Effect of Microbial Preparation and Biomass Incorporation on Soil Biological and Chemical Properties

Abstract

In order to meet the global nutritional needs of a growing population, attempts are being made to develop strategies that can effectively offset the negative effects of intensive farming. The aim of this study was to investigate the effect of Effective Microorganisms (EM) preparation and organic matter incorporation on the soil microbiological and chemical features. The analyses included the number of heterotrophic bacteria, fungi, actinobacteria, cellulolytic, amylolytic and proteolytic microorganisms, and bacteria of the genus Azotobacter. The content of organic carbon, the total and mineral nitrogen forms of phosphorus, potassium, magnesium, and the pH were also determined. The application of an EM of higher dose combined with the manure and straw resulted in the highest abundance of heterotrophic bacteria (165.1 × 10⁶ cfu g⁻¹), actinobacteria (43.2 × 10⁵ cfu g⁻¹), cellulolytic (17.2 × 10⁶ cfu g⁻¹), and proteolytic bacteria (82.0 × 10⁶ cfu g⁻¹). The highest content of chemical parameters was always observed in the experimental variant, including biomass incorporation, accompanied by EM use. The novelty of our research is the analysis of the synergistic effect of the experimental factors studied on the microbiological and chemical parameters of arable soils.

1. Introduction

Agriculture, as the area of the economy responsible for meeting humanity’s basic nutritional needs, is particularly vulnerable to the negative impact of human activity. The increasing knowledge about the causes of these disturbing phenomena motivates people to take action to minimize their negative influence, while the awareness of the risk of new threats resulting from irrational human decisions—to actively counteract them. The need to meet the global nutritional needs of the constantly increasing population often results in decisions whose consequences negatively impact many aspects of human life and the economy [1,2]. The intensification of agriculture, which was supposed to compensate for the limitations in food production that was restrained by the size of arable land, led to a significant deterioration of the natural environment, including the soil [3]. The effect of widely used practices based on deep interference in the soil structure by the application of excessive amounts of artificial fertilizers and pesticides is disrupting the functioning of the complex ecosystem of arable soils and reducing their fertility [4].

The strategy of sustainable agriculture developed as part of intervention activities and widely supported by legal regulations of individual countries and international organizations, sets out the directions of activities that are supposed to mitigate the negative impact of intensive agricultural production on the environment [5,6]. These include, among others, simplification of cultivation systems, rational management of organic matter generated on farms, and reduction in environmental pollution through the use of alternatives to traditional, pro-ecological fertilizers and plant protection products [7,8]. Various types of biomass, including agro-industrial residues, biochar, aquatic macrophytes, are applied as amendments to improve soil features. Biomass incorporation was shown to enrich the soils with carbon and nutrients. Due to the enhanced biological, chemical and physical conditions, these amendments allow the fast recovery of sandy, degraded, and lacking organic matter soils [9,10,11]. The introduction of straw, manure, or catch crop biomass into the soil is a way to manage the excessive amount of organic biomass produced in agriculture, while, at the same time, supplementing the deficits of organic matter in the soil, which is observed especially with a high share of cereals in the sowing structure (Table 1) [12]. The appropriate content of organic carbon in the soil is responsible for its proper structure, chemical properties, and biological activity [13,14]. It is also believed that as the amount of organic matter in the soil increases, so does its potential to sequester carbon from the atmosphere [15,16]. The benefits associated with the returning straw into the soil may be one of the arguments taken into account when making optimal decisions regarding its increasingly common non-agricultural use, e.g., as an energy resource [17,18].

Nutrients content is not the only soil parameter affected by the incorporation of biomass. Introduction of additional amounts of organic carbon stimulates the soil biological activity [19]. The role of the soil microorganisms is crucial in the proper functioning and high productivity of soil. Their importance is determined not only by their large number (the number of phylotypes of bacteria in 1 g of soil ranges from 10² to 10⁶) but mainly by their metabolic activity [20]. Microorganisms are involved in the formation of good soil structure and transformation of the most important elements for plants, i.e., carbon, nitrogen, phosphorus, potassium, sulfur, and iron. They produce various biologically active substances, support the growth and development of plants, and stimulate their resistance to biotic and abiotic stress factors [21,22,23]. Microbial biomass carbon and nitrogen are considered the most widely applied bioindicators of soil quality [24]. Specific groups of microorganisms isolated from the soil microbiome can also be used as components of ecological biopreparations used as part of the sustainable agriculture strategy [25].

Among the bioproducts available on the market containing active microbial cells, both farmers and scientists are interested mainly in EM (Effective Microorganisms) preparations (Table 1). They include bacteria with specific properties (photosynthetic, producing lactic acid) and fungi, both mold and yeast, but the exact proportion of individual species in the entire consortium is protected by the manufacturer [26,27]. The proven positive effects of using EM in agriculture concerned increasing soil fertility and related productivity, improving the health of crop plants, but also detoxification of the soil environment contaminated by heavy metals or with a high level of salinity [28,29,30,31,32,33]. Nonetheless, the effects of EM were also studied in various other areas of the economy, and the results obtained showed high variability, raising doubts among researchers dealing with this issue.

After analyzing the promising results of research indicating the synergistic effect of EM used complementary with the organic matter introduced into the soil, e.g., compost or biochar [34,35], an attempt was made to determine the effect of the EM preparation and the simultaneous enrichment of the soil with manure and straw on selected microbiological and chemical parameters of the soil. Microbiological analyses included the determination of the number of soil-culturable heterotrophic bacteria (B), fungi (F), actinobacteria (Ac), groups of microorganisms involved in biodegradation of cellulose (Ce), starch (Am), proteins (Pr), and bacteria of the genus Azotobacter (Az), while chemical analyses included the content of organic carbon (OC), total (TN) and mineral nitrogen forms, available phosphorus (P), potassium (K), magnesium (Mg), and pH in 1 M KCl (pH) in the soil in the spring wheat cultivation.

The object of the study was to investigate and evaluate the changes in soil properties and quality resulted from the co-application of microbiological preparation EM and organic amendments.

Table 1. Effect of various agricultural practices including manure, straw, and Effective Microorganisms preparation application on selected soil properties.

Treatments Applied	Experimental Variants	Effects of the Treatment on Soil Properties	References
5 straw utilization and fertilization modes	SM—straw mulching; SC—straw crushing; CM—cattle manure; NSR—control with no straw return; CK—control without fertilizers	Cattle manure—the most beneficial effects on soil fertility and bacterial diversity.	[36]
3 fertilization treatments	CK—no-fertilizer; NPK—nitrogen; phosphorus, and potassium fertilizers; NPKS—NPK plus straw	Straw addition—bacterial abundance unchanged, Actinomycetes abundance decreased, fungal abundance significantly increased.	[37]
2 planting patterns, 6 nitrogen fertilizer doses	SR—straw returning; TP—traditional planting (straw removed from the field after harvested) N fertilizer doses: 0, 100, 150, 200, 250, and 300 kg N ha−1	SR—significantly increased soil fertility, enzymatic activities, community diversity, and composition of bacterial and fungal communities compared to TP; TN, SOC—closely correlated with bacterial community composition.	[38]
3 mulching treatments	CK—no mulching; SM—straw mulching; PM—plastic film mulching	SM—no effect on bacterial diversity and richness; enhanced fungal diversity and richness compared to CK in subsoil layers; enhanced soil C and N fractions compared to PM and CK. Bacterial diversity correlated with soil C and N fractions.	[39]
4 fertilization treatments	CK—only chemical NPK fertilizers; the N substituted 20% by organic manure OM, straw SW, organic manure and straw (1:1) OMSW	Organic manure and straw application (especially in the OMSW) can increase soil N contents, but reduce N loss.	[40]
6 straw utilization and nitrogen fertilization modes	straw return rates (50%, 100%); N fertilizer doses (270, 360, 450, 540 kg N ha−1 yr−1)	50% straw return combined with 450 or 540 kg N ha−1 yr−1—increased soil nitrogen, available potassium, and available phosphorus contents. The long-term combined application of 100% straw returning and higher N fertilizer (>450 kg ha−1 yr−1)—not appropriate for soil health; the risk of disease and pollution in soil.	[41]
6 fertilization treatments	CK—unfertilized control; N—nitrogen fertilizer; NP—nitrogen and phosphorus (P) fertilizers; NP + S—straw plus N and P fertilizers; FYM—farmyard manure; NP + FYM—farmyard manure plus N and P fertilizers	Organic manure with inorganic fertilizers—increased SOC and TN contents; without straw or manure—soil available K content declined.	[42]
3 EM and fertilization treatments	EM compost treatment; traditional compost treatment; unfertilized control.	Soil organic matter, total N, available P, available K content—higher in the EM compost plot than in the traditional compost plot.	[43]
2 EM treatments	soil fertilization with EM; unfertilized control.	EM application—reduction in the content of organic carbon (TOC)	[29]
4 EM and fertilization treatments	T1 control—mineral fertilizers; T2—a diluted solution of EM sprayed on the soil surface; T3—green manure added as soil mulch above the ground; T4— mixture of EM1 and green manure.	T2 significantly increased the availability of phosphorous, potassium, and total nitrogen compared to T1. EM1 and green manure improved forage yield and soil properties.	[44]
7 EM treatments	(i) the spraying agent EMA; (ii) EMA with the EM enriched organic substrate Bokashi; (iii) EMA with Bokashi and farmyard manure; controls to (i)–(iii)—the same treatments with sterilized EM preparations; control without EM application.	(i)—no significant differences to the untreated control (treatment without EM application) for the investigated soil parameters. ((ii) and (iii)—significant differences to the untreated control for soil microbial parameters (not consistent throughout the parameters and sampling times). Treatments with living EM compared with its sterilised control treatments—no differences on any of the parameters. EMA did not improve soil quality.	[45]

Table 1. Effect of various agricultural practices including manure, straw, and Effective Microorganisms preparation application on selected soil properties.

Treatments Applied	Experimental Variants	Effects of the Treatment on Soil Properties	References
5 straw utilization and fertilization modes	SM—straw mulching; SC—straw crushing; CM—cattle manure; NSR—control with no straw return; CK—control without fertilizers	Cattle manure—the most beneficial effects on soil fertility and bacterial diversity.	[36]
3 fertilization treatments	CK—no-fertilizer; NPK—nitrogen; phosphorus, and potassium fertilizers; NPKS—NPK plus straw	Straw addition—bacterial abundance unchanged, Actinomycetes abundance decreased, fungal abundance significantly increased.	[37]
2 planting patterns, 6 nitrogen fertilizer doses	SR—straw returning; TP—traditional planting (straw removed from the field after harvested) N fertilizer doses: 0, 100, 150, 200, 250, and 300 kg N ha−1	SR—significantly increased soil fertility, enzymatic activities, community diversity, and composition of bacterial and fungal communities compared to TP; TN, SOC—closely correlated with bacterial community composition.	[38]
3 mulching treatments	CK—no mulching; SM—straw mulching; PM—plastic film mulching	SM—no effect on bacterial diversity and richness; enhanced fungal diversity and richness compared to CK in subsoil layers; enhanced soil C and N fractions compared to PM and CK. Bacterial diversity correlated with soil C and N fractions.	[39]
4 fertilization treatments	CK—only chemical NPK fertilizers; the N substituted 20% by organic manure OM, straw SW, organic manure and straw (1:1) OMSW	Organic manure and straw application (especially in the OMSW) can increase soil N contents, but reduce N loss.	[40]
6 straw utilization and nitrogen fertilization modes	straw return rates (50%, 100%); N fertilizer doses (270, 360, 450, 540 kg N ha−1 yr−1)	50% straw return combined with 450 or 540 kg N ha−1 yr−1—increased soil nitrogen, available potassium, and available phosphorus contents. The long-term combined application of 100% straw returning and higher N fertilizer (>450 kg ha−1 yr−1)—not appropriate for soil health; the risk of disease and pollution in soil.	[41]
6 fertilization treatments	CK—unfertilized control; N—nitrogen fertilizer; NP—nitrogen and phosphorus (P) fertilizers; NP + S—straw plus N and P fertilizers; FYM—farmyard manure; NP + FYM—farmyard manure plus N and P fertilizers	Organic manure with inorganic fertilizers—increased SOC and TN contents; without straw or manure—soil available K content declined.	[42]
3 EM and fertilization treatments	EM compost treatment; traditional compost treatment; unfertilized control.	Soil organic matter, total N, available P, available K content—higher in the EM compost plot than in the traditional compost plot.	[43]
2 EM treatments	soil fertilization with EM; unfertilized control.	EM application—reduction in the content of organic carbon (TOC)	[29]
4 EM and fertilization treatments	T1 control—mineral fertilizers; T2—a diluted solution of EM sprayed on the soil surface; T3—green manure added as soil mulch above the ground; T4— mixture of EM1 and green manure.	T2 significantly increased the availability of phosphorous, potassium, and total nitrogen compared to T1. EM1 and green manure improved forage yield and soil properties.	[44]
7 EM treatments	(i) the spraying agent EMA; (ii) EMA with the EM enriched organic substrate Bokashi; (iii) EMA with Bokashi and farmyard manure; controls to (i)–(iii)—the same treatments with sterilized EM preparations; control without EM application.	(i)—no significant differences to the untreated control (treatment without EM application) for the investigated soil parameters. ((ii) and (iii)—significant differences to the untreated control for soil microbial parameters (not consistent throughout the parameters and sampling times). Treatments with living EM compared with its sterilised control treatments—no differences on any of the parameters. EMA did not improve soil quality.	[45]

2. Materials and Methods

2.1. Experiment Location and Layout

The study was carried out from 2011 to 2014 on a spring wheat field, on a farm on the Inowrocław Plain, Kuyavian-Pomeranian Voivodeship in northern Poland (52°61′18.1″ N; 18°44′12.4″ E). The experimental plots were located on the soil Cambic Gleyic Phaeozem [46]. The texture of analyzed soils was silt loam: mean content of sand (2.0–0.05 mm)—38.2%, silt (0.05–0.002 mm)—50.78 and clay (<0.002 mm)—11.02% [47]. The Mastersizer analyzer (Malvern Instruments, Malvern, UK) was used to determine the texture of soil samples. The soil pH before the start of the experiment was 7.31 (in 1 M KCl); organic carbon content (OC) was 19.58 g·kg⁻¹, total nitrogen (TN): 1.26 g·kg⁻¹ d.m. soil; the available phosphorus (P), potassium (K), and magnesium (Mg) content was 16.1, 21.8 and 4.80 mg 100 g⁻¹, respectively (mean for all experimental objects).

The rainfall and temperature during the period of experiment were presented in the work of Lamparski and Kotwica [48].

A static (second and third year of spring wheat monoculture) three-way experiment was set up in a split-plot–split-block design in three replications (36 experimental plots, 12 objects in 3 repetitions). The area of a single experimental unit in each experiment was 96 m². The experimental factors included the following.

Factor A levels—biomass management method (manure and straw application):

A1—

No manure introduction and no straw incorporation;

A2—

No manure introduction and straw incorporation;

A3—

Manure introduction and no straw incorporation;

A4—

Manure introduction and straw incorporation.

Factor B levels—methods of applying biopreparation EM:

B1—

Without biopreparation EM application (EM × 0);

B2—

Biopreparation EM single application, added to the soil during post-harvest treatment in October; dose: 40 dm³·ha⁻¹ (EM × 1);

B3—

Biopreparation EM dual application: added to the soil the soil during post-harvest cultivation in autumn at a dose of 20 dm³·ha⁻¹ and EM sprayed on leaves; dose: 20 dm³·ha⁻¹ at BBCH 21–23 (EM × 2).

The spring wheat (Triticum aestivum L.) cv. Tybalt-qualified seed material was used in the experiment in March 2012 and April 2013 and 2014. Wheat sowing density was 450 grains·m⁻². The sowing depth was 4 cm, and the row spacing was 14.3 cm. Cattle solid manure was applied at a dose of 30 t·ha⁻¹. The stubble catch crop was white mustard (Sinapis alba L.) sown in August.

The commercial biopreparation EM “Naturally Active” is a suspension of microorganisms (Greenland Technologia EM sp. z o.o., Trzcianki, Poland) with certificate PZH/HT-1448/2002 and the Institute of Soil Science and Plant Cultivation qualification certificate No. NE/1/2004. EM is environmentally safe, without genetically modified organisms (GMO), and does not contain biotic and abiotic agents harmful for human or animals. According to an official statement of the biopreparation manufacturer (EMRO-EM Research Organization Japan, Okinawa, Japan), the main components of the product are various groups of bacteria (photosynthetic, lactic acid bacteria, actinobacteria, Azotobacter spp.), yeast, and cane molasses.

Nitrogen fertilization at a rate of 60 kg N·ha⁻¹ as well as phosphorus and potassium at doses of 15.7 kg P·ha⁻¹ and 76.4 kg K·ha⁻¹, respectively, were applied in spring before pre-sowing cultivation. Moreover, top-dressing with nitrogen was applied twice, once at the stage BBCH 32 at a rate of 60 kg N·ha⁻¹ and in the stage BBCH 51 at a rate 50 kg N·ha⁻¹. Agrotechnical treatments, as well as wheat yielding, were described in detail in the work of Kotwica et al. [49].

2.2. Soil Samples

Soil samples for microbiological and chemical analysis were collected from the cultivated layer from a depth of 0–30 cm. On each experimental plot for all the treatments 10 individual soil samples were collected. The samples of soil from a particular plot were mixed and homogenized to obtain a pooled sample. Microbiological soil samples analyses were performed in triplicate. The material was taken each year on three dates, in spring before sowing, in the earing phase BBCH 52–54, and immediately after harvesting spring wheat. Soil samples for chemical analyses were collected for the first time in 2011, at the beginning of the experiment (after harvesting the forecrop and before stubble cultivation), and the last time in 2014, after plant harvesting and before the beginning of post-harvest cultivation.

2.3. Microbial Analyses

The microbiological analyses of the soil samples collected involved determining the total count of heterotrophic bacteria (B), actinobacteria (Ac), filamentous fungi (F), and microorganisms participating in C and N transformations in soil (amylolytic—Am, cellulolytic—Ce, proteolytic—Pr, and bacteria of the genus Azotobacter—Az). Ten grams of each soil sample was added to 90 mL of Ringer’s solution. After homogenization for 25 min, tenfold serial dilutions were made (10⁻¹–10⁻⁶). Then, inoculations of the soil solutions prepared were made on proper culture media. The total heterotrophic bacteria were isolated on the standard nutrient agar (NA) and filamentous fungi on the Rose–Bengal agar with 30 µg mL⁻¹ streptomycin [50]. Actinobacteria were isolated on yeast extract–glucose medium (YGA) containing 100 nystatin μg mL⁻¹ [51]. Cellulolytic microorganisms were tested on the agar medium containing 0.1% sodium carboxymethylcellulose (Sigma) according to the method Gupta et al. [52]. The amylolytic microorganisms was determined on the selective medium with 0.2% of starch (Difco) and proteolytic microorganisms on the medium according to Alef and Nannipieri [53]. Azotobacter spp. were isolated on the nitrogen-free medium Ashby Sucrose agar [54]. The incubation of microorganisms was carried out at 25–28 °C for 4–5 days, and 10 days for actinobacteria. All analyses were performed in four replicates. After the incubation period, the colonies grown on Petri dishes were counted. The number of colony forming units (CFU) was determined per 1 g of soil dry matter (CFU g⁻¹ d.m. of soil).

2.4. Soil Chemical Analyses

In soil samples (air-dried disturbed) sieved through a 2 mm mesh, selected chemical properties were determined. The soil pH in 1 M KCl by potentiometric method was measured [55], organic carbon (OC), and total nitrogen (TN) concentrations using a Vario Max CN analyzer (Elementar, Analysensysteme GmbH, Langenselbold, Germany). The contents of available forms of phosphorus (P) [56] and potassium (K) were determined by the Egner–Riehm method [57], while the content of magnesium available to plants (Mg) was analyzed following the Schachtschabel method [58]. The content of forms available to plants was determined by atomic absorption spectroscopy and atomic emission spectroscopy using a Solaar S4 spectrometer. The forms of the mineral nitrogen, i.e., ammonium (N-NH₄) and nitrate (N-NO₃), were determined by flow colorimetry following soil extraction in 1% K₂SO₄ using the Skalar San Plus Analyzer. Based on the obtained values of soil chemical parameters (pH, content of OC, TN, and available forms P, K, and Mg) at two dates, before the start of the research and after its completion, the relative change index (Irc) was determined. The Irc index was calculated according to Kotwica et al. [59] guidelines. The values of the index above 1.0 indicate a favorable impact of a given combination of levels of the analyzed factors, and the values lower than 1.0 indicated the opposite.

2.5. Data Analyses

The final results are mean of three replications from each plot in each sampling time. The results of individual parameters were statistically verified using the variance of multiple experiments, according to the model appropriate for the randomized sub-block design. The analysis of variance (two-way ANOVA) was used, where the first factor (A) was the biomass management method (straw and manure application) and the second factor (B) was the Effective Microorganisms biopreparation use at various rates. To evaluate the significance of the factors’ influences and their interactions, a Tukey’s post hoc test was used with a 95% confidence interval, and the mean values of the parameters analyzed were compared. The differences between the objects were analyzed by principal component analysis (PCA) based on the mean data values of all of the tested soil features. The first two principal components (PC1 and PC2) were selected for the ordination of the cases. A cluster analysis was performed to determine the groups in the dataset based on a dendrogram. Due to the diverse ranges of absolute quantities of individual soil parameters, multidimensional analyses were performed on standardized data. The statistical analyses were performed using the StatSoft’s Statistica 12 PL software package [60].

3. Results

The concentration of heterotrophic bacteria (B) in the tested samples was highly dependent on the biomass introduced into the soil, with the highest number of this group of microorganisms found after the combined use of manure fertilization and incorporation of straw residues (A4). The maximum average number of heterotrophic bacteria (165.1 × 10⁶ cfu g⁻¹) was reported in the experimental object where the use of manure and straw was accompanied by EM double dose (A4B3). As for the soils from experimental plots where the highest EM dose (B3) was applied, the mean number of bacteria was significantly higher compared to treatments B1 (no EM) and B2 (single EM dose), but only when the soil was supplemented with an additional source of organic matter in the form of straw or manure (A2, A3, A4). The lowest number of bacteria (27.8 × 10⁶ cfu g⁻¹) was found in the experimental object without the use of any type of biomass or EM preparation (A1B1) (

Table 2. The number of the soil heterotrophic bacteria (B), filamentous fungi (F), and actinobacteria (Ac) depending on the manure and straw management and application of the biopreparation EM in spring wheat cultivation.

Levels of Factor A	Levels of Factor B
	B1 (EM × 0)				B2 (EM × 1)				B3 (EM × 2)
	Date of Analysis
	I	II	III	Mean	I	II	III	Mean	I	II	III	Mean
	Heterotrophic bacteria (106 cfu g−1)
A1	25.0 ± 4.1	41.7 ± 2.4	16.7 ± 1.2	27.8	22.3 ± 0.9	40.3 ± 2.4	22.3 ± 1.7	28.3	16.3 ± 3.3	44.0 ± 6.2	27.7 ± 2.6	29.3
A2	23.3 ± 6.8	39.7 ± 2.6	31.0 ± 1.4	31.3	20.0 ± 0.1	33.7 ± 1.9	48.0 ± 4.3	33.9	52.3 ± 2.6	71.0 ± 0.8	47.3 ± 3.1	56.9
A3	60.0 ± 4.2	26.0 ± 0.8	26.7 ± 0.9	37.6	22.7 ± 1.2	56.0 ± 3.3	36.0 ± 2.9	38.2	21.0 ± 2.8	113.0 ± 6.7	31.3 ± 1.9	55.1
A4	18.3 ± 1.2	42.0 ± 2.2	134.0 ± 9.4	64.8	20.7 ± 0.5	283.3 ± 7.0	74.0 ± 8.5	126.0	42.3 ± 1.7	390.7 ± 19.3	62.3 ± 4.0	165.1
Mean	31.7	37.4	52.1		21.4	103.3	45.1		33.0	154.7	42.2
LSD0.05 for Factor A = 13.44; Factor B = 3.37; Interaction A/B = 23.09; B/A = 15.73
Filamentous fungi (104 cfu g−1)
A1	22.3 ± 0.9	18.3 ± 1.6	10.0 ± 0.5	16.9	20.7 ± 1.9	18.0 ± 1.2	15.7 ± 0.9	18.1	28.7 ± 4.0	22.7 ± 4.1	12.7 ± 1.4	21.3
A2	16.7 ± 0.5	14.3 ± 0.5	15.0 ± 0.9	15.3	34.3 ± 2.6	15.0 ± 0.5	15.0 ± 0.5	21.4	67.3 ± 5.7	13.3 ± 0.5	8.7 ± 0.6	29.8
A3	36.0 ± 2.9	6.7 ± 1.1	14.3 ± 1.4	19.0	28.7 ± 2.3	20.3 ± 0.9	15.3 ± 0.9	21.4	32.0 ± 2.8	20.3 ± 0.9	13.0 ± 0.9	21.8
A4	33.3 ± 0.9	22.7 ± 1.7	17.7 ± 1.8	24.6	20.3 ± 2.1	57.0 ± 0.8	13.7 ± 1.1	30.3	31.7 ± 2.5	24.3 ± 0.5	28.3 ± 2.4	28.1
Mean	27.1	15.5	14.3		26.0	27.6	14.9		39.9	20.2	15.7
LSD0.05 for Factor A = 5.26; Factor B = 1.71; Interaction A/B = 8.88; B/A = 6.12
Actinobacteria (105 cfu g−1)
A1	24.7 ± 3.9	24.3 ± 3.3	14.3 ± 1.2	21.1	22.7 ± 2.7	24.3 ± 2.0	29.3 ± 2.9	25.4	27.7 ± 2.1	27.7 ± 2.5	19.3 ± 1.9	24.9
A2	21.7 ± 0.5	37.3 ± 5.2	23.0 ± 2.6	27.3	36.7 ± 2.1	29.5 ± 1.6	26.0 ± 3.6	30.7	28.3 ± 3.4	29.0 ± 2.2	27.0 ± 3.2	28.1
A3	23.3 ± 3.3	36.7 ± 2.4	14.0 ± 0.8	24.7	17.3 ± 0.5	34.0 ± 5.2	31.7 ± 3.6	27.7	56.0 ± 9.4	21.7 ± 1.2	23.0 ± 0.1	33.6
A4	39.3 ± 7.4	25.3 ± 3.1	32.3 ± 3.2	32.3	26.3 ± 3.5	34.3 ± 3.7	37.7 ± 4.5	32.8	46.0 ± 8.5	57.3 ± 4.2	26.3 ± 2.1	43.2
Mean	27.3	30.9	20.9		25.8	30.5	31.2		39.5	33.9	23.9
LSD0.05 for Factor A = 9.55; Factor B = 2.62; Interaction A/B = 14.38; B/A = 9.21

Factor A levels: A1 (no manure introduction + without straw), A2 (no manure introduction and straw incorporation), A3 (manure introduction + without straw), A4 (manure introduction + straw incorporation); Factor B levels: B1 (without biopreparation EM application), B2 (biopreparation EM single application), B3 (biopreparation EM dual application); LSD least significant difference, the significance of differences by Tukey’s test at p ≤ 0.05. Analysis date: I in spring before sowing, II—in the earing phase BBCH, III—after harvesting spring wheat.

).

The analysis of the obtained results proved the differentiated effect of soil enrichment with biomass on the average count of filamentous fungi (F), which varied from 15.3 to 30.3 × 10⁴ cfu g⁻¹. The addition of both biomass types, straw and manure, resulted in a significantly higher number of fungi in the treatments without EM application or with a single dose of the preparation. Experimental combinations with double EM application differed statistically, depending on the method of straw management; the number of fungi in the soil from plots with straw (A2, A4) was significantly higher compared to plots without the addition of this substrate (Table 2).

The use of manure and shredded straw on the soil surface had a positive effect on the increase in the number of actinobacteria (Ac). In the experimental objects combining the soil application of both types of biomass, the concentration of these microorganisms was usually significantly higher than in the treatments excluding their use; this trend was not observed with a single dose of EM (B2). The highest mean number of actinobacteria (43.2 × 10⁵ cfu g⁻¹) was found in the treatment variant with the application of two doses of the EM preparation, manure, and straw (A4B3) (Table 2).

The introduction of an additional source of biomass had a stimulating effect on the number of soil microorganisms characterized by the ability to decompose complex organic compounds. Even though the observed differences were not always statistically significant, in almost all analyzed variants the number of microorganisms hydrolyzing cellulose (Ce), starch (Am), and proteins (Pr) was highest when manure and straw were applied together, and the lowest when they were not used. The average number of cellulolytic microorganisms ranged from 7.1 to 17.2 × 10⁶ cfu g⁻¹, amylolytic from 10.1 to 24.1 × 10⁶ cfu g⁻¹, and proteolytic from 10.7 to 82.0 × 10⁶ cfu g⁻¹. The addition of the EM bacterial preparation did not affect the number of cellulolytic microorganisms, in contrast to the proteolytic microorganisms. The concentration of the latter ones in soils from the experimental objects with straw left on the surface (A2, A4) and a double dose of EM (B3) was significantly higher compared to the objects with other levels of factor A (

Table 3. The number of the cellulolytic (C), amylolytic (Am), proteolytic (Pr) soil microorganisms, and Azotobacter spp. (Az) depending on the manure and straw management and application of the biopreparation EM in spring wheat cultivation.

Levels of Factor A *	Levels of Factor B
	B1 (EMx0)				B2 (EMx1)				B3 (EMx2)
	Date of Analysis
	I	II	III	Mean	I	II	III	Mean	I	II	III	Mean
	Cellulolytic microorganisms (106 cfu g−1)
A1	1.7 ± 0.5	13.7 ± 2.4	6.0 ± 0.6	7.1	4.7 ± 0.0	13.3 ± 1.0	8.0 ± 0.5	8.7	13.5 ± 1.3	7.7 ± 0.9	7.0 ± 0.9	9.4
A2	5.7 ± 0.0	11.7 ± 1.1	11.3 ± 0.9	9.6	9.0 ± 0.9	13.3 ± 1.3	9.3 ± 0.9	10.6	6.0 ± 0.0	24.7 ± 1.8	10.3 ± 1.2	13.7
A3	5.3 ± 0.7	13.3 ± 0.9	9.0 ± 0.8	9.2	9.0 ± 1.1	11.7 ± 1.2	10.7 ± 0.9	10.4	12.0 ± 1.4	9.0 ± 1.0	8.7 ± 0.9	9.9
A4	7.7 ± 0.7	9.0 ± 0.9	28.0 ± 1.9	14.9	8.3 ± 0.6	20.0 ± 1.2	9.3 ± 0.9	12.6	13.7 ± 1.3	26.3 ± 2.3	11.7 ± 0.9	17.2
Mean	5.1	11.9	13.6		7.8	14.6	9.3		11.3	16.9	9.4
LSD0.05 for Factor A = 5.71; Factor B = 4.70; Interaction A/B = 9.93; B/A = 8.08
Amylolytic microorganisms (106 cfu g−1)
A1	12.0 ± 0.8	8.3 ± 0.7	10.0 ± 1.2	10.1	5.3 ± 0.2	19.0 ± 1.4	10.0 ± 0.8	11.4	11.5 ± 1.2	16.3 ± 1.2	7.7 ± 0.7	11.8
A2	8.0 ± 0.0	15.0 ± 1.0	14.0 ± 0.9	12.3	15.3 ± 1.6	13.0 ± 0.9	13.7 ± 1.2	14.0	22.7 ± 1.2	11.7 ± 0.9	7.7 ± 0.8	14.0
A3	8.0 ± 0.4	21.7 ± 2.4	15.0 ± 1.2	14.9	8.7 ± 0.7	23.7 ± 1.9	11.7 ± 1.1	14.7	14.7 ± 1.2	22.3 ± 1.9	14.3 ± 0.9	17.1
A4	7.7 ± 0.5	19.7 ± 2.1	21.3 ± 1.9	16.2	14.3 ± 1.1	95.3 ± 7.5	20.3 ± 1.2	43.3	13.3 ± 0.5	47.0 ± 4.2	12.0 ± 1.4	24.1
Mean	8.9	16.2	15.1		10.9	37.8	13.9		15.6	24.3	10.4
LSD0.05 for Factor A = 6.03; Factor B = 3.67; Interaction A/B = 8.47; B/A = 6.05
Proteolytic microorganisms (106 cfu g−1)
A1	7.3 ± 0.7	13.3 ± 1.1	11.3 ± 0.9	10.7	19.7 ± 0.9	21.7 ± 1.2	12.3 ± 0.8	17.9	10.7 ± 0.5	18.0 ± 2.2	13.7 ± 0.5	14.1
A2	9.3 ± 0.9	27.0 ± 1.7	12.0 ± 1.1	16.1	17.0 ± 0.8	17.3 ± 0.9	26.0 ± 2.2	20.1	31.0 ± 0.8	21.0 ± 0.8	17.3 ± 0.5	23.1
A3	18.0 ± 1.2	29.3 ± 3.3	13.7 ± 0.9	20.3	13.7 ± 1.1	25.0 ± 2.4	13.0 ± 1.2	17.2	26.3 ± 2.0	14.0 ± 0.8	15.7 ± 1.2	18.7
A4	9.0 ± 0.0	16.3 ± 1.2	42.0 ± 2.2	22.4	16.7 ± 1.2	41.3 ± 6.4	31.7 ± 2.8	29.9	21.0 ± 0.2	206.7 ± 9.4	18.3 ± 0.5	82.0
Mean	10.9	21.5	19.8		16.8	26.3	20.8		22.3	64.9	16.3
LSD0.05 for Factor A = 8.01; Factor B = 2.28; Interaction A/B = 14.49; B/A = 10.19
Azotobacter spp. (101 cfu g−1)
A1	6.7 ± 0.4	35.7 ± 1.3	5.3 ± 0.2	15.9	6.6 ± 0.0	13.7 ± 0.9	8.7 ± 0.7	9.6	16.7 ± 1.2	15.0 ± 1.1	9.3 ± 0.5	13.7
A2	5.0 ± 0.0	34.0 ± 1.5	45.7 ± 1.7	28.2	6.7 ± 0.4	28.7 ± 1.7	34.7 ± 2.3	23.3	26.7 ± 2.7	26.0 ± 2.5	7.7 ± 0.7	20.1
A3	10.0 ± 0.0	22.7 ± 1.4	15.0 ± 0.9	15.9	6.6 ± 0.0	30.5 ± 1.8	8.7 ± 0.9	15.3	8.3 ± 0.4	35.0 ± 1.8	6.7 ± 0.2	16.7
A4	23.3 ± 1.4	44.7 ± 4.7	18.0 ± 1.1	28.7	5.0 ± 0.0	46.0 ± 4.9	41.3 ± 3.9	30.8	25.0 ± 0.5	23.0 ± 0.9	17.7 ± 0.9	21.9
Mean	11.3	34.3	21.0		6.2	29.7	23.4		19.2	24.8	10.4
LSD0.05 for Factor A = 6.91; Factor B = 3.72; Interaction A/B = 12.92; B/A = 9.68

* Abbreviations: see Table 2.

).

The highest number of free-living diazotrophs of the genus Azotobacter (30.8 × 10¹ cfu g⁻¹) was reported in the experimental combination with the use of manure and straw. The number of Azotobacter spp. in the soils from all objects at this level of factor A was always significantly higher than those isolated from plots without any additional source of organic carbon (A1). The effect of using EM, despite the confirmed significant differences between the experimental objects, did not show a clear trend in terms of changes in the concentration of these bacteria in the tested soils (Table 3).

The pH of the soils tested during the three-year experiment was close to neutral and the pH value fluctuated only slightly in the range of 7.4–7.6 (Figure 1a). The values of the relative change coefficient show an increase in pH in soils with the addition of manure (

Table 4. Index of relative change (Irc) of the organic carbon (OC) and total nitrogen (TN) content depending on the manure and straw management and application of the biopreparation EM in spring wheat cultivation.

Levels of Factor A	Index Irc
	pH			OC			TN
	B1	B2	B3	B1	B2	B3	B1	B2	B3
A1	0.99	0.99	0.99	1.01	1.03	1.02	0.96	1.00	0.99
A2	1.00	1.03	1.03	1.04	1.09	1.12	1.04	1.06	1.09
A3	0.96	0.99	0.98	0.91	1.00	1.00	0.89	0.96	0.99
A4	0.99	1.00	1.00	1.01	1.04	1.06	1.01	1.03	1.03

).

The content of organic carbon (OC) in the soils from the experimental objects with the incorporation of manure and straw reached 37.24 g kg⁻¹ and was higher than in all other treatments. On the other hand, the values obtained from the soil without the addition of biomass were the lowest and ranged from 19.14 g kg⁻¹ to 23.90 g kg⁻¹. Moreover, only at this level of factor A, the values of the Irc coefficient did not exceed 1, which indicates the lack of a stimulating effect in terms of OC content in the soil (Table 4). The application of the EM bacterial preparation did not affect its amount, although, in most variants, a single dose of the preparation resulted in a higher concentration of OC in the soil than a double dose (Figure 1b).

Similar trends concerned the content of N in the analyzed soils, the amount of which was much higher in all levels of factor A including the introduction of manure and straw into the soil. The highest content of this compound (3.39 g kg⁻¹) was found in samples from a treatment where both of these forms of soil enrichment with organic matter and EM single dose (A1B2) were used. The values of the relative change index indicated an increase in the total amount of N after manure fertilization, both in combination with straw and without its addition (Table 4). The EM preparation moderately affected the direction of changes in nitrogen concentration in the tested soils (Figure 1c).

The highest and lowest content of assimilable forms of phosphorus (P) in the tested soils was 208.9 mg kg⁻¹ and 179.0 mg kg⁻¹, respectively (Figure 2a). In the treatments with manure and straw used together, these values were the highest, together with the values of Irc (

Table 5. Index of relative change (Irc) of P, K, and Mg available forms content depending on the manure and straw management and application of the biopreparation EM in spring wheat cultivation.

Levels of Factor A	Index Irc
	P			K			Mg
	B1	B2	B3	B1	B2	B3	B1	B2	B3
A1	0.99	1.02	1.01	1.01	1.01	1.04	0.99	1.00	1.03
A2	1.06	1.11	1.11	1.10	1.20	1.21	1.03	1.06	1.03
A3	0.97	0.99	0.99	0.99	1.03	1.03	0.98	0.99	0.99
A4	1.00	1.00	1.03	1.05	1.12	1.05	1.00	1.01	1.05

). In the remaining experimental combinations, also those without introducing biomass into the soil, no differences in the concentration of this element were reported. Likewise, the addition of EM also did not affect this parameter in a significant and unequivocal way (Figure 2a).

The amount of assimilable forms of potassium (K) was also the highest in variant A4, in which manure and straw were applied to the soil, and it exceeded even 400 mg·kg⁻¹. Unlike phosphorus, however, in the objects without the addition of organic matter, the concentration of potassium was lower. In the majority of experimental combinations, the values of Irc > 1 were observed, which proved a general increase in the amount of potassium in the analyzed soils (Table 5). The introduction of EM increased the concentration of available forms of soil K (Figure 2b).

As in the case of potassium, the content of magnesium (Mg) in the soil, ranging from 39.1 mg kg⁻¹ to 52.3 mg kg⁻¹, was always the highest when manure and straw were applied (A4), and the lowest in soils without their addition (A1). The lack of an additional source of organic matter also resulted in a decrease in the amount of assimilable Mg, which was confirmed by the values of Irc calculated for this experimental variant (<1) (Table 5). The content of this element after the use of each EM dose was higher than without it, especially in the soil from the objects where manure and straw were left on the plot (Figure 2c).

The total amount of mineral nitrogen (TMN) was the highest in soils from the objects fertilized with manure alone and in combination with straw. An almost two times lower content of mineral forms of nitrogen, not exceeding 30 mg kg⁻¹, was found in the variant without any addition of biomass. The EM application increased the value of this parameter, while the differences between the single and double doses of the preparation were relatively small. Very similar trends concerned changes in the concentration of individual forms of mineral nitrogen—N-NH₄ and N-NO₃. The analysis of the effect of manure fertilization and the method of straw management on the average content of these nitrogen forms in the soil proved that these practices during the three-year experiment increased the concentration of mineral nitrogen in the tested samples. The highest concentration of both ammonium and nitrate forms (31.9 mg kg⁻¹ and 26.5 mg kg⁻¹, respectively) was observed in the experimental variant A4B3 when using manure, straw, and a double dose of EM. The lowest content, on the other hand, was found when excluding from the cultivation system both the introduction of biomass and the application of EM into the soil (A1B1) and reached 15.6 mg kg⁻¹ for N-NH₄ and 10.4 mg kg⁻¹ for N-NO₃ (Figure 3a–c).

In order to evaluate the relationships between the analyzed soil parameters, i.e., bacteria (B); fungi (F); actinobacteria (Ac); cellulolytic microorganisms (Ce), amylolytic (Am), proteolytic (Pr), bacteria of the genus Azotobacter (Az), pH in 1 M KCl (pH); organic carbon content (OC); content of total nitrogen (TN), the content of available phosphorus (P), potassium (K), and magnesium (Mg); mineral nitrogen forms N-NO₃ and N-NH₄, and application of the EM preparation, as well as the applied method of soil enrichment with organic matter, the method of multifactor principal component analysis (PCA) was used (Figure 4a). From the available data, two main components PC1 and PC2 were extracted, which explained the total variance of 78.44%. The PC1 component accounts for 69.14% of the variability, indicating a significant negative correlation with Mg (−0.962), K (−0.921), B (−0.919), Ac (−0.898), OC (−0,879) N-NH₄ (−0.883), and N-NO₃ (−0.851). The PC2 component accounts for 9.30% and is negatively correlated with Az (−0.527), TN (−0.499), and positively with Pr (0.515). Consequently, most of the studied parameters were concentrated on the side of PC1. Therefore, this component can be identified with the impact of the applied EM biopreparation and the biomass incorporation.

The dendrogram generated based on the Ward cluster analysis and Euclidean distances enabled the separation of four clusters. All elements of cluster 1 (A1B1, A1B2, A1B3), corresponding to the experimental objects without the addition of organic matter, were characterized by the lowest number of heterotrophic bacteria, amylolytic microorganisms, and the content of organic carbon, total and mineral nitrogen, and magnesium. Cluster 2 included all objects fertilized with manure or straw, distinguished by a relatively high amount of P and mineral forms of soil nitrogen (N-NO₃ and N-NH₄). Cluster 3 consisted of two objects, in which only straw was introduced into the soil, and one of them, without the addition of EM (A2B1), was characterized by the lowest number of filamentous fungi (F). Noteworthy are all three objects of cluster 4, enriched with both manure and straw (A4B1, A4B2, A4B3), for which the highest number of heterotrophic bacteria (B) and the content of OC, TN, K, and Mg was found. Objects on which the EM preparation was used (B2, B3) were characterized by the highest concentration of amylolytic (Am) and proteolytic (Pr) microorganisms and P assimilable forms (Figure 5).

4. Discussion

Agricultural practices implemented to maintain or regulate the properties and functioning of the soil environment are aimed at reducing the number and intensity of treatments and replacing traditional fertilizers and pesticides with products based on natural substances or active microorganisms [61,62].

Replacing mineral fertilizers with natural ones, recommended as part of organic and sustainable agriculture, may affect the soil microbiome, its diversity and metabolic activity and, consequently, the growth, health, and yield of cultivated plants [63,64]. It is estimated that 80% to 90% of the processes taking place in the soil environment involve the participation of microorganisms [65]. The introduction of an supplementary source of organic carbon and other nutrients into the soil most often leads to the multiplication of autochthonous soil microorganisms and an increase in their activity, although it depends on the type of biomass and the way it is managed. According to Sun et al. [66], wheat straw did not have a significant impact on the soil microbiome, unlike manure, which restored the bacterial diversity to a level comparable to the unfertilized control. In the present study, both manure and straw stimulated the growth of heterotrophic bacteria and filamentous fungi, and the number of these groups of microorganisms was the highest in the variants with their combined application (Table 2). Metagenomic analysis of organic soil fertilized with farmyard manure and vermicompost showed a higher copy number of diazotrophs and phosphate solubilizers than in the soil cultivated with mineral fertilizers [67]. However, in the present study, it was found that the number of free-living diazotrophs of the genus Azotobacter was always significantly higher in the soil with manure and straw (21.9–30.8 × 10¹ cfu g⁻¹) than in samples from plots where no additional source of organic carbon was applied (9.6–15.9 × 10¹ cfu g⁻¹) (Table 3). The reported higher number of actinobacteria in samples from soils augmented with organic matter by Suyal et al. [67], resulted from their high metabolic activity allowing them to dominate in soils rich in organic carbon. Similar trends were confirmed in our research, revealing a higher number of this group of bacteria in soils with the addition of both manure and straw than in the other variants (although not always statistically significant) (Table 2).

Guan et al. [68] confirmed the correlation between the incorporation of straw and the increased enzymatic activity of soil microorganisms, which, in turn, results in an increase in the content of NPK forms available to plants, and ultimately a growth of crop yield. The highest count of soil microorganisms hydrolyzing cellulose (17.2 × 10⁶ cfu g⁻⁶), starch (43.3 × 10⁶ cfu g⁻⁶), and proteins (82.0 × 10⁶ cfu g⁻⁶) analyzed in this study was nearly always observed with the co-application of manure and straw, and the lowest without their use (Table 3). The activity of soil enzymes associated with the soil microbiome is considered an important indicator of the quality of this environment, and the metabolic activity of microorganisms stimulated by the incorporation of organic matter results in the intensified decomposition of biomass introduced into the soil [68].

Natural microbiological preparations are considered a safe alternative to traditionally used pesticides and fertilizers, providing plants with the right amount and form of nutrients without disturbing the structure and functioning of the soil ecosystem. Commercial preparations of effective microorganisms (EM) are one of the most commonly used in scientific research. According to Higa and Parr [69], the authors of the concept of using EM in agriculture, the microorganisms included in the preparation can have a positive effect on both the soil environment and crop plants. By adding to the soil or applying directly to the surface of plants, they improve the structure, fertility, and health of the soil, stimulate the processes of transformation of organic matter and other nutrients, and promote plant growth [70]. Even though the number of scientific publications on the impact of EM on the physical and chemical properties of soil is relatively high, there are just a few studies on the effect of their use on soil microorganisms. Pranagal et al. [71] suggest the prudent use of this type of product due to the risk of disturbance in the functioning of the soil environment as a result of the introduction of foreign microorganisms and their ability to decompose soil organic matter. Other authors [26], however, negate the possible permanent effects of EM on the composition and activity of indigenous soil microorganisms, assuming the short-term nature of possible changes in their numbers. In the present study, heterotrophic bacteria (165.1 × 10⁶ cfu g⁻¹), actinobacteria (43.2 × 10⁵ cfu g⁻¹), cellulolytic (17.2 × 10⁶ cfu g⁻¹), and proteolytic bacteria (82.0 × 10⁶ cfu g⁻¹) (Table 2 and Table 3) were the most numerous after applying a double dose of EM. A significant impact of EM on the soil microorganisms responsible for the decomposition of complex organic was found with proteolytic microorganisms in contrast to cellulolytic microorganisms, whose concentration generally did not change significantly after the application of the preparation (Table 3).

The synergistic effect of the higher dose of EM and the addition of organic matter, both manure and straw, used together or separately, was demonstrated in the case of heterotrophic bacteria. The number of this group of microorganisms was always significantly higher compared to the plots without or with a single dose EM preparation (Table 2). It should also be highlighted that the above-mentioned maximum number of individual groups of microorganisms, i.e., heterotrophic bacteria, actinobacteria, cellulolytic, and proteolytic, in the cultivation variant with a double EM dose always occurred in combination with the application of manure and straw. This phenomenon may confirm the hypothesis indicating the positive effect of organic matter and microbiological preparations on the soil microbiome. The results of the data meta-analysis performed by Gross and Glaser [72] revealed that the addition of manure increased the soil organic carbon amount in agricultural soils by an average of 35.4%. Suyal et al. [67] reported that due to fertilization with farmyard manure, the content of organic carbon, nitrogen, and available forms of phosphorus in the soil was increased by 56.97%, 3.24%, and 71.46%, respectively, than in conventionally fertilized soil. The direct relationship between the use of manure and a higher amount of organic carbon and available forms of N, P, K, and Mg was also confirmed in several other studies [73,74,75]. The increase in the concentration of organic carbon in soil enriched with straw can be from 12 to 14.9% [58,76]. According to Akhtar et al. [77], a 12% increase in the amount of total nitrogen was reported after introducing wheat straw to the soil. The use of straw can also lead to an increase in the concentration of assimilable forms of phosphorus and potassium by up to several dozen percent [78], although not all studies confirm this tendency [79]. The effect of straw incorporation also depends on the dose and the method of its management (mulch, mixing with soil) [78,80]. The results of the present study confirm the general tendencies—all of the chemical parameters determined (OC, TN, P, K, Mg, TMN) had the highest values in the variant with the addition of manure and straw. As for the assimilable forms of K, however, it was observed that the content of this element was not dependent on the enrichment of the soil with organic matter (Figure 1, Figure 2 and Figure 3). The substitution or supplementation of traditional mineral fertilization with manure and straw results in the prolonged effect of fertilization due to the gradual release of plant nutrients from slowly decomposing components of manure and straw [81].

Despite the use of EM being based on the activity of living microbial cells, and not on the chemical composition of the product, its application may affect the content of various elements and chemical compounds in the soil. In the study by Hu and Qi [29], the use of compost with the addition of EM led to an increase in the content of organic matter, total nitrogen, and available forms of phosphorus and potassium, compared to the soil from plots fertilized with traditional compost. This phenomenon may result from the activity of microorganisms contained in the preparation, which accelerated the decomposition of organic compounds and the release of nutrients from the soil. On the other hand, Szymanek et al. [82] reported a negative impact of EM on the amount of available forms of soil phosphorus, potassium, and magnesium. Likewise, long-term studies by Pranagal et al. [71] did not confirm the positive effect of EM on the physical properties of the soil, and the application of the preparation led to a decrease in the content of organic carbon in the soil. In our research, a moderate effect of the EM preparation on the content of OC, TN, P, Mg, and TMN in the soil was observed, although, usually, the values of these parameters were higher in the variants including their application. The introduction of EM into the cultivation system elevated the concentration of assimilable forms of K in the soil. In all the analyzed chemical parameters, their highest value was always found in the variant that included manure fertilization and the introduction of straw to the soil, accompanied by the application of EM, which may suggest a synergistic effect of these experimental factors (Figure 1, Figure 2 and Figure 3).

5. Conclusions

The development of cultivation strategies that effectively regulate disturbances in the physicochemical properties and biological activity of the soil environment concerns both the search for new solutions and the use of practices with known and predictable effects. The importance of the organic matter for the proper functioning of microorganisms inhabiting the soil is well documented, and the introduction of its additional sources, e.g., in the form of manure or straw, affects positively their growth, development, and metabolic activity. The results of our study confirm these observations. As for microbial preparations, it seems impossible to formulate clear conclusions on the rationality of their use, both on the basis of our own or other research results. Given the discrepancies regarding the values of EM preparations, it is necessary to take into account the influence of additional biotic and abiotic factors that can modify their effectiveness (climatic and soil conditions, use of other treatments, individual specificity of the preparation, etc.). Our results, suggesting the possibility of a synergistic effect of EM and organic matter on soil microorganisms, allow us to formulate the hypothesis that the use of EM should be considered mainly as a complementary treatment, aimed at enhancing or optimizing, but not replacing, the effects of other agrotechnical treatments.