Azospirillum brasilense INNOCULATION COMBINED WITH DIFFERENT LEVELS OF NITROGEN FERTILIZATION AND ITS EFFECTS IN THE GRAIN SORGHUM SUBJECTED TO WATER RESTRICTION

One of the major problems in the present scenario is the effect of climate changes and their consequences to the agriculture, mainly due to decreasing water availability. Water restriction is a limiting factor on plants growth, and it can lead to morphophysiological modifications as well as alterations in the plant’s development. Sorghum is a grass widely utilized in agriculture due to its outstanding characteristic of drought resistance, which is higher than the other grasses, therefore being the fifth most sowed grain in the world. The inoculation of growth-promoting rhizobacteria in plants can promote growth and remodel its root system, decreasing the impacts of water restriction. In this sense, this study aims to evaluate the effects of Azospirillum brasilense inoculation in mitigating the water restriction effects in the grain sorghum BRS 332 subjected to two different levels of nitrogen fertilization. Our experiment was carried out in a greenhouse under monitored conditions of temperature and moisture. Plants were treated with two distinct irrigation conditions, two A. brasilense inoculants, one treatment without inoculation, and two levels of nitrogen fertilization (high and low nitrogen fertilization). It was evaluated the differences in their ecophysiological and crop production characteristics. The results showed that plants subjected to drought and associated to rhizobacteria invested in shoot parts, which provided this hybrid higher efficiency between water absorption and loss, and consequently higher stomata efficiency during drought when compared to the control treatment. There was also an increment in the production of the grains, particularly flagrant under lower doses of nitrogen, minimizing the effects caused by drought and decreasing the need to utilize fertilizers.

Ejeta, 2020). The imbalance triggered by adverse meteorological conditions such as alterations in the temperature patterns, humidity and precipitation are capable of promoting significant alterations in the water availability of a given region, an element that is key to agriculture (Moncada;Petersen;Munkholm, 2021). Recent studies report that water restrictions can decrease plants growth season and development, leading to decreased patterns of plant productivity (Ashraf et al., 2021). In this scenario, studies aiming to increase food production under low water availability are essential.
Sorghum yields are capable of facing those climate adversities due to its high drought tolerance (Akman;Zhang;Ejeta, 2020). Sorghum Another current research aim is to decrease the utilization of fertilizers, once one of the major problems of using excessive amounts of fertilizers is the entrance of large quantities of nitrogen in the underground water, which leads to severe contamination of the water sources in certain area (Wang et al., 2021). One option to decrease the fertilizing dependency is to utilize growth promoting bacteria (GPB), which has great results in mitigating the adverse effects caused by water restrictions as well as increasing crop yields and plants growth (Reis, 2019).
Azospirillum brasilense is one of these bacteria whose role in drought tolerance has been documented in several studies. They have been described as enhancers of plants morphological characteristics, leading to individuals with increased number of root ramifications, increased root biomass, increased density of root hairs and resulting in an overall wider exploration of the soil profile when searching for water sources (Hungria, 2011). Azospirillum brasilense innoculation combined with...

I. Growth conditions and vegetal material
The

II. Description of the treatments and experimental design
The treatments were mainly constituted by: • Three kinds of inoculation: two based on Azospirillum brasilense (strains CMS11 + CMS1626, and one commercial inoculant), and a control treatment without inoculation. The treatments were identified as A1 (CMS11 + CMS1626), A2 (commercial inoculant) and A0 (control without inoculation); • Two kinds of irrigation: the first one was completely irrigated to field capacity (FC) and the other with water restriction (WR) at 50% field capacity from the moment that the plants were pre-flowering; • Two levels of nitrogen fertilization: high nitrogen (HN) (180 kg of N ha -1 ) applied at top dressing and low nitrogen (LN) (24 kg de N ha -1 ) applied for soil correction. Urea was utilized as a source of nitrogen fertilization (45% of N in its composition).
A completely randomized design (CRD) with factorial design of 3x2x2, totalizing 12 treatments and 5 repetitions was utilized in this experiment, leading to 60 experimental units. kPa, a value that corresponds to the soils specified.
The WR imposition was kept until the grains were harvested.

IV. Morphophysiological analysis and crop production components
Three morphophysiological evaluations were performed during the imposition of the water restriction, aiming to obtain data from the treatments fully irrigated (day 0), partially stressed (day 7 after the imposition of the water deficit) and at the highest stress (14 days after the imposition of the water deficit).
Plant height was measured by using a measuring tape and the stalks diameter by using a digital pachymeter. All physiological analysis was performed by using the last fully expanded leaf during the mornings, between 8am and 10am.
The stomata conductance was measured using a porometer (Leaf Porometer -Decagon Devices), the relative amount of chlorophyll was measured by using a SPAD (Minolta SPAD 502 Osaka, Japan) and the efficiency of the photosystem II (Fv/Fm) by using a FluorPen FP 100 device. The leaf water potential (Ψ w ) was evaluated by using the Shollander pressure bomb -(1000 Pressure Chamber) in the 7 th day of stress, performed at 12pm, and this water potential was kept until the 14 th day of stress.
The plants were harvested and divided in three parts: shoot parts (SP -leaves and stalks), panicles and roots. It was determined the total leaf area for each treatment by using a digital leaf area scan (LI-3100C, Licor, Nebraska, USA). After scanning, the SP, plants were placed in paper bags and dried in a dry oven at 65ºC for 72h to obtain their dry mass. The production components determined were: panicle mass, length and diameter; grain total mass and 100 grains mass.
The root system in each pot was carefully cleaned (with minimum loss of the plant material) using running water and stored in plastic pots containers with 70% (v/v) ethanol to preserve the samples until they were analyzed.
The morphometric analysis were performed using Brazil).

I. Morphophysiological analysis
The  during the 1st, 7th and 15th day of water restriction (DWR). Averages with the same lower case letters mean that there was no difference when using the Scott-Knott test at 5% probability.  On the 15 th day of water restriction the efficiency of the photosystem II in the sorghum plants was similar to those found on the 7 th day of water restriction (Table 3). There were also higher rates of stomata conductance in plants irrigated to FC irrespectively of the inoculant and nitrogen fertilization dose (Table 4). Treatments with low nitrogen fertilization (LN) associated Nitrogen -LN: 24 kg of N ha -1 ) subjected to two kinds of irrigation: WR (water restriction) and FC (field capacity). B) Photosystem II efficiency in plants inoculated with A1, A2 and with no inoculant (A0) in the interaction of irrigated and stressed water conditions. Averages with the same lower case letters mean that there was no difference when using the Scott-Knott test at 5% probability. Azospirillum brasilense innoculation combined with...

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Table 2 -Leaf water potential (Ψmd -MPa) in sorghum plants subjected to water restriction (WR), persistent irrigation and nitrogen fertilization, inoculated with Azospirillum brasilense (A1 and A2) and with no inoculant (control treatment A0). Averages values with the same letters mean that there was no difference when using the Scott-Knott test at 5% probability.

II. Sorghum yield data and root morphology
All the irrigated treatments had higher values of leaf area, shoot parts fresh mass, and shoots parts dry mass, than the treatments subjected to water restriction, regardless of the inoculation type (Figure 7). On the other hand, the inoculation with Azospirillum brasilense seemed to decrease the adverse effects caused by the water restriction in the production of sorghum grains.
The inoculation with Azospirillum brasilense had positive effects in the sorghum plants under water restriction, with increased panicle mass when compared to the control treatment ( Figure 8).
There was also a positive relation between water restriction (WR) vs. Azospirillum brasilense vs.
LN related to the panicle fresh mass (Figure 9 A) and panicle diameter (Figure 9 B).
The treatments subjected to field capacity   Averages values with the same lower case letters mean that there was no difference when using the Scott-Knott test at 5% probability. Azospirillum brasilense innoculation combined with...

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(FC) had a higher productivity of grains when compared to the treatments subjected to water restriction (WR). The total amount of fresh mass in the grains subjected to water stress was superior in the treatments that received the inoculant Azospirillum brasilense (Figure 10). Grains treated with low nitrogen (LN) fertilization had higher volume of grains than grains treated with high nitrogen (HN) fertilization when comparing the mass of 100 grains weight and total weight of the grains (Figure 11). When analyzing the morphometry of the root system, the root length, mean root diameter and root dry mass values were higher when associated to high levels of nitrogen (HN) fertilization and were independent of the presence of inoculants (Table 5). Regarding the root dry mass, the treatments inoculated with Azospirillum brasilense had lower mass than the control treatment. There were also no differences in the root morphology in the irrigated treatments and non-irrigated treatments, which is interesting because it is possible to assume that the rhizobacteria increased the root growth in plants subjected to water restriction.

DISCUSSION
Drought is a limiting event in plants life, and it can lead to morphophysiological and developmental modifications (Marques, 2019).
In this study, it was demonstrated that the gas exchange rates, the shoot parts (i.e. leaf area, leaf fresh mass and leaf dry mass) and the productivity indexes were affected by water restriction. It was also demonstrated that the inoculation with the Azospirillum brasilense rhizobacteria Nevertheless, there was no statistical difference  Figure 10 -Effect of the inoculation with Azospirillum brasilense rhizobacteria (A1 light grey bar and A2 dark grey bar) and without inoculation (A0 black bar) in the panicle total weight of sorghum grains subjected to water restriction. Averages values with the same lower case letters mean that there was no difference when using the Scott-Knott test at 5% probability.

Panicle total weight (g)
Weight of the grains (g)  (2018) and Kalaji et al. (2016), as the fluorescence of the chlorophyll a is a great indicative of the integrity of photosystem II. The results obtained using flourpen were under 0.75, and this result indicates that the quinone (primary acceptor of electrons in the photosystem II -PSII) is completely oxidized. Then, the center of reaction in FSII is open, which means that it is possible that the PSII reaction center was damaged or decreased its capacity to transfer energy for the other components (Konrad, 2005). Thus, a lower fluorescence indicates a lower level of stress and a higher photochemical efficiency.

CONCLUSIONS
Inoculating sorghum with Azospirillum brasilense strains increased the shoot parts growth as well as the production of sorghum grains (BRS 332 hybrid), particularly under low nitrogen doses. The inoculation also increased the root system growth, minimizing the adverse effects caused by the water restrictions.