Effects of Apatite Concentrate in Combination with Phosphate-Solubilizing Microorganisms on the Yield of Ryegrass Cultivar Izorskiy

Abstract

Soil microorganisms play a vital role in increasing the availability of phosphorus (P) for plants through mineralization of organic P and solubilization of precipitated P compounds. In this two-year study, we analyzed several P-solubilizing microorganisms (PSMs) of the genus Bacillus and their consortiums for the ability to release soluble P from apatite concentrates of various grinding degrees using ryegrass (Lolium multiflorum Lam.) as a model plant. The effects were accessed by analyzing plant growth and nutrient assimilation. The greatest effect on root system development and plant biomass accumulation (dry weight) was observed for the apatite concentrate of standard grinding in combination with Bacillus megaterium BI14 and Bacillus subtilis BI2 and Bacillus velezensis BS89 strains. Although the introduction of apatite concentrates led to an increase in the content of total strontium in soil, the levels of strontium did not exceed the maximum allowable concentration, and the accumulation of mobile strontium by plants was unchanged; importantly, the use of tested PSMs led to a decrease in the strontium content in the green biomass of ryegrass. Our results indicate that biologized apatite concentrates in combination with PSMs represent promising fertilizers that can provide a source of soluble P to be readily assimilated by plants.

1. Introduction

Most compounds of macro- and microelements in soil are not available to plants as they have poor water solubility and are not absorbed through the roots. For example, P and K are called plant macronutrients, but in many soil types they are found in hard-to-reach phosphorus and potassium minerals: iron and aluminum phosphates, apatite, phosphorite (P); aluminosilicate minerals—feldspars and micas (K). The majority of micronutrients are in oxidized form, and many of them are part of water-insoluble compounds: calcium carbonate or sulfate (Ca), Mg carbonates (Mg), aluminosilicate (Al), ferrum oxide (Fe), and so on. In total, soil contains about 0.05% of phosphorus (P), but only 0.1% of it could be absorbed by plants [1]. Since the availability of soil P is critical for agriculture, affecting the yield, net economic return, and quality of produce, considerable efforts are devoted to enhancing P absorption by crops.

In acidic soils, P is fixed in the form of insoluble iron and aluminum phosphates, whereas in alkaline soils it is found mainly as calcium phosphate. Therefore, at each cropping cycle, plants should constantly have P supplementation through chemical fertilization. Phosphate fertilizers are traditionally produced by high-temperature chemical treatment of phosphate rocks with sulfuric acid, a process that has potential risks to the environment and is not economically viable due to high energy consumption [2]. Apatite, a natural phosphate rock, is the main source of phosphorus, and about 85% of it is consumed by the phosphate fertilizer industry. Morocco has the largest reserves of phosphate raw, and the large ones have also been created in China (45% of total phosphate production, 2018), the USA, and Russia [2].

However, in accordance with “The International Code of Conduct for the Sustainable Use and Management of Fertilizers” [3], “perturbations to the biogeochemical flow of phosphorus due to its production for agricultural use has exceeded safe margins for human activities.” In the same document, FAO sets restrictions on the use of phosphate fertilizers containing heavy metals, in particular cadmium, while less than a third of the world’s proven reserves of phosphate rocks are phosphates with a content of less than 20 mg/kg. It should also be noted that P is a non-renewable resource, and its supplies are limited, creating concerns about its future sources.

However, only a small amount of additional P could be used, while most of it is deposited in soil. Therefore, the widespread use of P-based chemical fertilizers has a negative impact on soil fertility, plant development, and agricultural product quality, as well as on water resources, causing their eutrophication. The fertilization-associated environmental problems draw attention to the search of alternative approaches to meet the needs of plants for available P [4,5,6].

It is suggested that if P fixed in soil becomes bioavailable, additional P will not be required for almost 100 years [7]. Soil microorganisms play a vital role in increasing P availability for plants through mineralization of organic P and solubilization of precipitated P [7,8], and the positive effect of soil phosphate-solubilizing microorganisms (PSMs) as biofertilizers has been known since the 1950s. It has been shown that soil enrichment with PSMs can increase plant yield through solubilization of fixed and applied P [9] because these microorganisms are able to include P compounds in the biological cycle, thus mediating the availability of P to plants through trophic chains.

In this context, PSMs have emerged as the most promising approach to reduce the use of chemical fertilizers, improve agricultural sustainability, lower costs, and restore soil fertility and quality [4,5,6], and the development of plant-specific consortiums of phosphate solubilizers is in progress. It has been shown that bacteria of the genera Bacillus, Pseudomonas, Rhizobium, Aspergillus, and Penicillium could be the most effective PSMs for increasing the bioavailability of soil P [10] As P is absorbed by plant roots in the form of orthophosphate ions (H₂PO₄⁻ or HPO₄^2–), the objective is to increase the concentration of these ions in soil, which is normally <10 µM [11].

Previous studies indicate that PSMs are able to dissolve insoluble phosphates by producing organic acids [10,12,13]. The efficiency of solubilization depends on the strength and nature of the acid; thus, di- and tribasic carboxylic acids are more efficient than monobasic and aromatic acids and aliphatic acids—than phenolic, citric, and fumaric acids. Among the organic acids that solubilize phosphates are primarily citric, lactic, glycolic, 2-ketogluconic, oxalic, glyconic, acetic, malic, fumaric, succinic, tartaric, malonic, glutaric, propionic, butyric, glyoxalic, and adipic acids [10]. As were shown earlier, the rhizosphere PSM strains used here produced a number of organic acids: indolylacetic (Bacillus megaterium BI14) and indolyl-3 aldehyde (Bacillus subtilus BI2) and Bacillus velezensis [13].

Testing new fertilizers that combine minerals and microbial strains represents both theoretical and practical interests. The aim of this study is to evaluate the effectiveness of biologically modified apatite concentrate for root nutrition of multi-cut ryegrass (Lolium multiflorum Lam.) using PSM strains of Bacillus subtilis and Bacillus megaterium.

2. Materials and Methods

2.1. Experiment at the Artificial Climate Facility

The experiment was carried out at the artificial climate experimental facility (EUIK) (Federal Research Center of Biotechnology RAS, Moscow, Russia [55°45′07″ N, 37°36′56″ E, 144 m above sea level]) during a two-year period (2020–2021).

EUIK located in an open area of 144 m² represented a glass structure with a gable roof, consisting of a corridor and five isolated vegetative cabins with glazed partitions. In each cabin, the temperature regime (+10 °C to +35 °C), illumination (up to 100 klx), change of day and night, as well as plant watering and fertilization were automatically regulated according to the specified time and quantitative parameters. Cabins were heated through a central heating system (water temperature up to +100 °C) and cooled with cold water (temperature +4 °C) from a special circular cooler. EUIK was power supplied through AC electric power supply (380 V at 50 Hz; power consumption up to 193 kW) and equipped with 400 W DNAT lamps and an external weather station constantly recording air temperature, wind strength, illumination, and precipitation (rain or snow).

The experiment was carried out under controlled conditions in the cabin of EUIK with a round-the-clock temperature of +21 °C. Each sample was tested five times.

Soil Characteristics

The soil was soddy-podzolic sandy loam with a low content of mobile P. Every pot initially contained 5.5 kg of absolutely dry soil (ADS) to which 1.308 g of KCl and 1.768 g of carbamide were added. Liquid nitrogen and potassium fertilizers were applied at the rates of 0.75 g N and 0.75 g K₂O per 5 kg ADS, respectively.

2.2. Microorganism Cultures

Winter wheat grain endophyte B. subtilis strain BI2, grape endophyte B. megaterium strain BI14, B. velezensis strain BS89 (previously identified as B. subtilis Ch-13 [13,14]) isolated from wheat roots, and their consortiums were provided by PJSC PHOSAGRO, Russia.

B. subtilis BI2, a gram-positive sporulating bacterium with phosphate-mobilizing, cellulolytic, proteolytic, and amylolytic activity, increased the yield of spring wheat and barley grain by 5–9% (2.2–3.8 q/ha) and potato tubers by 25–44%.

B. megaterium BI14 showed phosphate-mobilizing, cellulolytic, and amylolytic activities and produced indolylacetic acid.

Inoculation of various crops (potatoes, spring and winter wheat, barley, sunflower, cabbage, carrots, and strawberry) with B. velezensis BS89 increased the yield from 12 to 40% [15,16].

Strain identification was carried out at the All-Russian Research Institute of Agricultural Microbiology.

2.3. Biologized Apatite Concentrates

Coarse apatite concentrates with mass fractions of P₂O₅ and moisture of 39.0% and 1.0 ± 0.5%, respectively, were provided by PJSC PHOSAGRO. The compositions are presented in

Table 1. Composition of coarse apatite (%).

Compound		Content (%)
TiO2		0.37
Fe2O3		0.37
FeO		1.89
Tr2O3		0.95
CaO		0.37
SrO		1.89
MgO		0.95
MnO		0.37
Na2O		1.89
K2O		0.95
Cd		3.1 × 10−6
Pb		4.1× 10−4
F		2.99
SiO2	total	1.89
	residue	0.95
Al2O3	total	1.89
	acid-soluble	0.95

.

Two apatite forms were used in the experiments:

-

Coarse concentrate ground and sifted under standard conditions, “residue on a sieve with mesh No. 016K, no more than 20% (≤20% of particles larger than 0.16 mm)” and used for the production of phosphate fertilizers;

-

Apatite crushed in a disintegrator to a particle size of about 20 μm, which should allow PSMs to more effectively decompose hard-to-reach P compounds from apatite.

Biologized apatite was obtained by mixing sterile diatomaceous earth with bacterial cultures and treating the apatite concentrate with the resulting bacterial suspension based on the titer required for the experiment. As a result, 12 × 10⁶ CFU/g of each strain (B. subtilis BI2, B. velezensis BS89, and B. megaterium BI14) were added to crushed and non-crushed apatite samples.

2.4. Characteristics of Ryegrass Variety Izorsky

Ryegrass (Lolium multiflorum Lam.) variety Izorsky (Russian Potato Research Centre, Moscow, Russia), which is frost- and disease-resistant, is used as a raw and green conveyor for lawn repair. The period from germination to the first cut is 51 days, that until seed ripening is 86 days, and that from the first to the second cut is 31 days, resulting in 2–3 cuts in total, which is important for our study. Ryegrass grows well on different soils, from sandy soil to drained peatland.

2.5. Analytical Methods

Plant weight was determined using a KERN EW laboratory balance (Balingen, Germany).

The following instrumentation was used for soil analysis: pH-meter (pH-150MI; Izmeritelnaya Tekhnika LLC, Moscow, Russia), non-automatic scales (HR-250AZG; AND, Tokio, Japan); electric resistance furnace chamber laboratory (SNOL 10/11; VNIIETO, Moscow, Russia); atomic absorption spectrophotometer (model A-7000; Shimadzu Europa GmbH, Tokio, Japan); inductively coupled plasma emission spectrometer (iCAP 6300, model iCAP 6300 DuoThermo Scientific, Waltham, MA, USA), and spectrophotometer PE-5400 VI (OOO EKROSKHIM, Moscow, Russia).

Soil pH was measured potentiometrically (GOST R 53381-2009), organic matter (humus) was determined gravimetrically (GOST 23740), and mobile P in P₂O₅ was measured photometrically (GOST R 54650). Mobile and total strontium were measured according to PND F 16.1:2.3:3.11-98 (ISP-AE). Mass fractions of calcium, potassium, strontium, phosphorus, and rare earth elements in solid samples (soils, rocks, etc.) were measured by inductively coupled plasma spectrometry (PND F 16.1:2.3:3.11-98).

Water-soluble fluorine was determined according to PND F 16.1.8-98. Mass concentrations of nitrite, nitrate, chloride, fluoride, sulfate, and phosphate ions in soil samples (water-soluble forms) were measured by ion chromatography.

Total nitrogen in soil was measured according to the national standard of the Russian Federation (GOST R 58596-2019 “Soils: Methods for determining total nitrogen”).

2.6. P Utilization Efficiency for Ryegrass Plants

The coefficient of P use from fertilizers by ryegrass plants was determined according to the following formula [17]:

PUE = (Ur − Uo/D) × 100%,

where PUE is P utilization efficiency, Ur is ryegrass yield (g), Uo is ryegrass yield in variants without P application (constant average), and D is the applied P dose (g per pot).

2.7. Experimental Scheme

We evaluated the effects of different degrees of grinding of biologized apatite concentrate and those of strains BI2, BI14, BS89 and their consortiums on the processes of phosphorite solubilization.

Samples of biologized apatite concentrates were thoroughly mixed with ADS at the rate of 2.1054 g per each 5.5 kg pot, and 0.5 kg ADS was taken from each pot for analysis of strontium content. Each variant was tested in five replicates; two control variants were used without the apatite concentrate: C-NK and C-0, which did or did not contain nitrogen and potassium fertilizers, respectively. The experimental scheme is shown in

Table 2. Experimental scheme.

Fertilizer Sample	Experimental Variant
	1	2	3	4	5
Soil analysis before the experiment	Base	Base	Base	Base	Base
Control without fertilizer	C-0	C-0	C-0	C-0	C-0
Control N + K	C-NK	C-NK	C-NK	C-NK	C-NK
Apatite concentrate of standard grinding − control	V.1.1	V.1.2	V.1.3	V.1.4	V.1.5
Apatite concentrate of standard grinding + strains BI2 and BS89 (6 × 106 CFU/g each)	V.2.1	V.2.2	V.2.3	V.2.4	V.2.5
Apatite concentrate of standard grinding + strains BI14 and BS89 (6 × 106 CFU/g each)	V.3.1	V.3.2	V.3.3	V.3.4	V.3.5
Apatite concentrate of standard grinding + strains BI2 and BI14 (6 × 106 CFU/g each)	V.4.1	V.4.2	V.4.3	V.4.4	V.4.5
Apatite concentrate of standard grinding + BI2, BI14, and BS89 (4 × 106 CFU/g each)	V.5.1	V.5.2	V.5.3	V.5.4	V.5.5
Apatite concentrate of fine grinding − control 2	V.6.1	V.6.2	V.6.3	V.6.4	V.6.5
Apatite concentrate of fine grinding + strains BI2 and BS89 (6 × 106 CFU/g each)	V.7.1	V.7.2	V.7.3	V.7.4	V.7.5
Apatite concentrate of fine grinding + strains BI14 and BS89 (6 × 106 CFU/g each)	V.8.1	V.8.2	V.8.3	V.8.4	V.8.5
Apatite concentrate of fine grinding + strains BI2 and BI14 (6 × 106 CFU/g each)	V.9.1	V.9.2	V.9.3	V.9.4	V.9.5
Apatite concentrate of fine grinding + strains BI2, BI14, and BS89 (4 × 106 CFU/g each)	V.10.1	V.10.2	V.10.3	V.10.4	V.10.5

.

2.8. Analysis of Plant Growth

Thirty ryegrass seeds were planted in each pot on 13 August 2020 and 17 June 2021. In order to maintain the uniformity of the experiment, after germination, the plants were thinned out to 20 per pot. Irrigation was performed with distilled water up to 60% of the field soil moisture capacity established in a laboratory experiment. The first cuttings were carried out at the tube stage on 23 October 2020 and 19 August 2021, and the second mowings were performed on 24 November 2020 and 8 September 2021, respectively; the third mowing was performed on 28 September 2021.

Plants were cut at a height of 6–7 cm from the soil level, and the weights of the wet and absolutely dry biomasses were determined. The characteristics of the absolutely dry biomass of the above-ground parts are presented in the Supplementary Material (Table S1).

The underground parts of the plants were removed on 25 November 2020 and 8 September 2021, thoroughly washed from soil, and analyzed for the wet and absolutely dry weights.

2.9. Statistical Analysis

All experiments were carried out in five replicates. Initial data were analyzed and visualized using the Microsoft Excel 2019 (Microsoft Corporation, Redmond, WA, USA), and statistical analysis was performed with Analysis ToolPak (Microsoft Corporation, Redmond, WA, USA). Data were subjected to analysis of variance (one-way ANOVA). Results with significant F-value were compared at the 95% probability level by the least significance difference (LSD) test. The results were expressed as the mean ± standard error (SE) and are shown in the Supplementary Material (Table S1) with F-value, p-value, and LSD (p < 0.05; p < 0.01) means.

3. Results

3.1. Biomass Accumulation

The accumulation of the aboveground biomass increased with the addition of the test preparations in all variants and in both years relative to the control (Figure 1). In contrast, there was a sharp decrease in the biomass of the control sample by the third cut. It should be noted that variant V5, which was treated with the apatite concentrate of standard grinding and a combination of the three strains: BI2, BI14, and BS89 (4 × 10⁶ CFU/g each), did not show a decrease in the total biomass with the cuts, and its root mass accumulation was the highest among the variants in 2021 (Figure 2 and Figure 3).

3.2. The Effect of Apatite and PSMs on the Accumulation of Microelements in Ryegrass Biomass

3.2.1. Phosphorus

In 2020, the use of microorganisms in combination with mineral fertilizers did not produce any effects (p-value 0.216) on the total P accumulation in dry biomass (Figure 4a), and in 2021, the total P accumulation in green biomass was below the control in all experimental groups and cuts (Figure 4b). A similar pattern was observed in plant roots, where all experimental variants had P levels below the control; the lowest level, 1480 mg/kg, was observed in the variants treated with the combination of the apatite concentrate with strains BI2 and BS89 (V1, V2, V5, and V10). The greatest difference with the control in total P accumulation in dry plant biomass was observed for the variant with apatite concentrate of standard grinding in combination with the three Bacillus strains, BI2, BI14, and BS89 (Figure 4b–g).

3.2.2. Calcium

The use of PSMs with mineral fertilizers in 2020 did not affect calcium accumulation (p-value 0.343) in the dry plant biomass (Figure 5a). However, in 2021, calcium accumulation was observed both in the green biomass (Figure 5e) and underground parts (Figure 5c) of the variants treated with the finely ground apatite concentrate. The highest level (up to 1640 mg/kg) was detected when the finely ground apatite concentrate was used together with strains BI2 and BI14 (6 × 10⁶ CFU/g each). Analysis of the total biomass confirmed the positive effect on calcium accumulation in ryegrass as all the experimental variants had higher gross calcium levels than the control; among the variants, V9 had the highest calcium content in the total and aboveground biomass.

3.2.3. Strontium

The content of strontium in the biomass of cuttings correlated with that of calcium. In 2020, V6 had the maximal strontium accumulation in the biomass of the second cut and the underground part. Increased strontium content in the soil relative to the base soil was detected for V4; however, the difference was not statistically significant (p-value 0.235) for any variant (Figure 6a).

The experiments performed in 2021 confirmed that the introduction of the apatite concentrates led to an increase of the total strontium content in the underground parts of the treated plants relative to the control; the highest levels were observed in the variants treated with the PSMs (V1, V2, V4, and V7–9) (Figure 6f). The contents of mobile strontium in the variants did not show statistically significant differences from that in the base soil.

In contrast, the use of PSMs did not lead to an increase of the strontium content in the aboveground biomass (Figure 6g). In V1 treated only with the apatite concentrate of standard grinding, the highest strontium content was observed in the second and third cuts, whereas in V9 treated with the finely ground apatite concentrate in combination with strains BI2 and BI14 (6 × 10⁶ CFU/g each), the strontium content in the green biomass decreased to a minimum. Although MPC standards for soil strontium have not been established, 600 mg/kg is considered to be the upper limit of the normal. In this study, the gross strontium content in the biomass of all variants did not exceed 30 mg/kg, i.e., was significantly below the upper limit.

3.3. The Effecst of Apatite and PSMs on Soil-Related Parameters

3.3.1. Soil Acidity

The results showed that the introduced biologized apatite, PSMs, or their combinations did not significantly change soil pH (p-value 0.618 in 2020, 0.577 in 2021). The soil acidity index before and after the experiment was in the acceptable pH range of 6.75–6.85.

3.3.2. Organic Matter

Similarly, the treatments did not have a significant effect on the content of organic matter (humus) in the experimental soil samples compared to the basic soil (p-value 0.761 in 2020, 0.508 in 2021); the average content was 3.064–3.256%.

3.3.3. Nitrogen

Photometric analysis of total soil nitrogen did not reveal significant differences between the experimental variants and the base soil (p-value 0.599 in 2020, 0.195 in 2021); the average nitrogen content was 0.09–0.158%.

3.3.4. Potassium

ANOVA indicated that there were no significant differences in potassium content between the experimental variants and the base soil, indicating that the introduction of apatite and PSMs had no effect.

3.3.5. Fluorine

The average fluorine content in soils worldwide is 321 mg/kg; for most soils, it is 140–400 mg/kg [18]. In this study, the total fluorine content in the control was 261 mg/kg, which corresponds to the average content in podzols of medium-loamy soils around the world, and mobile fluorine content was 120 mg/kg. In the experimental variants, the fluorine content differed from that in the base soil: the total fluorine content was the highest in the control (C-NK) and V1–V3 soils, similar to the mobile fluorine content (Figure 7). These results may indicate the binding of fluoride ions to the apatite concentrates, especially to the finely ground one.

3.3.6. Phosphorus

In 2021, the total P content increased in the soil of all experimental variants compared to the control and base soil, indicating the effectiveness of the strains used (Figure 8). In 2021, the variants treated with finely ground apatite (V6–V10) had higher soil P content than those treated with coarse apatite (V1–V5); the maximum P level was detected in V10 treated with all three PSM strains.

3.3.7. Calcium

The results indicated that compared to the control, the soil calcium content was slightly lower in the majority of variants in 2020 (Figure 9a) and in all of them in 2021 (Figure 9b). Overall, in all variants where apatite concentrate was used, the level of soil calcium was reduced, which may indicate its better assimilation by the plants.

3.3.8. Strontium

The introduction of apatite concentrate led to an increase in the total strontium content in soil (Figure 10), suggesting that strontium was released during apatite decomposition. In 2021, the content of mobile strontium increased only in a number of variants compared with the base soil (Figure 10c); V7, V9, and V10 treated with the finely ground apatite concentrate and strain BI2 in combination with the other PSMs opposite had the lowest levels.

3.4. The Efficiency of P Utilization by Ryegrass

Figure 11 shows the calculated efficiency of P use by experimental plants. The maximum efficiency, 13.09 ± 1.84%, was observed in V5 treated with the apatite concentrate of standard grinding in combination with all three PSM strains. There was also an increase in PUE of 7.19–9.18% in all variants with finely ground apatite (V6–V10).

4. Discussion

The results of this two-year study indicate that the application of apatite concentrates in combination with PSMs improves storage characteristics of ryegrass biomass and the soil fertility status.

The greatest increase in the P removal from the biologized fertilizer and its conversion into a form available for plants was observed for V5 treated with the apatite concentrate of standard grinding in combination with strains BI2, BI14, and BS89 (4 × 10⁶ CFU/g each) and for V8 treated with the apatite concentrate of fine grinding in combination with strains BI14 and BS89 (6 × 10⁶ CFU/g each). This result is in accordance with the previous study [19] of the effects on ryegrass of co-inoculation Pseudomonas fluorescens (MW165744) with apatite, which represented the significant increase of total P uptake. It can be assumed that analogous results of these two experiments reflect the similarity of experimental methods for analyzing the growth of Lolium multiflorum in sterilized soil conditions. It should also be noted that P. fluorescens, similar to our strain combination, produces some organic acids (citric, malic, tartaric, and gluconic [10]) and could use the same mechanisms for increase mobilization of insoluble phosphorous compounds.

When studying the papers on PSMs effect on different plants, we found that the use of PSMs promotes the development of the extended root system and increases root mass, thus helping plants capture P from a larger area [7,20]. We compared our results with the effect of strain Bacillus velezensis BS89 on the productivity of two strawberry varieties (Fragaria × ananassa Duch.) [15], which demonstrated that BS89 produced a large amount of indole-3-acetic acid (IAA) (about 494.1 mg/mL). These data put forward an idea that in the strain consortiums, which we identified in our study, the high IAA-producing strain BS89 is also responsible for the improved root architecture and increased root formation of ryegrass.

Our study is also consistent with the general idea that PSB–root system interactions play a large role in increasing the availability of P in the rhizosphere. Recent results [21] provide an example of more colonization of wheat plants root zone by several PMB genera (Arthrobacter, Bacillus, Massilia, et cetera) in response to organic acids present in root exudates. Additionally, for high level growth-promoting bacteria Bacillus velezensis BS89 (in model experiments on barley of Nur variety), the ability to move from mineral fertilizer granules to plant roots and colonize them effectively was also previously reported [16]. On the basis of these data, we then propose that the strains consortiums identified here play a significant role for improved P availability through root–microbe interactions.

Previously, it was reported [21] that PSB–root system interactions depend on the crop and the stage of plant development; therefore, the critical period for P absorption does not coincide in different crops. For example, in wheat, maximum P intake is observed in the budding and heading phases, in flax—in the flowering and ripening phases, and in perennial grasses—at the start of development. In this study, we added the biologized apatite concentrates in the pre-sowing period, and although the root system in the initial phases of ryegrass growth is poorly developed, this fertilization ensured the consumption of readily available P in the seedling phase and created good conditions for biomass gain during the growth season. It is known that the results of P fertilization can be observed for a long time; for example, Kirpichnikov et al. (2007) [22] had reported the aftereffects of applying large amounts of phosphate fertilizers to heavy loamy, soddy-podzolic soil for 22 years.

As it is well known, the increase in the availability of soil P for plants significantly improves their nutritional status. P is a key component of nucleic acids that define protein synthesis; it also regulates enzymatic activity and signaling pathways through protein phosphorylation/dephosphorylation cycles.

To sum up, we have demonstrated that the accumulation of the aboveground biomass (according to dry weight) was the highest in V8 (finely ground apatite in combination with BI14 and BS89 strains), whereas that of the total biomass was maximal in V5, where it was 17% higher than in the control.

Ryegrass, similar to other crops, needs enough nutrients for growing without decreasing its regenerative capacity, density, and productivity [23]. As a rule, most mineral fertilizers are obtained in granular form. Our study showed that the degree of grinding of apatite has a positive effect on the regenerative capacity of ryegrass. Thus, our results indicate that finely ground apatite concentrates (V6–V10) increased the soil P content compared to the controls (C0 and C-NK) and to the coarse apatite (V1–V5). Additionally, V9 treated with the fine apatite concentrate in combination with strains BI2 and BI14 (6 × 10⁶ CFU/g each) showed a minimal decrease in the yield from cut to cut. At the same time, in V5, uniform accumulation of green biomass was observed in all three cuts without a noticeable decrease from cut to cut, whereas in all other variants, the yield of green mass decreased significantly. From the practical point of view, these sustainable ryegrass production examples are the main goal of feed production and of pasture regeneration.

Access of the efficiency of P utilization from fertilizers according to PUE increased for variants with the finely ground apatite (Figure 11), indicating its correlation with the apatite grinding degree. However, the best performance was observed for V5 (13.09%), which appeared to have optimal parameters in terms of the grinding degree and combination and amount of PSMs. Our results on PUE, which was calculated considering the direct effect of P on ryegrass, are consistent with those obtained in one- and two-year field experiments at Rothamsted (UK), where the average P recovery calculated by a different method was 5–10% [19,24].

It should also be noted that the use of apatite concentrates reduced potassium content in the soil of all variants, which may indicate better potassium assimilation by the plants.

Opposite, reasonable concern is the possibility of strontium extracting from apatite concentrates and its accumulation in ryegrass biomass and soil. The alkaline earth metals interactions from apatite are complex and based on their content in the phosphate rocks. Earlier, the field trials [25] were carried out to compare concentrations of Ba, Sr, and Ca in plants growing in apatite–biotite–carbonatite soil. The trials demonstrated that Ca and Sr can compete with each other, but Ca biochemical functions cannot be replaced by Sr. Our pot experiment investigation showed that the combined use of apatite concentrates with PSMs significantly improved calcium accumulation in the green mass as evidenced by higher calcium content in V3, V5, V6, V9, and V10 (Figure 9). However, although the introduction of apatite concentrates increased the total strontium content in the soil, Sr levels did not exceed the MPC, and the accumulation of mobile strontium by plants was not affected. The strontium content also increased in the underground parts of ryegrass, but the use of PSMs decreased it in the aboveground parts. Furthermore, the highest strontium content was observed in the second and third cuts of V1 grown without PSMs. These benefit effects, as we propose, are connected with the increased level of Ca in growth ryegrass that may inhibit Sr uptake [25].

5. Conclusions

In this two-year study, we investigated growth characteristics and soil parameters of ryegrass treated with apatite concentrates of various grinding degrees in combination with Bacillus PSMs, known for their ability to release P from its insoluble forms. The results showed that microbial inoculation was strongly necessary to increase P availability to plants as P fertilizers are quickly immobilized in soil. The introduction of three PSM strains (BI2 + BI14 + BS89) in combination with the apatite concentrate of standard grinding had a positive effect on the assimilation of elements, such as Ca and P, by ryegrass. At the same time, we found the low uptake of Sr by ryegrass is advantageous for the future possibilities of apatite-containing ores for the agricultural use.

Our results revealed the potential of using these native PSM strains as microbial additives to fertilizers in agriculture. In conclusion, we demonstrated that biologized apatite may be a new priority for integrated crop nutrition through combinations with microbial strategies and without any risk of accumulation of strontium. These data are especially important for pasture crops, such as ryegrass, and for animal feed production.