Use of Siliceous Minerals as Natural Nitrification Inhibitors ^†

Abstract

The comparative study on the effects of nitrapyrin and the mixture of natural siliceous minerals on physiological processes in economically important C3 and C4 crops (viz. wheat and corn) grown under high nitrogen fertilization conditions, as well as on soil microbiota and nitrogen balance, was conducted in the laboratory and long-term field experiments. The siliceous minerals were shown to have significantly higher inhibitory effects on the nitrification process in different types of soils, amended with urea, the number of nitrifiers and micromicetes producing phytotoxic allelochemicals compared with nitrapyrin. Crops treated with siliceous minerals had higher photosynthetic pigment content and higher glutamic acid concentration, which indicated the intensification of photosynthetic processes. Nitrapyrin reduced the concentration of chlorophyll b and carotenoids, but slightly increased the content of chlorophyll a in the leaves of wheat and corn. The content of aspartate and some aromatic amino acids decreased, while arginine and lysine increased. Such metabolic changes suggested disintegration of nitrogen and phosphate metabolism in the studied crops. Thus, the use of siliceous minerals is more advantageous than nitrapyrin in terms of their effectiveness, persistence in various types of soils and beneficial effect on soil microbiota and crops’ functional state and productivity.

1. Introduction

Nitrogen (N) is an essential nutrient element and also a key limiting factor for the growth and development of plants in agricultural systems [1]. About 25% of nitrogen available to plants in the soil is presented in the form of ammonia and nitrate ions produced by microbiological transformation of residues and humus [1]. In the majority of agricultural soils, NH₄⁺ is rapidly converted to NO₃⁻ as a result of the biological oxidation of ammonia (NH₃) or ammonium (NH₄⁺) to oxidized nitrogen in the form of nitrite (NO₂⁻), and further to nitrate (NO₃⁻). This process is referred to as nitrification and takes a relatively short period of time [2]. The nitrate formed is susceptible to losses via leaching and conversion to gaseous forms via denitrification [2]. Often less than 30% of the applied N fertilizer is recovered in intensive agricultural systems, largely due to losses associated with the following nitrification [3]. NO₃⁻ leaching from intensive agricultural systems typically represents the major N loss. Unlike ammonium (NH₄)⁺, which is strongly held on soil particles, NO₃⁻ is a negative ion and weakly held by soil, making it susceptible to leaching under higher rainfall or irrigation, particularly if it is present in much greater quantities than plants can uptake [4]. Besides, nitrates are readily denitrificated by soil microorganisms to gaseous forms of nitrogen, mainly N₂O, which is a long-lasting greenhouse gas (lifetime—150 years), and is also the major source of ozone-depleting nitric oxide (NO) and nitrogen (N₂) [5].

In the next 20–30 years, a significant increase in the world’s population is expected, and, accordingly, it is necessary to produce food in sufficient quantities. To achieve this, the use of nitrogen fertilizers will have to double by 2050 [1]. For environmental reasons, this is not possible, as nitrate levels in drinking water, eutrophication of surface waters and greenhouse gas emissions have already reached critical levels in many countries of the world. The use of nitrification inhibitors can reduce the use of fertilizers and significantly increase their efficiency. Therefore, there is a need for environmentally friendly nitrification inhibitors, as well as methods for their study.

Approaches to the management of nitrification include those that control ammonium substrate availability and those that inhibit nitrifiers directly [2]. Strategies for controlling ammonium substrate availability include timing of fertilization, formulation of fertilizers for slow release and intensification of nitrogen cycling (immobilization). Another effective strategy is to inhibit nitrification directly with either synthetic or biological nitrification inhibitors [2,3]. The latter are chemical compounds temporarily reducing populations of Nitrosomonas and Nitrobacter bacteria in soil. Nitrification inhibitors protect against both denitrification and leaching by retaining fertilizer N in the ammonium form.

There are at least eight compounds commercially recognized as nitrification inhibitors, although the most commonly used and most studied are 2-chloro-6-(trichloromethyl)-pyridine (nitrapyrine), dicycanediamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP). These compounds inhibit microbial activity for several days to weeks depending on soil moisture and soil type, although there are differences in the way they are used. In general, nitrification inhibitors are more effective on sandy soils or soils with low organic matter content and low temperature effects.

Another group of chemicals used for regulation of the soil nitrogen include urease inhibitors. These chemicals block the activity of the enzyme urease. Urease is found in soil as well as in plant residues. Urease, along with water, will hydrolyze, or break down, urea into ammonium. The loss process that urease inhibitors protect against is ammonia volatilization. With high pH soil, or soil/residue environments that are poorly buffered against an increase in pH, the rapid hydrolysis of urea will result in an accumulation of ammonia (NH₃). The latter can be lost to the atmosphere as a gas. By keeping urea from hydrolyzing, urease inhibitors protect against ammonia volatilization, keeping nitrogen fertilizer in the urea form. Among commercially available urease inhibitors, (N-(n-butyl) thiophosphorictriamide (NBPT) and N-(n-propyl) thiophosphorictriamide (NPPT) are the most studied [6].

The use of the above-mentioned groups of chemicals allows reducing the use of fertilizers and significantly increasing their efficiency. However, the use of nitrification and urease inhibitors increases cost and the risk of environmental contamination [7]. Recently, nitrapyrin has been detected in streams, suggesting off-site transport of this nitrogen-stabilizing compound [8]. DCD residues were detected in milk in New Zealand, resulting in the suspension of DCD use in pastures [9]. Commercially available urease inhibitors NBPT and NPPT were shown to be phytotoxic to some sensitive crops [6]. Besides, chemical nitrification and urease inhibitors are not permitted in certified organic management systems. Therefore, it is necessary to use nitrification inhibitors which are environmentally safe, non-toxic, non-volatile and have a prolonged effect. In this regard, the natural siliceous minerals have been suggested as organic alternatives for the management of nitrification.

Natural siliceous minerals present promise as environmentally friendly regulators of the biogeochemical nitrogen cycle. In contrast to synthetic nitrification inhibitors, natural siliceous minerals do not impose any harm to soil microbiota and were shown to have beneficial effects on crop productivity [10,11]. They are stable in the soil environment, and, after having been applied once, express their effect for many years. Silicon was shown to increase nitrates’utilization by crops by stimulating photosynthetic rate, root activities and nitrate reductase activity in plants [12,13]. Rice grains accumulated 17–37% more N with an exogenous application of Si between 100 and 400 kg·ha⁻¹ SiO₂ compared to the control. Similar increases in N accumulation for rice straw and total biomass with the same Si doses were in the ranges of 19–29% and 18–33%, respectively [13]. Fertilization with sodium metasilicate (50–800 mg Si kg⁻¹) stimulated uptake of N and Ca by cowpea and wheat and improved nodulation and N₂ fixation in cowpea [14].

In the greenhouse study, NH₄-charged zeolite was shown to minimize NO₃-leaching from soil and to optimize water-saving soil capacity as well as corn growth and yield under different fertilizing conditions (i.e., standard, high or 70%, medium or 50% and low or 30% of conventional fertilization rate). The results suggested that plants may have a better response if NH₄-charged zeolite is used with a limited amount of conventional fertilizer, allowing a reduction of nitrate concentration in drainage [15]. The field studies on the effect of soil amendments (i.e., lime or zeolite (clinoptilonite)) on nitrous oxide (N₂O) and dinitrogen (N₂) emissions from pastoral soil conducted on Topehaehae silt loam soil (AericHaplaquent, a dairy catchment area at Toenepi, Hamilton, New Zealand) demonstrated that zeolite (clinoptilonite) significantly reduced total N₂O emissions by 11% from cow urine-treated soils, probably because of NH₄⁺ sorption by zeolite, while it had no such effect on N₂O emission in KNO₃-treated soils (both nitrogen fertilizers were applied at a rate of 200 kg N ha⁻¹). Lime did not have any effect on N₂O emission in either urine or KNO₃-treated soils [16].

The objectives of the given study were to compare the effectiveness of nitrapyrin and the mixture of natural siliceous minerals (analcite and tripoli) on the balance soil nitrogen, dynamics of the different functional groups of microorganisms in the soil, metabolism and productivity in economically important C3 and C4 crops (viz. wheat and corn) grown under high nitrogen fertilization conditions.

The mentioned siliceous minerals are readily available in Ukraine (Vinnytsia and Rivne regions) and present inexpensive and environmentally safe sources of fertilizers for agricultural needs. In addition, unlike nitrapyrin, these minerals are neither hazardous for human health nor explosive [17].

2. Experiments

2.1. Experimental Procedures

2.1.1. Pot Experiments

The effectiveness of nitrapyrin and a mixture of siliceous minerals (analcite and tripoli) as nitrification inhibitors was compared using different types of substrates (sand and soil mix) and crop species in the laboratory pot experiments. The latter were conducted in the plant growth chamber at the department of Allelopathy of the M.M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine (Kyiv, Ukraine). Wheat (Triticum aestivum L. cv. «Smuglianka») and corn (Zea mays L., Hybrid Pereyaslovsky 230 SV) seeds were obtained from the Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine.

The test plants were grown in 1 L plastic pots for 45 days under controlled conditions: air temperature of 20–22 °C and illumination of 1700 lux. The rate of application of nitrapyrin was 1 mL (1.58 g) per 1 L of soil mixture or sand, and a mixture of minerals of analcite and tripoli in the amount of 1 g per 1 L of sand or soil mixture, which included sand, peat, meadow soil and humus in proportion 1:1:1:0.2. As a source of nitrogen, a 0.5% urea solution was used.

Qualitative and quantitative analyses of soil microbiota and assessment of the content of nitrates in the sand and soil substrates were conducted on the 15th, 30th and 45th day of the test plant cultivation.

2.1.2. Field Experiments

Five-year field studies were conducted on the experimental plots (plot area—2 ha) situated at the Agricultural Research Station of the Institute of Bioenergy Crops and Sugar Beet of the National Agrarian Academy of Sciences of Ukraine (Kyiv region, KsaverovkaVillage, Kagarlytsky District during 2015–2019). The nitrification inhibitors were applied to winter wheat(Triticum aestivum L.) cv. «Samurai» and corn Zea mays L. hybrid «Adevei 4014»). The rate of the applied nitrapyrin was 1.7 L per 1 ha (2.69 kg per 1 ha), and the amount of siliceous minerals was 10 kg per 1 ha for winter wheat and 6 kg per 1 ha for corn. For winter wheat, the rate of urea application was 100 kg/ha, and for corn—60 kg/ha. Nitrification inhibitors were applied simultaneously with fertilizers (N₂₀P₆₀K₆₀): for winter wheat—at the stage of spring tillering (on frozen thawed ground), for corn—in the phase of 3–5 leaves under harrowing. The soil type was dark-gray podzol, sandy, slightly loamy, with the following content of the basic macro-elements: nitrogen—92 mg/kg, phosphorus—150 mg/kg and water-soluble potassium—1.8 mg/kg. The pH of the soil solution was 6.7.

The measurements of the content of nitrate nitrogen in soil were timed to crops’ phenological phases (tillering, first node, flowering, physiological maturity). Allelopathic activity of soil and the number of microorganisms producing phytotoxic allelochemicals were evaluated two months after the application of nitrification inhibitors. Changes in the amino acid composition and photosynthetic pigments’ content in the leaves of winter wheat and corn were evaluated 30 days after the application of nitrification inhibitors and urea.

2.2. Measurements

The macro- and micro-elements in soil samples were determined using inductively coupled plasma spectrometer iCAP 6300 DUO from Thermo Fisher Scientific, Waltham, MA, USA (2006). Their extraction was conducted with 1N HCl [18].

The photosynthetic pigments (chlorophylls a, b and carotenoids) were extracted from freshly collected leaves of the test plants with dimethylsulfoxide (DMSO) [19]. Their content was determined spectrophotometrically with a SPECORD 200 (Analytik Jena, Jena, Germany). Qualitative and quantitative content of free amino acids was determined by the amino acid analyzer Hitachi [20].

Allelopathic activity of the rhizosphere soil was assessed by direct bioassay (Grodzinski et al., 1990) on germination of radish (Raphanus sativus var. radiculata, cv. «Krasny s belym konchikon») seeds. The seeds (20 seeds per a Petri dish) were germinated at a temperature of +23–26 °C in darkness. The number of germinated seeds was counted on the second day, when in the control 50% of the seeds had germinated [21]. Phenolic allelochemicals were extracted from the soil samples with ethanol and 70% acetone in distilled water [22]. Their quantity was assessed spectrophotometrically using color reaction with Folin-Ciocalteu reagent with SPECORD 200 (Analytik Jena) [22].

Analysis of the number of microorganisms from freshly collected soil samples was carried out by sowing soil suspensions in appropriate dilutions on selective agar nutrient media: ammonifying bacteria (peptone ammoniation medium), nitrifying bacteria (Winogradsky medium), denitrifying bacteria (Trypticase soy agar supplemented with nitrate), micro-mycetes (Chapek’s medium), bacteria (meat peptone agar + wort agar) and actinomycetes (starch-ammonia agar). The count of the colonies was conducted visually using a light illuminated microscope Zeiss on days 3–7 after sowing [23].

2.3. Statistical Analysis

The data were subjected to the analysis of variance (ANOVA) appropriate to the randomized complete block design applied after testing the homogeneity of error variances using Levene’s mean-based F-test procedure, with modifications outlined by Sharma and Golam Kibria [24]. The significant differences between treatments were compared with the critical difference at the 5% probability level by the LSD (the least significant difference) test. The statistical operations were conducted using Statistica 10.0 software.

3. Results

3.1. Pot Experiments

The data presented in the

Table 1. The effect of exogenous nitrapyrin and a mixture of siliceous minerals (analcite and tripoli) on the content of nitrate nitrogen in the soil.

Treatment	Type of Substrate	pH of Soil Solution	Humus%	The Content of Nitrate Nitrogen, mg/kg
				Duration of Plant Exposure, Days
				15	30	45
Wheat
Control	Sand	7.2	0.03	115.7	193.6	298.1
	Soil mix	6.2	7.5	146.9	235.8	334.2
Nitrapyrin	Sand	7.2	0.03	97.5	83.9	92.4
	Soil mix	6.2	7.5	101.7	88.3	95.6
Analcite + Tripoli	Sand	7.2	0.03	71.3	64.9	61.7
	Soil mix	6.2	7.5	73.8	66.1	64.3
LSD (p = 0.05)		0.01	0.01	1.23	1.88	1.74
Corn
Control	Sand	7.2	0.03	99.3	168.4	255.8
	Soil mix	6.2	7.5	125.7	191.0	287.1
Nitrapyrin	Sand	7.2	0.03	81.2	73.7	77.6
	Soil mix	6.2	7.5	87.9	76.4	82.3
Analcite + Tripoli	Sand	7.2	0.03	52.4	43.9	42.5
	Soil mix	6.2	7.5	54.3	45.1	43.2
LSD (p = 0.05)		0.01	0.01	1.74	1.45	1.58

Once a week, a 0.5% solution of urea was added in an amount of 50 mL per 1 L of soil substrate.

clearly show that application of the 0.5% urea solution caused a gradual increase of nitrate concentration in both sand and soil mix, reaching maximum values toward the end of the experiment (45th day). The mixture of analcite and tripoli was the most effective in inhibiting the nitrification process in both types of substrates (sand and soil mix), irrespective of the test plant species. For nitrapyrin treatments, a minimum nitrates level in the substrates was observed on the 30th day after application. On the 45th day, the nitrates concentration slightly increased. Application of the mixture of siliceous minerals (analcite and tripoli) had been reducing the nitrates concentration from the beginning until the end of the experiments, reaching the minimum on the last (45th) day of the experimentation, indicating a higher persistence compared to nitrapyrin.

The decrease in pH of the soil solution and % of humus in the substrates stimulated nitrification processes in treatments with nitrapyrin more significantly compared with the similar treatments exposed to siliceous minerals.

Microbiological analysis of the soil samples of the pot experiment showed that application of urea caused an increase in the number of nitrifying and denitrifying microorganisms from 15 to 30 days of the test plants’ cultivation, followed by some decrease. When nitrapyrin was applied with urea, a sharp decrease in the number of nitrifiers and denitrifiers was observed on the 30th day of the experiment, with a gradual increase in their number on the 45th day (

Table 2. The dynamics of the different functional groups of microorganisms in the soil from pots where winter wheat was cultivated with different nitrification inhibitors.

Treatment	Microorganisms	Period of Winter Wheat Cultivation, Days
		15	30	45
Control	Total number, million/g	20.6	16.2	11.9
Nitrapyrin		21.4	24.7	14.3
Analcite + Tripoli		23.8	24.3	25.1
LSD (p = 0.05)		0.62	0.89	097
Control	Ammonifying, million/g	4.6	10.4	3.2
Nitrapyrin		9.7	17.5	12.0
Analcite + Tripoli		11.5	17.4	16.9
LSD (p = 0.05)		0.71	0.35	037
Control	Denitrifying, million/g	3.1	4.3	3.8
Nitrapyrin		1.8	1.3	1.5
Tripoli + Analcite		0.7	0.6	0.4
LSD (p = 0.05)		0.48	069	077
Control	Nitrifying, million/g	3.9	4.1	3.3
Nitrapyrin		3.0	2.1	2.3
Analcite + Tripoli		2.4	1.8	1.5
LSD (p = 0.05)		0.43	0.31	0.44

). Application of the analcite-tripoli mixture with urea demonstrated a more pronounced effect on the quantity of these two groups of microorganisms for the whole period of experimentation, with the minimum values registered on the 45th day, which indicated a more prolonged effect of silicon compounds on the inhibition of nitrification processes in the soil compared to nitrapyrin. Both nitrification inhibitors studied had a positive effect on the content of ammonifiers in the soil. However, the siliceous mixture again had a more prolonged effect on this group of microorganisms compared to nitrapyrin.

3.2. Field Experiments

The results of agrochemical analysis of the rhizosphere soil of field experiments were in good agreement with the tendencies observed in the laboratory tests (

Table 3. The effect of nitrification inhibitors on the content of nitrate nitrogen in the soil under winter wheat, mg/L.

Treatment	Phenological Phase
	Tillering	First Node	Flowering	Physiological Maturity
Control	78.3	51.8	28.3	15.9
Analcite + Tripoli	63.7	58.4	50.9	42.1
Nitrapyrin	71.3	50.9	37.4	28.2
LSD (p = 0.05)	2.12	1.87	124	1.77

). In the absence of nitrification inhibitors, the nitrate nitrogen content in the soil gradually decreased during the growing season, reaching, during the phase of physiological maturity of crops, the level of 20% of the initial level (tillering phase). The combined application of urea (100 kg/ha) and the analcite + tripoli mixture (10 kg/ha) to winter wheat had a positive effect on the nitrogen supply to plants compared with nitrapyrin treatment (2.69 kg per 1 ha).

Microbiological analysis of the root layer of the soil (depth 0–20 cm) showed a significant long-term positive effect of the analcite + tripoli mixture on the microorganisms producing phytotoxic allelochemicals (

Table 4. Allelopathic activity of soil and the number of microorganisms that produce phytotoxic allelochemicals 60 days after application of nitrification inhibitors.

Treatment	Wheat				Corn
	Germination of Radish Seeds, %	The Number of Phytotoxic Microorganisms, million/g			Germination of Radish Seeds, %	The Number of Phytotoxic Microorganisms, million/g
		Bacteria	Micro-Mycetes	Actinomycetes		Bacteria	Micro-Mycetes	Actinomycetes
Control	72.1	13.6	10.1	2.8	70.7	14.5	10.8	3.1
Analcite + Tripoli	92.2	3.3	4.5	1.7	91.0	3.8	5.1	1.9
Nitrapyrin	75.8	11.9	9.4	3.0	77.5	12.4	9.8	3.4
LSD (p = 0.05)	2.33	0.67	1.11	0.25	1.76	0.78	0.99	0.76

). The positive effect of nitrapyrin was significantly lower. Data from the microbiological analysis were in good agreement with the results of the assessment of the soil allelopathic activity using radish seeds as a bioassay.

The decrease in soil phytotoxicity is evidenced by a 1.2–1.9-fold decrease in the concentration of phenolic compounds (

Table 5. The effect of nitrification inhibitors on the content of phenolic allelochemicals in the soil.

Treatment	Phenolic Allelochemicals, mg/kg of Dry Soil
	70% Acetone Extract		Ethanol Extract
	Corn	Wheat	Corn	Wheat
Control	39.3	33.8	151.3	147.2
Analcite + Tripoli	22.7	19.4	112.7	104.9
Nitrapyrin	31.5	28.7	144.3	138.7
LSD (p = 0.05)	1.13	1.65	2.05	1.25

).

Application of the tested nitrification inhibitors caused significant biochemical changes, in particular in the composition of free amino acids (

Table 6. Changes in amino acid composition in the leaves of winter wheat and corn 30 days after the application of nitrification inhibitors, μg/100 mg of fresh weight.

Amino Acids	Wheat			Corn
	Control	Nitrapyrin	Analcite + Tripoli	Control	Nitrapyrin	Analcite + Tripoli
Aspartic acid	8.5	8.1	17.3	6.2	5.5	14.2
Threonine	15.0	9.8	7.4	26.9	22.1	19.2
Serine	2.3	1.8	0.9	2.1	1.3	0.5
Glutamic acid	6.4	6.7	9.1	11.2	11.8	15.1
Histidine	4.5	4.8	6.0	4.7	4.9	7.4
Tyrosine	2.3	2.4	3.9	1.4	1.6	2.5
Arginine	9.7	8.2	4.7	10.9	10.3	5.1
Lysine	2.5	2.2	1.5	2.3	2.0	0.8
Phenylalanine	1.3	2.1	2.6	1.8	1.9	3.3
LSD (p = 0.05)	0.04	0.01	0.01	0.01	0.02	0.01

) in the leaves of wheat and corn plants. Nitrapyrin treatment caused a decrease in aspartic acid as well as aromatic amino acids, while the content of arginine and lysine increased in both wheat and corn plants. In the test plants treated with the analcite + tripoli mixture, an increase in the content of glutamic acid was recorded, which indicated the intensification of photosynthetic processes.

The content of photosynthetic pigments in the leaves of winter wheat and corn plants 30 days after application of urea with nitrification inhibitors differed significantly between treatments. Application of the siliceous mixture contributed to a marked increase in the content of chlorophyll a and b, as well as carotenoids in the leaves of the studied crops, while nitrapyrin caused a decrease in the biosynthesis of chlorophyll b and carotenoids and a slight increase in the content of chlorophyll a in the leaves of the test plants (

Table 7. The content of photosynthetic pigments in the leaves of winter wheat and corn treated with the different nitrification inhibitors, mg/100 g of fresh weight.

Treatment	Wheat			Corn
	Chlorophyll		Carotenoids	Chlorophyll		Carotenoids
	a	b		a	b
Control	74.2	22.7	92.5	80.9	37.6	105.7
Nitrapyrin	76.9	21.8	91.7	96.3	34.2	88.2
Analcite + Tripoli	82.5	40.3	112.6	95.1	58.9	121.3
LSD (p = 0.05)	1.12	0.84	0.95	0.98	0.86	0.92

). A significant increase in the concentration of chlorophyll b in the leaves of wheat and corn plants treated with the analcite + tripoli mixture should be noted.

The data in

Table 8. The effect of nitrification inhibitors on the yield of winter wheat.

Treatment	Yield, Quintal/ha	Protein Content, %	Fiber, %
Control	68.4	12.6	24.3
Analcite + Tripoli	82.5	13.5	25.1
Nitrapyrin	75.3	12.9	24.7
LSD (p = 0.05)	0.97	0.54	0.48

clearly show that the highest yield of winter wheat was observed on plots where the mixture of tripoli and analcite was applied. Grain was characterized by the highest content of protein and fiber. Nitrapyrin showed significantly less efficacy compared to the siliceous mixture.

4. Discussion

Despite a great interest in nitrification inhibitors to date, only a few compounds have been adopted for agricultural use. The main problems are the high cost and contamination of the environment [6,7,8,9]. There is need to continue efforts to develop nitrification inhibitors that are inexpensive, readily available locally and effective at reasonable rates of application.

The effectiveness of zeolite in minimizing NO3-leaching from soil and optimizing crops’ growth and yield under different fertilizing conditions was shown in a number of studies [15,16].

The results of our laboratory and field experiments confirmed the good potential of the mixture of the natural siliceous minerals (analcite and tripoli) to reduce nitrification processes and NO₃-leaching from different types of agricultural soils under sowings of winter wheat and corn. In our study, application of a mixture of the natural siliceous minerals (analcite and tripoli) was noticeably more effective in altering soil inorganic N content, composition of microbial community and nitrogen metabolism in crops, as compared to nitrapyrin. The duration of the observed effects for the siliceous mixture was more lasting as compared to nitrapiryn.

In particular, the mixture of tripoli and analcite more efficiently preserved nitrogen in the soil in comparison with the synthetic nitrification inhibitor (nitrapyrin). In addition, the use of the mixture of tripoli and analcite provided a longer preservation of nitrogen in the soil. At the same time, it was found that a decrease in pH and quantitative parameters of humus of soil substrates stimulated processes of nitrification in treatments with nitrapyrin more essentially in comparison with treatments with siliceous minerals.

The results of microbiological analysis are in good agreement with the data of agrochemical evaluation of soil. The application of a siliceous mixture caused a more prolonged decrease in the number of nitrifiers and denitrifiers, as well as an increase in the number of ammonifiers in the soil compared with nitrapyrin.

In addition, the siliceous mixture more effectively inhibited the development of microorganisms that produce phytotoxic allelochemicals. In particular, this applies to bacteria, micromycetes and actinomycetes. It is known that long-term cultivation of wheat and corn causes changes in the microbiota composition of the rhizosphere soil, which significantly affects the properties of the soil. In particular, the development of beneficial microorganisms that produce vitamins, enzymes and organic acids is inhibited, while microbiota producing phytotoxic allelochemicals prevail. All of the above affect the yield of these crops [25]. Application of a siliceous mixture contributed to improving the composition of the microbiocenosis and allelopathic regime of the rhizosphere soil.

Similar results were obtained by Ellanska et al. [26]. The authors studied the effect of analcite applied at a rate of 50 kg·ha⁻¹ on the soil microbial community under sugar beet plantations and showed that growth of soil streptomycetes was almost 2-fold, as much as in the rhizosphere soil without analcite amendment. This led to the increased activity of enzymes involved in transformation of nitrogen and carbon compounds, as well as increased titer of microorganisms assimilating inorganic nitrogen. The increase of the mineralization index up to 2.53 (compared to 0.81 in control) indicated the enhancement in soil mobilization processes and associated mineralization of organic compounds. Abundance of Azotobacter chroococcum was 100% in both cases. However, application of analcite stimulated their faster development as compared to the control [26]. In another study, conducted by Mali and Aery [14], exogenous silicates promoted nodule formation in legume plants and hence promotion of N₂ fixation. The authors reported an increase in absorption of N and Ca for cowpea and wheat fertilized with increasing doses of sodium metasilicate as well as an improvement in nodulation and N₂ fixation in cowpea.

In our study, the tested nitrification inhibitors demonstrated different effects on nitrogen metabolism in winter wheat and corn leaves. In particular, nitrapyrin application decreased aspartic acid content in the test plants’ leaves, indicating impaired nitrogen metabolism caused by suppression of amination and reamination reactions. In turn, with unsatisfactory nitrogen supply, a significant part of inorganic phosphorus accumulates in the vacuoles, resulting in a decrease in its entry into the cytoplasm and chloroplasts. Disorder of phosphate metabolism is also indicated by an increase in the content of arginine and lysine and a decrease in the content of amino acids of the aromatic series [27]. In particular, the synthesis of phenylalanine, histidine and tyrosine, which is associated with the formation of the benzene ring and the starting material, which are phosphorylated sugar compounds, decreases not only due to inhibition of photosynthetic carbon sequestration, but also as a result of disorder of phosphate metabolism and respiration processes. On the other hand, stimulation of biosynthesis of glutamic acid by the analcite-tripoli mixture indicates the activation of photosynthesis in the test plants.

The analysis of the pigment complex of the test plants allowed for assessing the degree of variability and stability of their photosynthetic apparatus under the condition of application of nitrification inhibitors, as well as factors promoting the intensity of the photosynthetic process. In particular, it was shown that the content of photosynthetic pigments in the leaves of winter wheat and corn plants significantly depended on the type of nitrification inhibitor used. Application of the siliceous mixture contributed to a significant increase in the content of chlorophyll a and b, as well as carotenoids in the leaves of the tested crops. This testifies to a better supply of nitrogen to plants and activation of photosynthetic processes. The increase in the content of chlorophyll b and carotenoids indicates an increase in plant resistance to environmental stressors. At the same time, application of nitrapyrin reduced the biosynthesis of chlorophyll b and carotenoids and showed a slight increase in the content of chlorophyll a in the leaves. These biochemical changes were associated with higher grain yield and quality.

Thus, the study of general patterns of plant metabolism in the laboratory and field experiments allowed revealing the mechanisms of adaptation of plants to nitrogen fertilization, to develop optimal techniques for application of nitrification inhibitors and to implement environmentally friendly natural siliceous minerals into agricultural production for regulated nitrogen up-take by plants.

The use of nitrogen-containing fertilizers with nitrification inhibitors in agriculture provides a number of advantages in agronomic and economic terms. Considering this, of particular importance for the environment are siliceous minerals, which are characterized by high biological activity and persistence of action, harmless to both soil microbiota and higher plants. Their production is simple and economical as they are readily available in many regions of the worlds and have beneficial effects on components of agroecosystem (i.e., soil, microbiota, water balance, etc.).

5. Conclusions

The comparative study on the effects of nitrapyrin and the mixture of natural siliceous minerals (analcite + tripoli) on nitrification processes in different types of agricultural soils under wheat and corn sowings under high nitrogen fertilization conditions clearly indicated higher effectiveness of the siliceous mixture as compared to nitrapyrin. In particular, the analcite + tripoli mixture had a more profound effect in slowing down nitrification processes for a prolonged period of time and strongly inhibited the number of nitrifiers, denitrifiers and micromycetes producing phytotoxic allelochemicals compared with nitrapyrin. Crops treated with siliceous minerals had higher photosynthetic pigment content and higher glutamic acid concentration, which indicated the intensification of photosynthetic processes. Nitrapyrin reduced the concentration of chlorophyll b and carotenoids, but slightly increased the content of chlorophyll a in the leaves of wheat and corn. The content of aspartate and some aromatic amino acids decreased, while arginine and lysine increased. Such metabolic changes suggested disintegration of nitrogen and phosphate metabolism in the studied crops. Thus, the use of siliceous minerals is more advantageous than nitrapyrin in terms of their effectiveness, persistence in various types of soils, beneficial effect on soil microbiota, crop’s functional state and productivity.