Optimizing Nitrogen Application for Enhanced Barley Resilience: A Comprehensive Study on Drought Stress and Nitrogen Supply for Sustainable Agriculture

By inergency On Feb 29, 2024

[ad_1]

1. Introduction

The emerging impacts of climate change present a growing threat to agricultural crops, especially in tropical and sub-tropical regions. Drought stress stands out as a critical factor negatively influencing crop growth and productivity [1]. Simulation models play a crucial role in predicting the changing scenario of drought conditions due to climate change. The effort to enhance the resilience of strategic crops against the harmful effects of drought stress, inevitably resulting in reduced crop productivity, is essential for more sustainable agricultural practices [2].

The success of the Green Revolution can be primarily attributed to the development of superior plant varieties and the extensive application of fertilizers, particularly synthetic nitrogen fertilizers, to realize the increased yield potential of these new varieties. Approximately 50% of the nitrogen fertilizer applied to fields is assimilated and utilized by the designated crop, with the remainder being lost to the environment [3]. A small percentage is converted to the potent greenhouse gas N₂O, which contributes substantially to the total greenhouse gas emissions from agriculture. By the way, Green Economy thinking aims to promote sustainability through N₂O emission reduction in the context of the wider nitrogen cycle, with an emphasis on improving full-chain nitrogen use efficiency, optimal dose use for each crop plant and region, and exploiting a combination of technical measures in agriculture and other combustion sources [4,5]

Barley, a widely cultivated and economically valuable crop, stands at the forefront of the agricultural landscape [6]. The correlation between agronomic yield and nitrogen supply under well-watered conditions is well established [7]. Nitrogen, when efficiently managed, possesses the potential to alleviate water stress in crops by sustaining metabolic activities even under low tissue water potential. Establishing optimal fertilizer regimes is therefore essential to enhance metabolic and regulatory processes during kernel development in cereal crops [8]. Nitrogen application has been shown to influence starch synthesis and grain quality, particularly under drought stress conditions [9].

In the quest for sustainable solutions, the use of nitrogen supply not only improves water use efficiency but also supports the antioxidant system. This includes crucial enzymes like superoxide dismutase and catalase, which play a role in mitigating the stress associated with deficit irrigation [10]. Nitrogen serves as a regulator influencing the impact of short-term heat, drought, and combined stresses on diverse aspects of wheat physiology, encompassing photosynthesis, yield, nitrogen metabolism, and nitrogen use efficiency [11,12].

Despite extensive global research on plant responses to drought stress, studies on the role of nitrogen in mitigating drought stress in barley compared to other cereals crops are comparatively rare [13]. Barley, a resilient crop cultivated in both highly productive agricultural systems and marginal subsistence environments, holds the position of the fourth most important cereal crop globally, following wheat, maize, and rice [14,15]. Although its direct contribution to human food may be minor, the potential for new applications that leverage the health benefits of whole grains and beta-glucans is significant [16].

Drought stress significantly impacts barley physiology, affecting soluble and insoluble sugar levels, as well as the uptake of grain nitrogen (N), phosphorus (P), and potassium (K) [17]. The pursuit of climate-resilient and high-yielding barley varieties is ongoing, with researchers focusing on understanding the genetic controls influencing morphological and physiological responses to drought at different stages of plant growth [18].

In tandem with breeding strategies, exploring locally sourced barley genotypes becomes crucial to harness the potential of plant biodiversity in mitigating drought stress with the aid of nitrogen supply. Parameters such as relative water content, photosynthetic rate, stomatal conductance, mesophyll concentration of CO₂, total leaf nitrogen, grain yield, total organic nitrogen content, starch content, and macronutrient concentrations (N, P, K, Ca, Mg) are significant for optimizing agricultural practices. The current study aimed to investigate, through a long-term vegetation pot experiment, the above-mentioned parameters in spring barley measured under the influence of drought stress and different dosages of nitrogen supply. A focus on the potential of nitrogen supply in mitigating drought stress is important in developing more sustainable agricultural practices.

2. Materials and Methods

2.1. Plant Experiment

This three-year field experiment involved the cultivation of spring barley (variety Kompakt) plants in cylindrical plastic containers measuring 290 mm in diameter and 260 mm in height. Each container, as detailed in the preceding section, received 15 kg of soil, which was meticulously homogenized through thorough mixing. Soil was introduced into the containers ten days before sowing to facilitate proper settling. Before sowing, any germinating weeds on the soil surface were eliminated, and the top 30 mm of the soil was aerated by loosening.

The experiment aimed to investigate the impact of three different nutrition strategies under two levels of water regimes during three distinct growth phases of spring barley (shooting, stalk elongation, and budding flowering). An optimal water regime was set up and maintained at the level of 50 to 60% of field water capacity. Under drought stress, soil humidity was reduced to 15–20% of field water capacity.

The following fertilization methods were employed:

1st variant—without N fertilization (control);
2nd variant—N fertilization to the level of 1 g per pot + 0.33 g P + 1.1 g K (near 200 kg N hectare⁻¹);
3rd variant—N fertilization to the level of 2 g per container + 0.33 g P + 1.1 g K (near 400 kg N hectare⁻¹).

In the context of the presented pot experiment, we consciously opted to maintain the original values of 1 g and 2 g of nitrogen per container, thereby preserving the experiment’s initial framework. Simultaneously, we introduced the conversion to kg N hectare⁻¹, acknowledging its dependency on various factors in field experiments. This assumption was made with the intention of fertilizing one hectare of soil to a depth of 0.2 m. Considering an approximate weight of 3,000,000 kg per hectare of land and assuming uniform soil density (1.5 g/cm³) in the Slovak Republic, this value was employed for calculations.

The nitrogen fertilizer utilized in this study was DAM 390 (containing 30% nitrogen). For phosphorus fertilization, we employed triple superphosphate (containing 20% phosphorus), and for potassium, we used potassium salt with a potassium content of 60% (equivalent to 49.8% K). These fertilizers were incorporated into the soil in predetermined amounts during the filling of the experimental containers. Each combination of experimental factors, defined by the interaction between moisture regime and fertilization, was replicated four times. The biological replication for each variant was n = 10 (10 containers for each variant, 29 plants in one container), and the analytical replication for the studied experimental parameters was n = 3.

The investigation focused on different fertilization variants applied under two distinct soil moisture regimes:

The growth and development of the spring barley plants was monitored during the growing season, and the onset of the main growth phases were recorded by date. The growth phases were rated using the DC scale:

1st branching (DC 21–DC 29);
2nd stabling end (DC 30–DC 49);
3rd blooming end (DC 50–DC 69).

DC is decimal code for the development stages of cereals (wheat, barley), corresponding to Zadoks et al. (1974) [19].

2.2. Relative Water Content

The relative water content (RWC), expressed as a percentage, reflects the relationship between the water content within a plant organ (e.g., leaf) and its water content under full turgor pressure conditions, as described by Turner (1981) and Olsovska et al. (2016) [20,21]. RWC values were calculated both before initiating the drought treatment (control, representing turgid plants) and after specific periods or stages of water deprivation (drought).

To determine RWC, leaf discs were extracted from the central region of an experimental leaf. Fresh weight (FW) was measured immediately after conducting gas exchange analyses. Turgid weight (TW) was obtained after hydrating the leaf disc in distilled water at 4 °C in the dark for 12 h. Dry weight (DW) was determined after drying the leaf disc at 80 °C for a 24 h period.

FW—fresh weight of the leaf segment;
TW—weight of the leaf segment in full turgor;
DW—dry weight of the analysed leaf segment.

2.3. Net Assimilation Rate of CO₂

The net assimilation rate of CO₂ (Pn—μmol·m⁻²·s⁻¹) was determined by measuring the CO₂ consumption in intact photosynthesizing plant leaves enclosed within an assimilation chamber of the CIRAS-3 DC (PP Systems International, Inc., Amesbury, MA, USA) non-dispersive, open-type infra-red gas analyser, which contained, in the Ciras-3 console, four independent gas analysers simultaneously measuring absolute CO₂ and H₂O for both reference and analysis gas streams. All measurements were corrected for temperature and pressure. The external CO₂ concentration was maintained at 370 μL·L⁻¹, and the illumination level was set at 800 μmol·m⁻²·s⁻¹.

2.4. Stomatal Conductance and Internal CO₂ Concentration

Stomatal conductance, denoted as g_s in mmol·m⁻²·s⁻¹, and internal CO₂ concentration (Ci) in (μmol.m⁻².mol⁻¹) were derived from the same measurements of the CO₂ and H₂O flows in the CIRAS-3 photosynthetic system as Pn, as outlined by Olšovská and Brestic in 2001 [22].

2.5. Monitoring of Crop-Forming Elements

The following crop-forming elements were monitored in the container experiment:

(a): Grain yield per container (g);
(b): Thousand kernel (grain) weight—HTZ (g) (used DIPOS grain counter).

2.6. Assessment of Spring Barley Grain Quality

Following post-harvest maturation, the barley grain underwent evaluation based on the following parameters:

(a): Starch content (%): Determined using the Ewers polarimetric method.
(b): Measurement of total nitrogen: This was accomplished through Kjeldahl analysis, with nitrogen content being multiplied by 6.25 to calculate total nitrogen levels as per the method outlined by Kjeldahl in 1883 [23].

2.7. Agrochemical Analyses of Plants

Plant yield and the concentrations of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) were evaluated annually throughout the duration of the experimental study. The spring barley yield was determined by manually harvesting plants from two adjacent central rows at various growth stages: shoot emergence or when the second stem node became visible (DC 29), the second phase of stem elongation (DC 49), and the third phase of flowering (DC 69).

For the analysis of nutrient concentrations, six randomly selected spring barley plants (including both control and drought treatment groups) were divided into stem, leaf, husk, and grain components. These plant samples were dried at 65 °C to a constant weight and subsequently ground for further analyses. Nitrogen concentration in the plant material was determined using the Kjeldahl method [23]. Phosphorus and potassium concentrations were assessed in ground plant material after mineralization at 550 °C for 6 h. The resulting ash was mixed with 2 cm³ of diluted HNO₃ (a 1:1 mixture of concentrated nitric acid and distilled water). Phosphorus was quantified calorimetrically using vanadium–ammonium molybdate, while potassium and calcium concentrations were determined via flame photometry (SpectrAA-250Plus, Varian, Markham, ON, Canada). Magnesium was quantified using atomic absorption spectrophotometry.

All macronutrient concentrations (N, P, K, Ca, Mg) were expressed as percentages based on their dry weight representation, and macronutrient uptakes by above-ground biomass were recalculated per kilogram per hectare (kg·ha⁻¹). This paper will present the results of the statistical analysis of the investigated agrochemical parameters.

2.8. Statistical Analyses

Statistical analyses were carried out using the Statistica v. 10 software (StatSoft Inc., Tulsa, OK, USA) and the graphics software SigmaPlot version 11.0 (Systat Software Inc., San Jose, CA, USA).

3. Results

Over the span of three experimental years, in conditions of optimal soil moisture, a positive impact of nitrogen fertilization on Nitrate Reductase Activity (NRA) was observed in all growth phases, regardless of the nitrogen dosage. NRA displayed a slight upward trend as the growth phases progressed, signifying an increase as the stand aged. The highest NRA value, reaching 45.8 nmol N-NO²⁻ g⁻¹ fresh mass.min⁻¹, was observed during the scallion growth phase, when 2 g N per container was applied (Table 1).

Under drought conditions, significantly lower NRA values were consistently recorded at all growth stages and under various nutrition levels in comparison to plants grown under optimal soil moisture conditions. Among the stressed plants, NRA values decreased during the seedling phase in fertilized variants but increased during the tillering stage, particularly when a higher fertilization level (2 g N per container) was applied, compared to NRA values recorded during the tillering phase.

When averaged over three years, as well as within individual years, the relative water content (RWC) of the leaf tissues in optimally irrigated plants was found to be statistically significantly higher than that of leaves from plants subjected to soil drought.

In the nitrogen-fertilized treatments, a reduction in relative water content (RWC) was observed in all growth phases except for the tillering stage under optimal moisture conditions when compared to the unfertilized control. Over the average of the experimental years, net assimilation (Pn) in the main stem leaves of barley showed statistically significantly higher values in all monitored growth phases when grown under optimal irrigation conditions compared to the Pn values measured in the barley plants subjected to drought stress conditions. In all experimental variants with different nitrogen supplies (0 g, 1 g, 2 g of N), a significant decrease in Pn values was observed under the effects of drought stress, especially during the shooting phase of barley growth. Drought stress led to decreases in Pn values of 84% in the 0 g N supply variant, 72% in the 1 g nitrogen supply variant, and 61% in the 2 g nitrogen supply variant compared to the control (without N supply).

Under optimal soil moisture conditions, barley leaves displayed the highest intercellular CO₂ concentration (Ci) values on the non-fertilized treatments (ranging from 120 to 216 μmol CO₂·m⁻²·s⁻¹) in the individual growth phases, in contrast to the nitrogen-fertilized treatments, where a significant decrease in Ci values was evident (statistically supported). Under stress conditions, the barley plants responded differently to nitrogen fertilization concerning this parameter. The lowest Ci values were recorded in the unfertilized control treatment in all three growth phases. A particularly significant decrease of 25% in Ci value was observed during the shooting growth phase in the variant with 0 g N under drought stress. Nitrogen fertilization significantly increased the intercellular concentration of CO₂ in barley leaves, reaching peak values (438.49 or 382.00 μmol CO₂·m⁻²·s⁻¹) during the shooting growth phase, with a greater impact observed at the lower nitrogen dose (1 g N). At the lower N dose (1 g per container), the Ci values increased by 35% during the tillering growth phase, by 66% during the shooting growth phase, and by 50% during the blooming phase. Interestingly, higher increases in Ci values were observed in the variant with a higher nitrogen dose (2 g per container)—by 48% during the tillering growth phase, 67% during the shooting growth phase, and 52% during the blooming phase, respectively.

Overall, the stomatal conductance (g_s) values were higher in the unfertilized, optimally irrigated plants (ranging from 73.35 to 137.37 mmol·m⁻²·s⁻¹) compared to the values obtained for the plants grown under stress conditions (ranging from 34.09 to 85.43 mmol·m⁻²·s⁻¹), indicating that drought reduced stomatal conductance (through stomata closure). However, there was a substantial 84% reduction in stomatal conductance observed in the variant with a 2 g N supply during the early growth phase (tillering). In the variant with a 1 g N supply under drought stress, the barley plants exhibited a 65% decrease in stomatal conductance compared to the control variant without drought stress. As the growth stages progressed, both variants with nitrogen supply demonstrated noteworthy reductions in stomatal conductance values. Notably, the early stage of barley growth proved to be more sensitive to drought stress, displaying a significantly higher difference in the response between the variant with a 1 g N supply and the variant with a 2 g N supply—a 19% difference.

Nitrate reductase activity (NRA) was significantly decreased under drought stress in both experimental variants with N supply. At the tillering growth stage, NRA values were reduced by 58% in the variant with a nitrogen supply dose of 1 g and by 49% in the variant with a N supply dose of 1 g. At further growth stages, the variant with a nitrogen supply dose of 1 g under drought stress showed a significant decrease in NRA by 83% and 78% at the shooting and blooming growth phases, respectively. In the subsequent growth stages, the barley plants in the variant receiving 2 g of N supply under drought stress exhibited a noteworthy reduction in NRA, with decreases of 67% during the shooting growth phase and 57% during the blooming growth phase.

Across an average of three experimental years, all monitored factors, including year, growth phase, and fertilization, had a statistically significant effect on all of the observed physiological characteristics of barley, namely NRA, RWC, net CO₂ assimilation rate (Pn), intercellular CO₂ concentration (Ci), and stomatal conductance (g_s), under both optimal and stress moisture levels.

All experimental variables demonstrated a statistically significant influence on the nutrient concentration (N, P, K, Ca, Mg) within the dry matter of barley’s above-ground biomass and the nutrient uptake by the above-ground plant biomass (Table 2). However, there were exceptions to this pattern. Notably, the impact of the year on the concentration and uptake of potassium (K) and magnesium (Mg) during barley cultivation under optimal moisture conditions did not reach statistical significance. In contrast, under drought stress conditions in barley plants, both the year’s effect on Mg uptake and the impact of fertilization on the dry matter concentration of Mg were found to be statistically significant.

Over a three-year period, the nitrogen fertilization of spring barley resulted in a twofold increase in nitrogen (N) concentration within the dry matter of the above-ground biomass, both under optimal soil moisture conditions and in the plants subjected to stress compared to the unfertilized control. As the growth phases progressed, a dilution effect led to a reduction in N concentration in the dry matter for both the fertilized and unfertilized variants (Table 3).

In the final observed growth phase (heading), the N concentration in dry matter was higher for the fertilized variants of the stressed plants (2.19% and 2.71%, respectively) compared to the same fertilized plants grown under optimal conditions (1.85% and 2.41%, respectively). Nitrogen fertilization, whether under optimal or stress conditions, increased N uptake by the above-ground biomass; however, the rate of this increase was notably lower under stress conditions when compared to the plants grown under optimal moisture conditions. The application of 2 g N per container proved to be more effective in all cases than the half-dose of 1 g N per container.

Throughout all three years, the total harvested yield (HTZ) in the unfertilized treatments under stress conditions remained relatively consistent, occasionally even surpassing that of the unfertilized treatments under stress-free conditions (Table 3). Nitrogen fertilization further exacerbated this reduction. Conversely, when stress was applied during the different growth phases, nitrogen fertilization had the opposite effect, decreasing HTZ. At the tillering growth phase in the variants with N supply under drought stress, the HTZ values were mostly at the control level (without N supply). The HTZ values decreased under drought stress in barley plants of both nitrogen treatment variants (1 g and 2 g N) by 18% and 15%, respectively. This decreasing tendency regarding the HTZ level also continued at the shooting and blooming growth phase.

Nitrogen enhanced crude protein levels, yet both drought stress and nitrogen application reduced starch content. A significant increase in crude protein levels was observed in both the shooting and blooming growth phases. In the variant with a dose of 1 g of N supply, it increased by 43% in the shooting growth phase and by 34% in the blooming growth phase. Similar results with an increasing crude protein level were shown for the variant with a dose of N 2 g—by 43% in the shooting growth phase and by 33% in the blooming growth phase.

5. Conclusions

In summary, our study reveals the complexity of nitrogen’s role in sustainable barley cultivation. The additional nitrogen supply during drought stress heightened CO₂ concentration throughout the barley growth period, affecting stomatal conductance. Positive effects on magnesium uptake enhanced barley productivity, but the temporal dynamics varied across growth phases.

The thousand kernel weight (HTZ) responded inconsistently over three years, influenced by nitrogen and stress timing. While nitrogen boosted HTZ during tillering under stress, the complexities in the outcomes highlight the challenge of balancing nitrogen, water stress, and barley yield. Further research is essential for refined, sustainable crop management strategies.

Our findings underscore the intricate interplay between nitrogen fertilization, drought stress, and crop yield. The benefits observed during the early exposure of the plants to stress contrasted with the adverse effects in the later growth phases, emphasizing the need for a nuanced approach in sustainable crop management. These insights contribute to evolving strategies for effective and sustainable barley cultivation in changing environmental conditions.

[ad_2]