Soil bacteria and fungi play an integral part and are irreplaceable within the “soil-microorganism-plant” system (Curl and Truelove, 1986; Hamilton and Frank, 2001; Herman et al., 2006; Landi et al., 2006; Bulgarelli et al., 2013; Zhalnina et al., 2018; Berg et al., 2020). An optimal set of rhizospheric microorganisms provides plants with the proper mineral nutrition, protection from pathogens, and improved yield potential of the crops (Watt et al., 2006; Marschner, 2012; Fasusi et al., 2021). This indicates that crop-growing technologies should foresee the optimization of microbiological processes in the soil, including the adaptation of the composition of groups of microorganisms in the root spheres of plants. It is especially relevant for modern agriculture, which is significantly affected by the anthropogenic load on the soil with the subsequent negative impact on the soil biota.

Pre-sowing seed inoculation with the selected strains of agronomically-valuable microorganisms named as Plant Growth-Promoting Rhizobacteria (PGPR) (Kloepper and Schroth, 1978; Bashan and Holguin, 1998), has become a significant technological mean used for the optimization of the processes of formation of groups of soil microorganisms and improvement of the agricultural crops' technologies. Today many countries and companies are producing microbial preparations based on nitrogen-fixing, phosphate-mobilizing, growth-stimulating microorganisms and antagonists of phytopathogens. There is no doubt about their positive influence on the growth and development of crops, possessing either direct or indirect influence on their productivity. The effect of PGPR is largely associated with the processes of molecular nitrogen fixation, solubilization of phosphorus compounds, production of siderophores, physiologically active substances, including plant hormones, enhancement of nutrient uptake by plants, improvement of the general plant health by the synthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which contributes to the crop tolerance to drought, soil salinity and heavy metal contamination (Bashan and Holguin, 2002; Volkogon et al., 2006; Lugtenberg and Kamilova, 2009; Babalola, 2010; Martínez-Viveros et al., 2010; O'Callaghan, 2016; Di Benedetto et al., 2017; Olanrewaju et al., 2017; Di Salvo et al., 2018; Jeyanthi and Kanimozhi, 2018; Khanna et al., 2019; Ilyas et al., 2020; Trivedi et al., 2020; Santoyo et al., 2021; Yasmin et al., 2021, 2022; Adedayo et al., 2022; Figueiredo et al., 2022; Volpiano et al., 2022). However, most studies of the PGPR do not question their effect on soil fertility in terms of the formation of nutrient reserves, as it is a priori believed that inoculation contributes to the improvement of soil fertility. There are practically no reports that could characterize the transformation of biogenic nutrients in the soil profile during the cultivation of bacterized plants.

Lysimeters provide a unique opportunity to study these issues, however, they are not often used due to the limitations of the relevant installations. The results obtained in lysimeters can be highly indicative and informative when determining the influence of various factors on the character of the vertical migration of compounds of biogenic elements.

Using the lysimeters we studied the effect of PGPR during the cultivation of various agricultural crops, namely spring barley, maize, and winter rye on their productivity and extent of the losses of biogenic nutrient compounds, namely nitrogen (NO₃/NH₄), phosphorous (P₂O₅), potassium (K₂O), calcium (CaO) and magnesium (MgO) as well as water-soluble organic matter losses with the water drainage down the soil profile.

The research was conducted in the stationary lysimeter installed at the Institute of Agricultural Microbiology and Agro-Industrial Production of the National Academy of Agrarian Sciences of Ukraine (Chernihiv). The lysimeter site is located in the zone of the washing water regime (Northern Ukraine, Polissya climate zone) and has 48 sections-lysimeters, placed in two parallel rows of 24 lysimeters each. Receiver vessels are installed underneath the lysimeters to collect infiltrate. Lysimeters are concrete, bulk type in design and simulate the profile of sod-podzolic sandy soil [organic matter content−1.1%; pH_salt−5.0; hydrolytic soil acidity−2.5 mg-eq./100 g; easily hydrolyzed nitrogen-−45 mg/kg of soil; phosphorous (P₂O₅) content−170.0 mg/kg of soil, potassium (K₂O)−62.0 mg/kg of soil], starting from the mother rock and considering the sizes of genetic horizons (see Figure 1). The soil was tested for the depth of the arable layer of 0–23 cm, using the standard soil analysis methods (Fomin and Fomin, 2000). The planting area of each lysimeter is 3.8 m², the soil depth is 155 cm, the soil weight is 10.5 tons/lysimeter.

Significant losses of mineral nitrogen under the spring barley cultivation on sod-podzolic soil in washing water regime conditions were observed in all variants, including control. The application of mineral fertilizers caused additional leaching of nitrate nitrogen. However, the use of A. brasilense 410 for the pre-sowing treatment of barley seeds reduced nutrients leaching beyond the root zone of the soil by 21%−33%, subject to the fertilization norms. Seed inoculation led to the reduction of potassium washing down the soil profile by 11%−41%. No significant difference in phosphorous (P₂O₅) loss reduction was observed, except for the variants with the highest dose of mineral fertilizers. The substantial drop in leaching of calcium (by 24%−34%) and magnesium (by 25%−50%) compounds was observed under the use of A. brasilense 410 on various fertilizers backgrounds. Losses of water-soluble organic matter also decreased by 25%−43% (Table 1).

The yield of spring barley increased with the introduction of mineral fertilizers. Pre-sowing inoculation promoted a significant increase in the productivity of the crop grown without fertilizers and with the mineral fertilizers applied at N₆₀P₆₀K₆₀ rate (Table 2). No significant response in crop yield was obtained from inoculation under the increase of fertilizers norms to N₁₂₀P₁₂₀K₁₂₀.

Inoculation of maize seeds with the phosphate-mobilizing bacterium P. polymyxa KB had a similar effect: depending on the agricultural background, the reduction of nitrate loss compared to controls was 8%−25%, CaO-−12%−39%, MgO-−8%−18% (Table 3).

Noteworthy is the lack of effect of maize seeds inoculation in the test plots with cattle manure and the significant efficiency of inoculation in the variants with green manure (lupine).

The highest losses of water-soluble organic matter were observed in the test plots with mineral fertilizers and cattle manure (Table 3). The use of the intermediate green manure crop (narrow-leaved lupine) reduced the losses of water-soluble organic matter to values lower than the ones observed in the control (variant without fertilizers). Moreover, the combination of green manure with mineral fertilizers in the N₉₀P₉₀K₉₀ norm also significantly limited the leaching of water-soluble organic compounds. The lowest losses were observed in the plots with seeds inoculation with P. polymyxa KB and the use of mineral fertilizers. The combination of factors “green manure + N₉₀P₉₀K₉₀ + P. polymyxa KB” was shown to be the most efficient, ensuring the reduction of water-soluble organic matter leaching lower than the absolute control values.

Maize grown under the use of green manure and mineral fertilizers background had the highest grain productivity both in variants with and without seeds treatment with P. polymyxa KB, while seeds inoculation with P. polymyxa KB had no significant effect on maize yield under the crop cultivation on cattle manure background (Table 4).

With the increasing doses of mineral fertilizers the productivity of winter rye had also increased up to the application of the N₆₀P₆₀K₆₀ rate. However, further increase of fertilizers norms (N₉₀P₉₀K₉₀) had shown less return in yield per applied fertilizer norms (Table 5).

Pre-sowing treatment of winter rye seeds with A. brasilense 18-2 increased winter rye grain productivity in all test plots, including control without fertilizers (13.5% yield increase). Although the seeds treatment with A. brasilense 18-2 resulted in a reliable yield increase the increment was the lowest across the experiment (Table 5).

Improvement of crop yield in variants with seeds inoculation with A. brasilense 18-2 is supported by the analysis of the lysimeter drainage waters showing the reduction of nutrients leaching from the soil profile (Table 6). Seeds treatment reduced the losses of all studied compounds, as well as water-soluble organic matter.

Modern approaches to agriculture foresee the extensive use of fertilizers to provide plants with the necessary nutrients. However, the excessive use of fertilizers can cause significant environmental impacts, as certain nutrients can be leached from the soil with downstream water runoff caused by rain or irrigation, resulting in eutrophication and water pollution (Adesemoye and Kloepper, 2009; Yu et al., 2019). These threats designate the need to develop measures for effective management of crop nutrition systems, taking into account environmental protection. A promising approach aimed at increasing the efficiency of fertilizer use and environmental protection is the use of PGPR for the inoculation of agricultural crops.

All studied crops grown on a mineral fertilization background following pre-sowing seeds inoculation with PGPR had a similar pattern effect preventing nutrients from leaching down the soil profile. Statistically significant reduction of nutrient losses were observed in most of the studied plots. Variants, shown no reliable changes, still had a clear tendency to decrease nutrients leaching with the drainage waters compared to the corresponding controls. These findings confirm the significant influence of PGPR on the degree of nutrients assimilation by plants, including those from fertilizers, described previously in the literature (Lin et al., 1983; Okon et al., 1998; Zaidi and Khan, 2005; Abbasi et al., 2011; Sharma et al., 2011; Pii et al., 2015; Backer et al., 2018). Higher assimilation levels are accompanied by increased removal of biogenic elements with the crop, resulting in a reduction of nutrients losses from the soil. And although the mechanisms of influence of the studied PGPR on the production process of agricultural crops may be different, the consequences of the functioning of artificially created bacterial-plant associations were similar.

Part of the nutrients is retained in the crops' root system, which in turn has increased as a result of seed inoculation. It is known that physiologically active substances produced by inoculants contribute to the optimization of rhizogenesis (Jacoud et al., 1998; Dobbelaere et al., 2001; El Zemrany et al., 2006). The bacteria used in our research, in addition to the ability to fix nitrogen and mobilize phosphate, are also active producers of auxins, cytokinins and gibberellins, which promote changes in the architecture of the root system and its scopes. Accordingly, the enlarged root system can temporarily retain significant amounts of nutrients in organic form. After the root residue mineralization, these nutrients can benefit the following crops in the crop rotation or be used at de novo synthesis of soil organic matter.

The reduction of calcium and magnesium compounds' leaching down the root zone of bacterized plants is another essential characteristic of the mechanism of the effect of biological preparations on both the productivity of agricultural crops and soil fertility. With calcium playing an important role in the processes of soil formation (Mazur, 2008) and metabolic regulation in both eukaryotes and prokaryotes (Smith, 1995), the retention of calcium compounds in soil from leaching is a positive indicator. Magnesium takes an active part in the chloroplasts' biogenesis and is part of certain enzymes in the plant organism (Kwon et al., 2019; Alejandro et al., 2020). Magnesium deficiency in plants results in leaf chlorosis, while severe cases result in stunted growth (Bang et al., 2021). A decrease in magnesium compounds' losses under the use of PGPR for seed inoculation is an unambiguously confident consequence. However, the observed significant reduction of calcium and magnesium compounds losses as a result of seed inoculation cannot be explained only by the increase in the level of their assimilation by bacterized plants and requires additional research.

The decrease in the leaching of water-soluble organic matter in test plots with pre-sowing seed inoculation was somewhat unexpected. Subject to the fertilization background, barley seed inoculation with A. brasilense 410 contributes to the reduction of the water-soluble organic matter losses by 25%−43% compared to the corresponding variants without inoculation. Maize growing in control variants (without fertilizers) and on a mineral background, N₉₀P₉₀O₉₀ followed by sed inoculation with P. polymyxa KB ensured a drop of water-soluble organic matter leaching by 35% and 30%, respectively. Slightly lower values were observed for the winter rye crop, with seeds inoculated with A. brasilense 18-2.

It can be assumed that part of water-soluble organic matter is mineralized and assimilated by plants due to the bacterial hydrolytic enzyme activity. Mineralization processes can be enhanced in the rhizosphere of plants due to the phytostimulatory effect of PGPR and an increased amount of readily available carbon compounds, which contributes to the growth of the number and activity of heterotrophic microorganisms (Oger et al., 2004; Blagodatskaya et al., 2010). At the same time, the production of hydrolytic enzymes can be conditioned not only by the development of the bacteria introduced into the rhizosphere but also by the indirect effect of inoculation on the development of the resident microbiota in the rhizosphere (Hu et al., 2021). Recent studies using molecular biological methods (Ramakrishnan et al., 2017; Jiménez et al., 2020) confirm the effect of PGPR inoculation on the development of resident microbiota in the rhizosphere of plants.

The ability of plants to absorb both mineral and organic substances can also be affected by the reduction in the intensity of water-soluble organic matter leaching. Information about the assimilation of organic compounds with low molecular weight by plants is not new (Hutchinson and Miller, 1912), however, it was questioned until recently when the importance of this process for crop yield formation has been shown (Farzadfar et al., 2021). The size of the root system is considered the most promising mechanism for the absorption of organic substances by plants (Farzadfar et al., 2021), so the ability of PGPR to influence the rhizogenesis of plants can stimulate the assimilation of not only mineral compounds, but also organic compounds.

Altogether the prevention of the nutrient and water-soluble organic matter losses in the rhizosphere of crops inoculated with PGPR and the potential involvement of increased plant biomass in the processes of “synthesis-mineralization” indicate the importance of the PGPR on soil fertility.

The results obtained relate in the first place to the conditions of the washing water regime, typical for the soil-climatic zone of Polissia of Ukraine. However, it may also be important to take into account these PGPR effects on the processes of nutrient migration in the soil under irrigation.

Fertilization had a significant influence on the PGPR effect on crop yield. Relatively low doses of mineral fertilizers increased the efficiency of seed inoculation. In our opinion, low rates of fertilizers can lie within the physiological optimum both for the development of plants and bacteria (Volkogon, 2013). It is under such conditions that biological and mineral resources can be synergistic in providing plants with nutrients and biological functions congenial to PGPR.

High doses of mineral fertilizers applied to spring barley and winter rye crops had significantly reduced the effect of inoculation on crop productivity, indicating their redundancy within the course of biological processes in the “soil-plant-bacteria” system. The obtained results confirm the conclusions of Ozturk et al. (2003), who conducted research with wheat and barley. A similar situation is also described for other artificially created plant-bacterial associations. Thus, Shaharooma et al. (2008) reported an increased nitrogen use efficiency by wheat crops in response to Pseudomonas fluorescens inoculation, but to the different extents subject to mineral fertilizer rates, which provided 115, 52, 26, and 27% increases in yield compared to the uninoculated control with nitrogen, phosphorus, and potassium applied at 25, 50, 75, and 100% of the recommended rates, respectively.

The results of our research support the conclusion on seed inoculation as an important strategy for sustainable soil management and lessening environmental problems due to the reduction of the chemical fertilizers load (Santa et al., 2008; Adesemoye et al., 2009; Hungria et al., 2010). However, in contrast to publications promoting PGPR as the direct replacement for mineral fertilizers, in our assessments of the prospects of inoculation, we share the views focused on the effective combination of mineral and biological factors for agricultural crops fertilization.

Noteworthy is the lack of effect of PGPR P. polymyxa KB used for pre-sowing seed inoculation of maize against a manure background. It can be explained by non-specific soil bacterization with a vast amount of microorganisms introduced into the soil with cattle manure, which largely neutralizes the effect of pre-sowing seed inoculation due to the creation of a competitive environment. A similar effect was also observed in our studies of the PGPR effects (Volkogon et al., 2021). Recent research conducted in our laboratory on the peculiarities of the development of genetically marked PGPR strains in the root zone of agricultural crops grown on different mineral fertilization backgrounds and application of cattle manure confirms these findings. After manure application, the introduction of selected agronomically valuable bacteria into the root zones of plants was ineffective (Sydorenko et al., 2020).

At the same time, significant effectiveness of seed inoculation is observed when growing crops on green manure background. Unlike cattle manure, the sideral mass is less contaminated with microorganisms, which do not interfere with the formation of effective interaction of introduced bacteria and plants. Besides the mineralization of green manure biomass can serve as an additional source of nutrients for P. polymyxa KB.

Lupine, as green manure, promotes nutrient loss reduction when used in combination with mineral fertilizers. Under these conditions, P. polymyxa KB shows significant effectiveness when applied as a seed treatment. Considering the high grain productivity achieved in test plots for this combination and the preservation of soil resources, the combination of seed inoculation with P. polymyxa KB, mineral fertilizers and green manure is considered to be the most promising for maize cultivation on sod-podzolic soils.

PGPR promote the reduction of biogenic elements and water-soluble organic matter losses with the drainage water flow down the soil profile upon the cultivation of spring barley, maize, and winter rye on mineral and organic fertilization backgrounds. However, the application of cattle manure decreases the observed effect. The observed peculiarities are particularly useful for crop cultivation in zones with a washing hydrological regime, as well as under irrigation.

As shown by the results seed inoculation with PGPR stimulates nutrient assimilation, thus explaining the mechanism of the positive effect of pre-sowing seed bacterization on crop productivity and soil fertility.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

VV substantiated the research idea, carried out the microbiological part of the work, analyzed the experimental data, and worked on the manuscript. LP worked with the lysimeters. MV assisted with data interpretation and manuscript preparation. All authors read and approved the final manuscript.