MALAVOLTA et al. 1997). Ca and Mg content were determined by atomic absorption spectrophotometry (AAS) with acetylene/air. P was assessed by the vanadate yellow colorimetric method, S by turbidimetry, and K by flame photometry. After sulfuric acid digestion, N was determined by the micro-Kjeldahl method (MALAVOLTA et al. 1997 , and the thousand grain weight was determined. The test weight, which involves a physical analysis of the grain, was measured, with the mass of 100 liters of wheat expressed in kg hl ; GYwn represents grain yield with nitrogen fertilizer; GYon is grain yield without nitrogen fertilizer; and QNa is the quantity of applied N in kg.
The data were initially tested for error normality and homogeneity of variance by the Shapiro-Wilk test and Bartlett's test, respectively. There was no need for data transformation, and the Anova assumptions were met. Analysis of variance by Snedcor's F distribution at 5% significance level was performed using the data. Regression analysis was applied to test the linear and quadratic effect of the quantitative factors. The Pearson correlation coefficient was also used to measure the association between the response variables.

RESULTS AND DISCUSSION
Weather conditions were not ideal for the wheat crop's full development (Figure 1). The water balance revealed that the absence of precipitation events during July and September culminated in a water deficit during the flowering and grain-filling stages (Figure 2). The water requirement for the wheat crop is 450 to 600 mm, depending on the climate and the duration of the cycle (DOORENBOS & KASSAM 1979), with an average consumption of 3.0 mm day -1 (LIBARDI & COSTA 1997). Although cumulative precipitation during the cultivation period reached 351 mm, the precipitation events were poorly distributed. There was excess water at the beginning of crop development ( Figure 2) and only 122 mm between the tillering stage, when the treatments were applied, and the grain ripening stage. The water deficit limited the average yield of the experimental area (1125 kg ha -1 ), which was lower than Paraná's state average (2308 kg ha -1 ) (CONAB 2018).
There was no interaction between A. brasilense and N for any of the response variables in question (p<0.05). None of the treatments had an effect on spike length, the number of spikelets per spike, the number of spikes per m², thousand grain weight, test weight, or on the leaf's N, P, K, Fe, Mn, and Zn content (Table 2). However, A. brasilense had an isolated effect on the leaf's Ca and Mg content, and N had an isolated effect on yield, the number of grains per spike, and the leaf's Ca, Mg, and Cu content.
Spraying leaves with A. brasilense increased their Ca and Mg content, while the maximum dose for each was 283 and 380 ml ha   It is assumed that these results are due to root growth stimulus. NOZAKI et al. (2014) observed that seed inoculation with Azospirillum increased the wheat plant's dry mass, fresh mass, and root size. The Millet root morphology changed when plants were inoculated. The number of secondary roots increased, and all the lateral roots were densely covered with root hair (TIEN et al. 1979). Moreoever, root system development is important for the absorption of elements that meet the root by root interception, such as Ca and Mg, which, although largely absorbed by mass flow, are also absorbed by root interception.
Root growth is due to the synthesis of phytohormones produced by bacteria, mainly indoleacetic acid, gibberellins, and cytokines (TIEN et al. 1979). In addition, the application of Azospirillum is also responsible for higher plant water and mineral absorption rates (OKON & KAPULNIK 1986, CASANOVAS et al. 2002 and an increased tolerance to abiotic stresses such as drought (CASSÁN et al. 2009, KIM et al. 2012).
According to DOBBELAERE et al. (2002), the benefits of Azospirillum on plant growth are mainly due to morphological and physiological changes in inoculated roots.
However, bacteria of the genus Azospirillum are capable of synthesizing substances such as cadaverine (CASSÁN et al. 2009). All-natural polyamines, including cadaverine, strongly inhibit the opening and closing of stomata by regulating potassium channels in guard cells, an important effect under abiotic stress conditions (LIU et al. 2000), such as the water deficit between July and September. Plant growthpromoting bacteria such as Azospirillum may play a strategic role in stress conditions due to the activation of various physiological and biochemical mechanisms of tolerance in plants, called induced systemic tolerance (YANG et al. 2009, KIM et al. 2012. Therefore, we can expect a higher probability of favorable responses to A. brasilense when plants are affected by biotic or abiotic stresses. In a study by GALINDO et al. (2015a) with irrigated wheat, the duration of the leaf application of A. brasilense did not influence the leaf's nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, boron, copper, iron, manganese, and zinc content or the yield or yield components (GALINDO et al. 2015b).
The positive effects of inoculation with Azospirillum in wheat plants are more pronounced when there is low or moderate fertilization, which points to the importance of the bacteria's role in stimulating root development rather than the fixation of atmospheric N itself (DOBBELAERE et al. 2002). In this sense, without aluminum, a high availability of nutrients (P, Ca, Mg, and K), and organic matter, which is indicative of the availability of N, the high soil fertility is remarkable (Table 1).
Increased doses of N increased the leaf's Ca, Mg, and Copper content quadratically, with maximum values at 61, 47, and 49 kg N ha -1 , respectively (Figure 4). ESPÍNDULA et al. (2010) observed N rates and an increase in the Ca and Cu content of wheat seeds but saw no effects for Mg. The results suggest that the increased Cu content in leaf tissue may be related to higher nutrient absorption as a function of nitrogen fertilization. The use of amide or ammonium fertilizers, which generate ammonium by hydrolysis, intensifies soil acidification because, in the nitrification process, each NH 4 + molecule that is oxidized to NO 3 releases two protons (H + ). Thus, soil acidification increases the availability of cationic micronutrients such as Cu (MORAGHAN & MASCAGNI Jr. 1991).
As for the isolated effects of the application of N, there was an increase in the number of grains per spike (NGS) and in yield, with a quadratic model adjustment, where the highest NGS and the maximum yield corresponded to the doses 56 and 54 kg N ha -1 , respectively ( Figure 5). In a study by BESEN et al. (2018), N application increased the number of grains per spike, spike length, and the number of spikes per m². RONSANI et al. (2018) examined N in doses of 0, 30, 60, and 120 kg ha -1 and noted a linear increase in spike length, spikelets, grains, thousand grain weight, and yield. The influence of N on wheat yield components may be related to a higher interception of solar radiation and to an increase in leaf area index, as indicated by the plant's growth in height (HEINEMANN et al. 2006), thereby resulting in a greater yield (BESEN et al. 2018). Furthermore, N performs vital functions for the plant in the structural functions of amino acids, proteins, glycols, lipoproteins, vitamins, and it is also active in the constitution of all enzymes and is responsible for activating a multitude of them (MALAVOLTA et al. 1997).
SILVA & PIRES (2017) analyzed the same N rates and also identified a quadratic response in grain yield. In addition, the authors found that the supply of N with urea was not effective in increasing the effects of inoculation with Azospirillum on wheat yield and that inoculation with A. brasilense does not substitute nitrogen fertilization. RIBEIRO et al. (2018) also reported that leaf spraying with A. brasilense had no effect on yield or yield components. However, PEREIRA et al. (2017)   Yield had a positive correlation with the number of grains per spike and the Cu and Mn content in the leaf (Table 3). In a no-till system, MOREIRA et al. (2019) also found that wheat grain yield was associated with the availability of Cu. With the exception of N and P levels, the other nutrients (K, Ca, Mg, Fe, Zn, Cu, and Mn) showed a positive association, with the highest correlation coefficient between Mg and Mn (0.95).    There was a negative correlation between the test weight and Mn (Table 3). Other studies also reported a negative association between Mn and variables related to grain quality. In a study by WILSON et al. (1982), the seed's protein content correlated negatively with the plant's Mn content. COELHO et al. (2001) also reported a negative correlation between the Mn and protein content in wheat grains.
Therefore, the benefits of leaf inoculation were restricted to the improved absorption of different cationic macronutrients (Ca 2+ and Mg 2+ ). On the other hand, N supply favored increases in grain yield, which was correlated to the number of grains per spike and to the leaf's Cu and Mn content.

CONCLUSION
Despite the water deficit in the experimental conditions, leaf application of A. brasilense did not allow the dose of N to decrease and even improved absorption of Ca and Mg. The best responses were between 283 and 380 ml ha -1 . Similarly, topdressing wheat with nitrogen fertilization altered the Ca, Mg, and Cu content in the leaves. The best responses were observed between doses 47 and 61 kg ha -1 . N supply was responsible for increases in grain yield that were related to the increased number of grains per spike and to greater Cu and Mn content in the leaves.
Nitrogen use efficiency decreased as the dose of N increased, regardless of leaf spraying with A. brasilense.