Responses of Soil Enzymes Activities to Sprinkler Irrigation and Differentiated Nitrogen Fertilization in Barley Cultivation

Abstract

Our study aimed to assess the impact of sprinkler irrigation on the activity of selected soil enzymes in terms of nitrogen metabolism and oxidation–reduction processes in soil with different doses of inorganic nitrogen fertilizers. An Alfisol was sampled from an experimental field of spring barley within the University Research Center in the central part of Poland, namely the village of Mochełek with a moderate transitory climate, during the growing seasons of 2015–2017. The soil resistance (RS) was derived to recognize the resistance enzymes during drought. In the maturity phase, nitrate reductase activity was 18% higher in irrigated soil and the activities of other enzymes were higher than in the non-irrigated plots by 25% for dehydrogenase, 22% for peroxidase, 33% for catalase, and 17% for urease. The development phase in the barley influenced nitrate reductase activity. Enzymatic activities changed throughout the research years. During the maturity stage, a lower ammonium nitrogen content in the soil resulted from a higher spring barley uptake due to drought stress. Irrigation probably contributed to increased leaching of nitrate in the soil. The highest index of resilience was found in the soil catalase activity.

1. Introduction

The forms of mineral and organic nitrogen (N) undergo several transformations throughout the N cycle. This element is easily transformed from a reduced form to an oxidized form, which results in a free migration of nitrogen in hydrological and atmospheric processes. The amount of nitrogen available to plants is positively correlated with the mineralization of organic matter in the soil, biological nitrogen fixation, fertilization, and the total and distribution of atmospheric precipitation [1]. However, due to such processes as immobilization, harvesting and removal, denitrification, volatilization, leaching, runoff, and erosion, nitrogen loss from the soil occurs. The intensity of these processes is influenced by environmental factors such as the soil’s pH, the soil’s texture, its density, aeration, the water content, and thermal conditions, but also the management of crop residue, the method and timing of fertilization, agricultural treatments such as irrigation, and changes in land use. It is assumed that in most cases, less than five percent of nitrogen in the soil is directly available to plants from the total nitrogen content. This nitrogen is mainly in the form of nitrates NO₃⁻-N and ammonium NH₄⁺-N, with organic N being the residue, which gradually becomes available due to mineralization [2,3]. What is characteristic of arable soils is exceptionally high dynamics of mineral nitrogen forms in the growing season, which results from the microbiological nature of nitrogen transformations in the soil. Nitrogen occurs in many forms, covering the range of valence states from –3 (in NH₄⁺) to +5 (in NO₃⁻-) in both agricultural and natural ecosystems. The change of one valence state into another is mainly biologically mediated and depends primarily on environmental conditions [4]. Soil oxidoreductase enzymes take part in oxidoreductive processes. Dehydrogenases (E.C.1.1.) are extracellular enzymes that can be considered a helpful indicator of microbial activity and oxidative metabolism in soil [5]. Another intracellular enzyme from the oxidoreductase class is catalase (EC 1.11.1.6), which manages oxidative stress in the soil by catalyzing the decomposition of hydrogen peroxide into water and oxygen [6]. Peroxidases (EC 1.11.1) use H₂O₂ as an electron acceptor, and their activity in soil results in the depolymerization of lignin [7]. Urease activity (EC 3.5.1.5) can result in an increase in soil pH and loss of nitrogen to the atmosphere due to the release of NH₃ as a result of the hydrolysis of urea to CO₂ and NH₃ [8]. The activity of this enzyme can be viewed as a desirable indicator of soil quality due to its role in regulating plant nitrogen supply. In turn, the enzyme responsible for catalyzing the reduction of NO₃⁻ to NO₂⁻ in anaerobic conditions in soil is nitroreductase (EC 1.7.99.4) [9]. It has been proven that changes in soil use and management affect soil enzymes that actively participate in metabolic processes [10,11]. Enzymes indicate the metabolic level of the microbial community in soil and catalyze specific reactions in the carbon and nutrient metabolism cycle [12,13]. Free enzymes excreted by plants and animals and associated mainly with or within cellular structures are called exoenzymes. Later, they are released into soil after cell lysis and death [14]. Therefore, if soil use and management influence the soil’s microbial environment, changes in the activity of soil enzymes can also be observed [15]. The biochemical properties of soil, which are indicators of its quality, are highly variable depending on climatic, weather, and geographical conditions; pedogenic factors; fertilization; and irrigation. Microorganisms living in soil are important factors that determine the nutrient metabolism cycle. They also interact intricately with plant organisms. Land-use systems that improve soil microbiological properties can result in higher yields with better raw material quality while reducing production costs. Similarly, by limiting the use of mineral fertilizers and plant protection agents, these systems support the sustainable development of agricultural areas. Therefore, to improve the condition of soil, it is necessary to constantly monitor and evaluate the physicochemical and biological processes in the soil and to examine changes in its physicochemical properties. Diverse soil use in agricultural systems in terms of crop rotation and plant protection treatments results in changes in soil properties, both physical and chemical, but mostly affects biological activity. This, in turn, affects both productivity and environmental quality, and hence affects human and animal health. Multi-year studies on the impact of agriculture on the biological and biochemical properties of soil bring valuable information on the transformation of nutrients in soils [16,17]. The definition of soil quality indicates the ability of soil to operate within an ecosystem, as well as its ability to support biological productivity, to maintain the quality of the environment, and to encourage the sanitary conditions of plants and animals [16].

The stability (resistance and action) of a soil system is a consequence of the influence of microorganisms on the properties and processes in the ecosystem. To define different systems, it is important to select appropriate indicators that will quantify the relative value of how the system will respond to specific soil-use scenarios. In our paper, we compare our indices with previously published stability indices and test their performance against a real dataset. One of the indicators that quantifies the relative value of the microbiological response is the resistance index, according to Orwin and Wardle [18].

In this study, we aimed to evaluate the following: (1) the responses of N-related properties of an Alfisol, such as the forms of N in the soil and the activity of enzymes involved in the metabolism of nitrogen in the soil; (2) the reaction of enzymatic activity related to the transformation of soil nitrogen depending on soil moisture under sprinkler irrigation during a growing season in spring barley in a warm temperate climate zone; (3) the impact of irrigation on the activities of enzymes related to nitrogen metabolism and oxidation–reduction processes in soil during varied growth stages with various doses of inorganic nitrogen fertilizers; and (4) whether the calculated resistance ratio (RS) can be used to find an effective solution to enzymatic stress.

2. Materials and Methods

2.1. Study Area and Soil Sampling

A carefully controlled field experiment was conducted at the Research Center of the Bydgoszcz University of Science and Technology in the village of Mochełek (53°130 N, 17°510 E). The experiment site was located in the Kujawsko–Pomorskie province, in central Poland. The plant that was investigated in this experiment was spring barley cv. ‘Signora’, cultivated in three consecutive growing seasons, 2015–2017.

The soil, according to the USDA soil taxonomy, was defined as a typical Alfisol made of sandy loam (clay 6%, sand 79%, loam 15%) [19]. It was found that the reaction of the topsoil was slightly acidic: the pH in 1 M KCl was 5.7–6.1. The topsoil showed relatively low contents of total organic carbon (TOC) (7.60–7.70 g·kg⁻¹) and total nitrogen (TN) (0.70–0.76 g·kg⁻¹). The contents of other available nutrients were as follows: the phosphorus (P) (64.0 mg kg⁻¹) and sulfur (S) (13 mg S kg⁻¹) contents were average, and the potassium (K) content was high (126.0 mg⁻¹). The subsoil comprised light loamy sand on shallow medium loam. The soil properties were determined before the experiment and they are presented below (in

Table 1. Properties of soil in the experimental plot.

Soil Property	Content
TOC	7.60–7.70 g·kg−1
TN	0.70–0.76 g·kg−1
pH KCl	5.8–6.2
Available P	64.0 mg·kg−1
Available K	125.0 mg·kg−1
SO42−	12 mg·kg−1

). The water content in the soil, corresponding to the water content in 1 m of the soil layer for field capacity, was 215 mm.

2.2. Experimental Design and Weather Conditions

This study had a two-factor split-plot design with four replications. The first factor (i) was sprinkler irrigation (where W₀ meant no irrigation, and W₁ meant optimal irrigation with 100% coverage of the water requirements of plants in the period of high water needs). The second factor (ii) was a differentiated level of nitrogen fertilizer application in the crystalline form of ammonium nitrate (three doses: N₁, N₂, and N₃; see

Table 2. Description of experimental factors.

Irrigation Factor	Fertigation Factor	Nitrogen Fertigation Level
W0—no irrigation W1—optimal irrigation	N0	Control
	N1	30 kg⋅ha−1 pre sowing
	N2	60 kg⋅ha−1 pre sowing
	N3	90 kg⋅ha−1 (60 kg⋅ha−1 pre sowing and 30 kg⋅ha−1 top-dressed during the shooting stage)

). This fertilizer was applied before sowing at different doses for all groups except N₀ (control) (Table 2). For N₄, an additional dose of fertilizer was top-dressed during the barley’s shooting stage. The second factor was static and remained constant throughout the whole experiment. However, the first factor, irrigation, was dynamic and was scheduled according to weather conditions. The spring barley was provided with optimal irrigation. Throughout the period of high water requirements in plants in the rhizosphere, there was a constant reserve of readily available water (RAW). The number of single irrigation doses and the total number of seasonal doses (

Table 3. Weather conditions and irrigation doses applied in the growing seasons of 2015–2017.

Growing Season	t (°C)	P (mm)	Irrigation Application Dates	Irrigation Doses (mm)
2015	13.8	193.3	26 May	30
			3 June	30
			10 June	25
			1 July	30
			6 July	20
			total applications:	135
2016	14.3	386.7	24 May	35
			8 June	32
			total applications:	77
2017	13.1	474.8	29 May	20
			9 June	20
			28 June	15
			total applications:	55
Average for 1991–2020	14.8	324.5	–	–

) were established based on the amount and distribution of atmospheric precipitation, according to Żarski et al. [20].

The climate conditions of this study area represent a temperate transitory zone in Central Europe. The mean annual temperature and rainfall conditions for the growing season from April to September are 14.8 °C and 324.5 mm, respectively. In the growing season of 2015, classified as dry, as much as 135 mm of water was applied in 4 single doses. For the other two seasons, classified as moist, a total of 77 mm was applied in two doses in 2016 and only 55 mm was applied in three doses in 2017. For the whole experimental period of 2015–2017, the temperature conditions in the area were similar to the climate norm for 1991–2020 (Table 3) (Figure 1). However, the atmospheric precipitation totals from April to September were considerably higher in 2016 and 2017 when compared to the many-year average (Table 3) (Figure 1). The dates of barley sowing were as follows: 23 March 2015, 1 April 2016, and 31 March 2017. Barley was grown according to the recommendations of the State Plant Health and Seed Inspection Service regarding the optimization of phosphorus and potassium fertilization and chemical plant protection. The doses of macroelements for the barley were 60 kg P₂O₅⋅ha⁻¹ for phosphorus and 75 K₂O⋅ha⁻¹ for potassium, and these were applied before sowing. In all of the study years, due to the use of properly selected herbicide mixtures, weed infestation was minimal. The herbicides used from the 3-leaf stage until the end of tillering (BBCH 1–29) were Pike 20 WG at a dose of 30 g/dm³·ha⁻¹ (metsulfuron-methyl) and Aurora 40 WG at a dose of 50 g/dm³·ha⁻¹ (carfentrazone-ethyl). To combat fungal diseases in the T1 and T2 periods, a factory mixture of fenpropimorph and epoxiconazole in the Duett Star 334 SE preparation was used in the amount of 1 l·ha⁻¹, and on the ears, we used epoxiconazole with kresoxim-methyl of the Tocata Duo preparation in the amount of 0.8 l·ha⁻¹. In all of the study years, grain moths (Oulema melanopus L.) were observed on the barley, which was controlled with Bi 58 Nowy (dimethoate) at a dose of 0.5 l·ha⁻¹. The harvesting area covered 10 m². Grain was harvested on 3 August 2015, on 23 July 2016, and on 8 July 2017.

2.3. Irrigation System and Schedule

For the irrigation, a portable sprinkler irrigation system equipped with low-pressure Nelson-type sector sprinkler heads was used. The unit efficiency was 200 dm³·h⁻¹. The irrigation system was connected to the municipal waterworks network.

We scheduled the dates of irrigation treatments based on weather monitoring from an automatic weather station set in the vicinity of the experimental plot. Daily precipitation and the content of readily available water (RAW) in the soil were established. The soil water storage from the topsoil to a one-meter depth of the soil profile was 215 mm according to the field capacity. To conduct constant rhizosphere moisture monitoring, we applied the method of readily available water balance, commonly used for irrigation scheduling [20]. Moreover, direct measurements of the soil water content at the depth of 20 cm were conducted with the TDR method using the portable Fieldscout TDR 300 Soil Moisture Meter (Spectrum Technologies, Inc. 3600 Thayer Court, Aurora, IL 60504, USA) for each plot every day. Barley water requirements were met by maintaining soil moisture in the range of RAW in the plant rhizosphere. For the barley grown on irrigated plots, soil moisture in the rhizosphere was maintained in the RAW range from 0 to 30 mm according to field capacity.

The method for establishing irrigation deadlines was based on simple weather measurements. It involved balancing the income and expenditure of water from the soil layer with controlled moisture (rhizosphere) during the period of high water requirements in plants that covers May–July. The income side of the balance sheet included the following elements: effective water retention of soil for crops (for barley grown on light soil in compact substrate, this is 30 mm); precipitation; and net water doses from irrigation. On the expenditure side, there was the amount of daily water consumption, which depended on the average daily air temperature; the type of crop; and the type of soil. This balance method is beneficial for controlling irrigation at the point scale. It was tested many times in carefully controlled field experiments, in which it almost perfectly signaled the need to start irrigation, and it was consistent with the methods directly determining soil moisture. A balance was struck during the barley’s phase of high water requirements by assuming a value of initial effective water retention of the soil equal to the value of total atmospheric precipitation in the 10 days preceding the beginning of the critical crop period, which was established as 20 May.

2.4. Chemical and Biochemical Analyses

Soil was sampled from 0 to 20 cm of the topsoil three times at the following development phases: I—during spring germination (BBCH 9–19); II—after fertilization/ripening (BBCH 71–78); and III—before harvest/maturity (BBCH 86–87). At each development phase, soil was sampled in four replications for all of the treatments. Material from the field-sampled soil was sieved (2 mm mesh) and kept in a plastic box at 4 °C. After two days, the microbial activity stabilized in the soil and the enzymatic activity was studied.

N-NO₃⁻ and N-NH₄⁺ contents were extracted from moist field soil samples using KCl and K₂SO₄, respectively. The nitrate nitrogen content was determined using the phenol disulfonic acid method and the ammonium nitrogen content was determined with the indophenol method [21].

Urease activity (UR activity; EC 3.5.1.5) in the soil was assayed according to Kandeler and Gerber [22]: An amount of 1 g of soil was incubated with 4 mL of borate buffer (pH 10.0) and a 0.5 mL solution of urea at 37 °C for 2 h. Later, it was filtered after adding 6 mL of 1 M KCl and the solution and then diluted with water. With the spectrophotometric method, the urease activity was evaluated 30 min after adding NaOH salicylate and acid dichloroisocyanide at 690 nm. The UR activity is presented in mg N-NH₄⁺ kg⁻¹·h⁻¹.

Nitrate reductase activity (NR activity; EC. 1.7.99.4) was assayed as described by Kandeler [23]: Soil samples were incubated with KNO₃ (substrate) and a solution of 2,4-DNP at 25° C for 24 h. These samples were provided with KCl solution and filtered, and 5 mL of solution was provided with 3 mL of ammonium chloride buffer and reagent for staining; then, the samples were mixed and measured at 520 nm. The unit of NR activity was mg N-NO₂⁻ kg⁻¹·24 h⁻¹.

The activity of dehydrogenase (DH; EC 1.1.) is presented in mg TPFg·⁻¹ h·⁻¹, and was determined according to Thalmann [24]. Soil samples were mixed with buffered tetrazolium salts (TTC) and glucose and incubated at 30◦C for 24 h, and the activity of the DH oxidoreductase was assayed with the spectrophotometric method at 546 nm.

The catalase activity (CAT activity; EC 1.11.1.6) was determined using the method by Johnson and Temple [25]. The soil was incubated for 20 min with hydrogen peroxide and then, in an acidic environment, titrated with potassium permanganate. The catalase activity was calculated against the control samples in µmol H₂O₂·g·⁻¹·min⁻¹.

Peroxidase activity (PER activity; EC 1.11.1.7) was quantified in accordance with Ladd [26]. The substrates consisted of pyrogallol and hydrogen peroxide, and the peroxidase is presented in mmol of purpurogallin g⁻¹·h ⁻¹.

2.5. Data Analyses

The resistance of the soil (RS) was derived from the formulas suggested by Orwin and Wardle [18]:

$${\mathrm{R}\mathrm{S}(\mathrm{t}}_{0)}=1-{\displaystyle \frac{{2\mathrm{D}}_0}{{(\mathrm{C}}_0+\left|{\mathrm{D}}_0\right|)}}$$

(1)

where |D₀| is the difference between the control soil (C₀) and performing soil (P₀) at the end of irrigation (t₀).

The enzymatic activity results and chemical analysis results were subjected to analyses of variance via Tukey’s test with a 5% level of significance using statistics software analysis of variance for orthogonal experiments of the Bydgoszcz University of Science and Technology: ANALWAR–5.1. FR software package,. Pearson’s linear correlation coefficients of the biometric feature were calculated using Statistica 13.1 for Windows 10 software.

3. Results

The NO₃⁻-N and NH₄⁺-N contents in the Alfisol and their dynamics during the growing season significantly depended on the conditions of the experiment from irrigation to nitrogen fertilization (

Table 4. Contents of nitrate, ammonium, and mineral nitrogen in soil under barley (mean values for 2015–2017).

		NH4+			NO3−			Nmin
Date	N dose	IRR	NIRR	Mean	IRR	NIRR	Mean	IRR	NIRR	Mean
Germination	N0	6.107b	6.107b	6.107	2.657	2.657	2.657a	39.437	39.437	39.437a
Ripening	N0	4.310a	3.95a7	4.133	10.023	6.497	8.260a	64.502	47.040	55.771a
	N1	4.513a	4.383a	4.448	7.703	13.147	10.425a	54.977	78.885	66.931a
	N2	5.217b	8.040b	6.628	21.930	29.850	25.890b	122.16	125.51	123.83b
	N3	6.060b	6.357b	6.208	23.777	19.637	21.707ab	134.27	116.97	125.62b
Average		5.025	5.684	5.355	15.858	17.283	16.570	93.976	92.100	93.038
Maturity	N0	4.413a	3.777a	4.095	6.553	6.937	6.745a	32.683	31.545	32.114a
	N1	4.667a	2.960a	3.813	3.150	20.260	11.705a	35.175	104.49	69.833a
	N2	3.030a	4.397a	3.713	10.763	20.953	15.525ab	67.235	106.10	86.670b
	N3	5.603b	3.573a	4.588	25.330	19.753	22.542b	101.87	118.31	110.09b
Average		4.428	3.677	4.053	11.449	16.976	14.213	59.240	90.111	74.676

IRR—irrigation, NIRR—no irrigation, different letters following the mean values indicate significant differences with the Tukey test at p ≤ 0.05.

). The content of NH₄⁺ depended on the interaction of irrigation during the development phases (Table 4). At the second date (after fertilization), during ripening, the content of ammonium ions was higher; in no-irrigation plots, it was on average 13% less than in the irrigated plots. Before harvest, a higher content of the ions was observed in irrigated plots, especially with the N₁ and N₃ doses. In the plots fertilized with nitrogen, the lowest content of NO₃⁻-N^- occurred in spring (germination). After applying mineral fertilization, the content of those ions increased considerably, and then slightly decreased at the end of vegetation. The content of mineral nitrogen N_min depended also on nitrogen fertilization. Differences in contents were found depending on the irrigation before the harvesting of the spring barley. In the plots without irrigation, on average, 34% more mineral nitrogen was recorded than in the plots with irrigation.

The contents of NH₄⁺-N and NO₃⁻-N^- during the experimental years depending on nitrogen fertilization and irrigation are shown in Figure 1. The content of NH₄⁺-N ranged from 1.187 to 6.867 mg·kg⁻¹ of soil and it did not depend on irrigation; it increased only slightly with increasing doses of the nitrogen fertilizer. However, the content of NO₃⁻-N^– fell within a wider range from 1.50 to 33.23 mg·kg⁻¹ of soil (Figure 2). In all of the experimental years, a higher content of this nitrogen fraction was found in samples from non-irrigated plots when compared to samples from the irrigated ones, and this difference in each year chronologically was 50%, 30%, and 12%.

However, at maturity, only NR activity was 18% higher in irrigated soil (

Table 5. Enzymatic activity at ripening and maturity in barley in 2015, 2016, and 2017.

Treatment		Ripening					Maturity
		DH ‡	PER	CAT	NR	UR	DH	PER	CAT	NR	UR
Irrigation	N0	13.55	7.717	3.192	4.800	5.032	18.21	4.819	2.887	6.260	8.144
	N1	33.38	9.394	3.641	4.884	4.030	18.03	4.606	2.825	5.248	6.370
	N2	29.51	8.205	3.783	3.174	3.646	24.35	5.643	3.103	4.077	4.551
	N3	23.60	8.266	4.799	5.471	4.980	57.86	3.843	4.261	6.067	7.758
	Mean	25.01	8.395	3.853	4.582	4.422	29.61	4.728	3.269	5.413	6.706
No irrigation	N0	27.25	7.198	3.574	7.134	4.364	16.92	6.710	2.284	2.486	6.623
	N1	27.58	9.242	3.368	2.970	5.959	37.26	4.209	1.897	5.211	7.808
	N2	27.33	8.601	4.310	7.901	3.195	55.62	8.235	3.069	3.888	8.040
	N3	53.08	7.473	3.843	7.927	3.242	45.51	5.185	2.595	4.568	9.758
	Mean	33.79	8.128	3.774	6.483	4.190	38.83	6.085	2.462	4.038	8.057
Development phase		ns	ns	ns	ns	ns	ns	ns	ns	1.013	ns
Irrigation		ns	ns	ns	ns	ns	ns	1.813	0.970	0.132	ns
N fertilization		ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
Development phase× irrigation		ns	ns	ns	1.559	ns	ns	ns	ns	1.724	ns

^‡ DH—dehydrogenase activity per mg TPFg ⁻¹ h ⁻¹. PER—peroxidase activity per mmol of purpurogallin g⁻¹·h⁻¹. CAT—catalase activity per µmol H₂O₂·g⁻¹·min⁻¹. NR—nitroreductase activity per mg N-NO₂⁻ kg⁻¹·24 h⁻¹. UR—urease activity per mg N-NH₄⁺ kg⁻¹·h⁻¹, ns—not significant.

). The activities of the other enzymes were higher in no-irrigation treatments by 25% for DH, 22% for PER, 33% for CAT, and 17% for UR compared to the irrigated soil. The statistical analysis showed the effect of irrigation on the PER, CAT, and NR activity. As for NR, the activity was influenced, apart from the influence of irrigation, by the barley’s development phase. A significantly higher NR activity was found in the soil sampled in 2016—on average about four times higher compared to the average activity determined for the samples taken in 2016 and three times higher compared to the average soil activity in 2017. However, the activities of the other oxidoreductases developed differently over the experimental years. The highest catalase activity was found in the samples from 2015, where it was 29% higher compared to the average from 2017.

Peroxidase, on the other hand, showed 70% higher activity in samples taken in 2017 when compared to that in the 2015 samples. Regarding the influence of fertilization on the enzymatic activity, DH and CAT activity increased with increasing fertilizer doses. As for PER, the highest dose of fertilizer resulted in a 14% reduction in its activity when compared to N₂. The activities of enzymes involved in nitrogen metabolism in the soil were different compared to the oxidoreductases (Figure 3). The activities of both enzymes were highest at the third date of soil sampling. As for UR, the activity in this period was on average 43% higher than at the beginning of the season (germination), and nitrogenase showed 27% higher activity when compared to the lowest activity at the second sampling date (

Table 6. Enzymatic activity during germination in barley in 2015, 2016, and 2017.

Year	Germination
	DH ‡	PER	CAT	NR	UR
2015	6.930	4.340	5.120	0.311	4.780
2016	25.30	4.490	2.420	3.452	6.890
2017	18.40	8.970	2.021	7.890	6.590
Mean	16.88	5.930	3.187	3.884	6.087

^‡ designations the same as in Table 5.

). The influence of nitrogen fertilization on the activities of these enzymes was also recorded, and on average, the activity of UR was reduced by 13% when fertilized with a dose of N₂ and that of NR was reduced by 7% when fertilized with a dose of N₁, as compared to the control.

Sprinkler irrigation applied in the barley experiment did not have a significant impact on soil enzymatic activity. This was proven by the non-significant coefficients of correlation between the content of RAW and the enzymatic activity both for the irrigated and non-irrigated plots (

Table 7. Coefficients of correlation (r) between soil enzymatic activity and the content of readily available water (RAW) and differentiated N fertilization level.

Type of Soil Enzyme	RAW	N Fertilization
Catalase	−0.0712	0.2001
Dehydrogenase	0.0735	0.2576
Peroxidase	−0.1652	−0.0087
Urease	0.0532	0.0000
Nitroreductase	0.0676	0.0711

RAW—readily available water in soil, N fertilization—nitrogen fertilization.

). The most sensitive enzyme to soil water content was peroxidase (r = −0.1652), while the other ones demonstrated a similar level of response (r between −0.0712 and 0.0735). As for the second factor, there was no response of urease to the nitrogen fertilizer level, while catalase and dehydrogenase responded with positive, yet non-significant, values (r = 0.2001 and r = 0.2576, respectively). Importantly, the values of coefficients were bidirectional, depending on the type of enzyme, which confirms that the reactions of these enzymes to irrigation and N fertilizer dose were ambiguous.

Regarding the contents and enzymatic activities determined in this study (

Table 8. Relationship between selected soil properties.

Dependent Variable (y)	Independent Variable (x)	Equation	Correlation Coefficient (r)
Catalase activity	NH4+	y = 2.8422 + 0.64010x	0.299
Dehydrogenase activity	NO3−	y = 3.8906 + 0.28549x	0.523
Peroxidase activity	NH4+	y = 3.6836 + 0.54160x	0.331
Urease activity	NH4	y = 7.6386 – 0.3937x	−0.297
Nitroreductase activity	Urease activity	y = 6.2506 − 0.2875x	−0.340
Peroxidase activity	NO3−	y = 3.6337 + 1.2621x	0.336
Urease activity	Peroxidase	y = 6.9388 − 0.1986x	−0.245

), catalase, peroxidase, and urease activities were significantly correlated with the NH₄⁺-N content (r = 0.299, r = 0.331, and r = −0.297, respectively). However, dehydrogenase and peroxidase were positively correlated with the NO₃⁻-N content in the soil. The urease activity was significantly negatively correlated with the soil enzymatic activities of nitroreductase (r = −0.340) and peroxidase (r = −0.245).

The resistance of soil (RS) data are presented in

Table 9. Resistance of soil (RS) for enzymatic activities depending on nitrogen doses during vegetation in spring barley.

N Doses	Resistance of Soil (RS)
	NR	UR	CT	PX	DH
N0	−0.206	0.627	0.934	0.560	0.859
N1	0.986	−0.597	0.991	0.828	0.319
N2	0.907	0.395	0.580	0.521	0.425
N3	0.506	0.660	0.444	0.589	0.573

. Differences in resistance to irrigation across the enzymes were observed for nitrogen doses. Oxidoreductases (PER, CAT, DH) with the highest RS value were observed for N₀ and N₁. The highest RS values (0.991 and 0.934) were calculated for CAT activity and for N₀ and N₁. For these N doses, high values of RS for DH activity (0.859 for N₀) and PX activity (0.828 for N₁) were recorded. For UR, the highest RS values were found for N₁ (0.986) and N₃ (0.907). The RS values for the activities of UR and NR were negative: N₁ (−0.597) and N₀ (−0.206).

4. Discussion

Water and nitrogen are in charge of limiting rural production in most parts of the world [27]. Transforming nitrogen in soil is essential for nitrogen metabolism and crop tolerance to drought stress, and it is engaged in nearly all of the physiological transformations in plants and microorganisms [28]. According to Wang et al. [29], NH₄⁺-N uptake is universally enhanced in the majority of plants during drought stress, and superior nitrogen uptake may increase plant drought hardiness. The outcome of the present experiment identified the impact of irrigation on the development phases in spring barley. During barley vegetation, it was found that with the development of plants, the NH₄⁺-N content in lessive soil showed a decreasing trend, especially in non-irrigated soil; the ammonium content decreased significantly, which could have been due to the fact that at maturity, spring barley has a higher NH₄⁺-NNH₄⁺-N uptake due to drought stress. This result is consistent with the work of Lawlor et al. [30], who recorded an increasing effective NH₄⁺ nitrogen uptake and increasing activity of NR in plants during drought stress. As compared to the non-irrigated soil, the content of NO₃⁻ and N_min in soil exposed to irrigation decreased during spring barley vegetation. The lowest contents of NO₃⁻-N and N_min at the third sampling date suggest NO₃^−—N leaching. Similar results were obtained by Wu et al. [31], who reported that the mineral nutrient content in soil changed depending on irrigation and nitrogen fertilization and that a high irrigation water content can increase nutrient leaching and reduce the soil nutrient content. Muhammad et al. [32] show that the mechanisms of NO₃⁻-N leaching depend on the physical properties of soil, especially its water capacity. A higher amount of N (300 kg N ha⁻¹) resulted in a higher soil SOC and higher total and mineral N under low (60%) irrigation. Nitrogen in the form of nitrate is highly mobile in soil and its content depends on soil water conditions [33]. Irrigation probably contributes to increased leaching of nitrate in soil. The results of the present experiment show that doses of nitrogen fertilizers have an impact on the contents of NO₃⁻-N and N_min. These findings are consistent with those of Jia [34], who report that NO₃⁻-N leaching increases even with the same N fertilizer rate due to a vast amount of total irrigation. The effects of temperature and moisture on enzyme diffusion and substrate availability are all critical factors influencing soil enzymes’ activities [35]. Drought greatly influences almost all of the physiological and biochemical transformations of plants: growth, development, and productivity. The nitrogen content and transformation in soil are decisive during drought stress for plant and microorganism metabolism. The present study demonstrates that enzymes are soil components that are strictly connected with the physicochemical and biological properties of soil. The reactions of enzymes depend on their origin and features [36]. In this study, we demonstrated a lack of responses of all five types of soil enzymes to different levels of nitrogen fertilization in barley. Cui et al. [37] suggest that monoculture and fertilization processes can increase enzyme activity by improving soil nutrients and microbial richness. In 2020, Zhang et al. [38] determined that nitrogen fertilizer lowered both β-1,4-glucosidase and acid phosphatase, while water from irrigation inhibited acid phosphatase only. Additional nitrogen and irrigated water affected enzyme activity mainly by affecting the soil microbial biomass carbon and NH₄⁺-N. In general, the addition of nitrogen or water over a long time did not affect β-1,4-glucosidase, which implies that this enzyme is resilient and stays unaffected by modifications in the environment. In contrast, acid phosphatase showed sensitivity to nitrogen and irrigation and responded to seasonal fluctuations in precipitation. This suggests that this enzyme could be an indicator of transformations in soil nutrient cycling. Many field studies have examined the effects of added nitrogen on the activity of enzymes in soil. The results of those studies were inconclusive. Some results suggested that the addition of a nitrogen fertilizer caused soil acidification and inhibited soil enzymatic activity [39]. Other studies indicated a stimulating effect of nitrogen on enzyme activity, or no such effect at all [40,41,42,43,44].

Urease hydrolysis of small organic substrates containing nitrogen into inorganic compounds (ammonia) is used to supply nitrogen for the normal growth and development of plants [45]. In our study, the development phase, irrigation, and N mineral fertilization showed no statistical impact on urease activity. Similar results were obtained by Zhao et al. [46] in their research: single-nitrogen nor mixed-nitrogen applications did not affect urease activity significantly. However, the present study reveals that the activity of hydrolase in the soil increased and later decreased the urease activity, and it hit its maximum at the maturity phase and increased with the increasing doses of N fertilizer, especially in non-irrigated plots. Weng et al. [47] and Gong et al. [44] note that mineral nitrogen fertilizer often increases urease activity. Fortification of urease activity due to natural or organic nitrogen addition was observed by Nayak et al. [15] and Iovieno et al. [48]. Higher hydrolase activities may be due to an increase in carbon and nitrogen in the soil and improvements in the soil physicochemical properties, as well as a more appropriate soil environment for microbial growth and proliferation, which stimulates microbial and enzymatic activity. Negative effects resulting from lower pH have also been observed with the long-term use of nitrogen fertilizers [49].

The activity of enzymes depends on several factors, especially on the presence of a substrate; as for NR, the substrate is nitrate in soil. Nitrate reductase is an enzyme that controls and reduces nitrate assimilation in plants, which is not only responsive to external nitrogen but also indirectly creates a difference in the uptake and utilization of nitrogen by plants [50]. Waraich et al. [51] and Sardans and Peñuelas [52] report on drought stress reducing plant N uptake and assimilation by reducing both nutrient diffusion and N supply via mineralization [53]. In our study, the lower NR activity at maturity in the plots with no irrigation may have resulted from a reaction of the plants and microorganisms to long drought stress. The NR activity increased at ripening and then decreased at maturity when not exposed to irrigation. At maturity in the spring barley, the CAT activity increased in irrigated soil. However, PER activity presented a different reaction and reached its highest in non-irrigated soil and depended significantly on the rates of water. Peroxidase is an enzyme that is expressed for a variety of reasons, including carbon and nitrogen and protection. This enzyme moves into soil via excretion or lysis, where it mediates the ecosystem functions of lignin degradation, humification, carbon mineralization, and dissolved organic carbon release [7]. A higher PER activity in non-irrigated soil indicates high oxygen availability, optimal pH conditions, and optimal mineral activity, and hence a high oxidative activity and limited accumulation of organic matter in the soil [7].

We present interesting observations regarding dehydrogenases, which are one of the most important oxidoreductases, and which are used as an indicator of the total soil microbial activity since they are closely linked with microbial oxidoreduction processes, as they occur in all living microbial cells and as such can indicate the microorganism activity in soil [54]. Our research has shown that soil moisture influences dehydrogenase activity. The high DHA activity observed in the soil during spring barley vegetation in 2016, which was the highest-rainfall year throughout our experiment, coincides with the results reported by Gu et al. [55], who observed an increase in DH in high-moisture soil. A high dehydrogenase activity can be due to two factors: flooding, with the release and spread of soluble organic compounds in the soil, which contributes to the development of a larger number of bacteria that secrete dehydrogenases; and/or the change of oxygen conditions to anaerobic conditions and the proliferation of anaerobic microorganisms [56]. Also, Dora [57] indicates that dehydrogenase and catalase activities are higher in irrigated soil. Tan et al. [58] found that long-term mulched drip irrigation (8, 12, 16, and 22 years) tends to accumulate soil nutrients and rebuild enzyme conditions. Soil enzymes such as catalase and urease were more active in the subsoil than in the topsoil. Also, Liang et al. [59] confirmed that long-term irrigation strongly increased the activity of dehydrogenase as well as urease in soil. Núñez et al. [60] indicated that a reduction in enzyme activity after irrigation termination in corn may point to changes in biogeochemical cycling and even a potential reduction in the decomposition of leftovers [11,61]. However, enzyme activity can also be affected by changes in soil environmental circumstances [61,62], such as reduced water availability, which can increase enzyme immobilization and decrease the diffusion rates, decreasing enzyme efficiency and affecting residue decomposition independent of changes in potential enzyme activity [63]. A negligible effect of irrigation on the activity of soil enzymes was also reported in grassland ecosystems [64]. Also, it has been shown that additional water application can mitigate the effects of nitrogen enrichment on microorganisms by leaching or reducing the accumulation of inorganic nitrogen [65,66], and it can also have a significant effect on soil enzyme activity.

The present study identifies that catalase, peroxidase, and urease were correlated significantly with NH₄⁺-N content (appropriately, r = 0.299,  r = 0.331, and r = 0.297; p = 0.05), and dehydrogenase and peroxidase activity were correlated significantly with NO₃⁻-N content  (r = 0.523 and r = 0.336; p = 0.05). Nitroreductase was negatively correlated significantly with the activities of urease (r = −0.340; p = 0.05) and peroxidase (r = −0.254; p = 0.05), indicating that some enzyme activities may affect and induce other enzyme activities in the soil considerably.

The resistance of the enzymes to drought was different depending on the doses of nitrogen fertilization. Catalase showed the highest resistance to drought stress, followed by NR and PER. Urease and the dehydrogenases demonstrated a lower resistance to soil drought. The resistance of the enzymes to drought was different depending on the doses of nitrogen fertilization. Catalase showed the highest resistance to drought stress, followed by NR and PER; urease and the dehydrogenases showed a lower resistance to soil drought. The results reported by Lemanowicz [67] point to catalase activity having a strong resistance also to salinity stress.

5. Conclusions

In conclusion, our results suggest that no irrigation influences the NH₄⁺-N content in Alfisols with spring barley at maturity due to its low uptake being the consequence of drought stress. Irrigation may contribute to increased nitrate leaching in the soil profile. The results of our experiment show that different doses of nitrogen fertilizer influence the contents of NO₃⁻-N and N_min. The nitrogen fertilization of 60 t ha⁻¹ was optimal for achieving the ideal contents of NO₃^-−-N and NH₄⁺-N available to plants. The present study indicates that enzymes are sensitive to soil properties, which are closely related to the contents of NH₄⁺-N and NO₃⁻-N in the soil. The enzymatic activity changed over the studied years, depending on the weather conditions. The resistance of soil could be used for an enzymatic water stress solution. The highest index of resilience was presented by catalase. These results suggest a need for further research on selected physicochemical and biochemical parameters, as well as on other types of soil and under other crops, especially in areas that have a moderate transitional climate with varied weather conditions.