Response of Phaseolus vulgaris to the Use of Growth-Promoting Microorganisms Associated with the Reduction of NPK Fertilization in Tropical Soils: Clayey Oxisol and Sandy Ultisol

Abstract

This research analyzed the effects of inoculation with Rhizobium tropici, combined with beneficial microorganisms, on the reduction of NPK fertilization and its impacts on common beans. Conducted in two types of soil (clayey Oxisol and sandy Ultisol), the experimental design was a randomized block design under a factorial scheme 4 × 4, with different combinations of inoculation (Rhizobium tropici alone or in combination with Azospirillum brasilense, Bacillus subtilis, and Trichoderma harzianum) and fertilization doses (0%, 33%, 66%, and 100% of the recommended dose). The results showed that inoculation with R. tropici, especially in combination with Trichoderma harzianum, increased nodule formation and improved agronomic parameters such as leaf chlorophyll, dry matter of the plant, number of pods, and grains. Co-inoculation with Azospirillum brasilense resulted in a significant increase in grain yield, particularly in clayey soil with 33% NPK. Inoculation with lower doses of NPK was sufficient for good yield, suggesting the feasibility of reducing the use of mineral fertilizers. This study indicates that soils with corrected fertility, in a no-tillage system, can contribute to the reduction of fertilizer use due to the cycling of organic matter and improvement of soil health. Additionally, the use of microorganisms is an effective ally for the sustainability of agroecosystems.

1. Introduction

The common bean (Phaseolus vulgaris L.) is a plant of the Fabaceae family of extreme social and economic importance in many countries. It is present in 50% of the leguminous grains supplied to thousands of people. It is among the three most important legumes in the world, along with soybeans (Glycine max L.) and peanuts (Arachis hypogaea L.) [1,2].

Common bean is of great socioeconomic importance because of the processes involved in their cultivation until harvest. They are responsible for generating jobs and income, especially for low-income populations. They are essential protein, iron, phosphorus, magnesium, and manganese sources. In addition to being low in fat and sodium, they do not contain cholesterol, are rich in B vitamins, and are a food with a high content of fiber and complex carbohydrates [3].

It is a plant that can grow in different soil and climate conditions. It has a wide variety of grains of various colors and sizes. In many places, it is considered the main accessible source of protein, vitamins, and minerals. It is the staple food of the population, as is the case in Brazil, where it is a major producer and consumer of this legume in the population’s basic diet [4,5]. According to Conab [6], Brazil has 2.7 million hectares cultivated, producing 3036.7 million tons and an average yield of 1125 kg ha⁻¹. The crop requires adequate management in terms of fertilization. This plant is considered demanding nutrients because of its short cycle and low volume of soil explored by the roots due to its superficial and poorly developed root system [7,8]. Proper fertilization management with primary macronutrients at sowing, such as nitrogen, phosphorus, and potassium, is essential for the initial growth of the root system and plant viability throughout the vegetative cycle [9,10].

These nutrients are directly related to critical physiological processes, such as photosynthetic and respiratory processes, biological nitrogen fixation by legumes, and the activation of enzymatic sites [11]. The low availability of these nutrients in the soil is one factor limiting common bean yield [12,13].

The NPK recommendation for common bean crops for tropical agriculture considers the class of nutrient contents in the soil analysis and the expected yield. Nitrogen is recommended to be applied partially at sowing, with the rest applied as a top dressing, at a dose of 10 to 40 kg ha⁻¹ at sowing and 80 to 120 kg ha⁻¹ as a top dressing. Phosphorus is recommended at doses of 20 to 140 kg ha⁻¹ at sowing, and potassium can also vary from 30 to 140 kg ha⁻¹ at sowing [14]. The main sources of mineral fertilizers that provide these nutrients are expensive; in some cases, they are poorly assimilated by plants, require high energy consumption in their manufacturing, and are related to environmental pollution [15,16,17,18]. Brazil is responsible for approximately 8% of global fertilizer consumption, ranking fourth behind China, India, and the United States [19,20].

As a leguminous plant species, common beans can associate with diazotrophic microorganisms present in the soil, called rhizobia [21]. Through this interaction between rhizobia and leguminous plants, there is an exchange of molecular signals and gene expression that results in infection and the formation of typical structures in the roots called nodules, whose structures are responsible for biological nitrogen fixation [22].

The use of growth-promoting microorganisms can be used as a technology to benefit plants; it is an alternative to increase the yield and efficiency of fertilization and consequently reduce yield costs, in addition to contributing to sustainable management in agriculture [23,24,25].

Co-inoculation is the combination of two or more species of beneficial microorganisms that produce synergistic effects, stimulating each other through biochemical mechanisms [23]. This agricultural practice is based on exploring their different mechanisms of action, in addition to biological nitrogen fixation (BNF), such as phosphate solubilization, biocontrol, and growth regulators, which can positively influence crop development [26].

Considering the observed benefits, according to Hungria et al. (2013) [23], the inoculation of common bean seeds with Rhizobium tropici increased the grain yield by 98 kg ha⁻¹ (8.3%), and when associated with Azospirillum brasilense, the yield increased from 285 kg ha⁻¹ (19.6%). Other authors, such as Mortinho et al. (2022) [27], also reported increases in grain yield with the co-inoculation of R. tropici + A. brasilense, reducing the sowing fertilization dose with NPK by 50%. In addition, Silva et al. (2023) [13] reported that the inoculation of Trichoderma harzianum with a 50% reduction in the fertilizer dose had positive effects on the phenology and biomass of P. vulgaris through the conversion of root exudates into plant growth-promoting substances.

Considering that common beans are a crop of economic importance, these species deserve to be studied due to the lack of research, facilitating the understanding of the dynamics of interactions between microorganisms and the environment. Since their use can not only increase yield but also reduce the use of NPK fertilizers, thus lowering yield costs [21,23]. This fact is extremely important for making the crop more profitable and attractive, as the ability of microorganisms to assist in biological nitrogen fixation (BNF) can reduce the use of nitrogen fertilizers to zero doses, as happens with Brazilian soybeans [18,21]. Bacteria such as R. tropici, A. brasilense, and B. subtilis are already commonly studied, and their mechanisms of action are well explained in the literature. The fungus T. harzianum has been used in biological control, with some studies mentioning its benefits as a growth promoter and stress reducer under water deficiency [13]. Due to its complexity and the lack of information in the literature, it deserves to be a focus of study.

Given the benefits observed for R. tropici alone or in association with other beneficial microorganisms through inoculation and co-inoculation and the lack of information on its effects on common bean crops, the hypothesis of this study is that co-inoculation with beneficial microorganisms allows for the reduction or partial replacement of NPK fertilization in common bean crops in sandy and clayey soils. Another hypothesis is that soil texture or granulometry influences biological nitrogen fixation and the beneficial actions of multifunctional microorganisms that are reflected in the vegetative and productive performance of common beans.

Given the above, the objective of this study was to evaluate the effects of Rhizobium tropici inoculation, both alone and when co-inoculated with beneficial microorganisms, associated with a reduction in the base fertilization dose with NPK on the productive components and grain yield of common bean, in sandy and clayey soils under long-term no-tillage system. At the end, it will be possible to verify the influence of soils and their texture, as well as whether management practices can assist in the response to management.

2. Materials and Methods

2.1. Characterization and Location of the Experimental Area

The study comprised two experiments conducted under field conditions during the fall–winter period at two different locations on soils belonging to two distinct classifications and with contrasting textures (clayey and sandy). The first location is located at the Teaching, Research, and Extension Farm of the Ilha Solteira School of Engineering, São Paulo State University, in the municipality of Selvíria, Mato Grosso do Sul (51°22′ West longitude and 20°22′ South latitude), at an altitude of 335 m (Figure 1A). According to the Köpen classification, the location’s climate is humid tropical with a rainy season in summer and a dry season in winter, which are the fundamental type Aw, with average rainfall and average annual temperature of 1332 mm and 23 °C, respectively. The soil at this location is classified as a dystrophic Red Latosol with a clayey texture [28], corresponding to a clayey oxisol, according to soil taxonomy [29]. The experimental area has been under a consolidated no-tillage system for 17 years and has a history of crop rotation of soybeans, corn, common bean, and wheat, with millet having been cultivated in the previous harvest (summer). The second location is located in the experimental area of the educational institution, the Faculty of Agricultural Sciences and Technology, Universidade Estadual Paulista, which is located in the municipality of Dracena, São Paulo (51°52′ West Longitude and 21°29′ South Latitude), with an altitude of 420 m (Figure 1B). The average annual temperature is 24 °C, and the average annual rainfall is 1261 mm. According to the Köppen classification, the climate type of the region is humid tropical (Aw), classified as dry winter and hot, rainy summer. The soil at this location is considered a dystrophic Red–Yellow Argisol with a sandy texture [28], corresponding to a sandy ultisol, according to soil taxonomy [30]. The area has a common bean and corn cultivation history, with corn being grown in the previous harvest (summer). The climatic data on rainfall and maximum and minimum air temperatures for the two locations, which occurred during the experimental period, are shown in Figure 2A,B.

The common beans are commonly produced in regions of Brazil such as the states of São Paulo, Paraná, Minas Gerais, Mato Grosso, Goiás, Bahia, Ceará, and Pernambuco, with the Northeast region being responsible for 75% of the national yield. The common bean crop prefers well-drained soils rich in organic matter (OM) [1,3]. In the country, yield occurs in various locations with different soil textures, which impacts management and cultivation practices. In the Northeast region, there is a predominance of sandy and sandy-loam soils, which are shallow, making the crop’s response to sandy soils crucial for improving yield in these areas [1,6]. Due to the complexity of management and response, especially in sandy soils, the research focuses on the crop’s response in different locations and soil textures, considering the history of national yield.

Before the installation of the experiments, the chemical and physical attributes of the soil were characterized at both locations by collecting a composite sample from 20 simple samples of disturbed soil throughout the experimental area, according to the methodology proposed by Raij et al. (2001) [30], in the 0.00–0.20 and 0.20–0.40 m layers. These analyses allowed for the identification of soil fertility, as well as the response of soil management in a no-tillage system over long periods, thus understanding the dynamics to which these microorganisms would be exposed and the response that could be observed in the end, due to the influence of fertility. In the location of Selvíria, MS (clayey soil), the following results were presented for the 0.00–0.20 m and 0.20–0.40 m layers: 24 and 13 mg dm⁻³ of P (resin); 20 and 18 g dm⁻³ of M.O.; pH 5.4 and 5.3 (CaCl₂); K⁺, Ca²⁺, Mg²⁺, H + Al, SB and CTC = 2.6 and 1.3; 43 and 20; 13 and 12; 25 and 25; 61.6 and 33.3 and 86.6 and 58.3 mmolc dm⁻³; S-SO₄, B, Cu, Fe, Mn and Zn = 7 and 12; 0.07 and 0.08; 5.6 and 5.5; 21 and 19; 24.6 and 24.7; 1.2 and 0.7 mg dm⁻³, respectively; and 71 and 57% base saturation, respectively. In Dracena, SP (sandy soil), the results presented in the 0.00–0.20 m and 0.20–0.40 m layers were as follows: 13 and 6 mg dm⁻³ of P (resin); 11 and 10 g dm⁻³ M.O.; pH 5.9 and 5.5 (CaCl₂); K⁺, Ca²⁺, Mg²⁺, H + Al, SB and CTC = 1.4 and 1.2; 14 and 10; 7 and 5; 11 and 12; 22.4 and 16.2 and 33.4 and 28.2 mmolc dm⁻³; S-SO₄, B, Cu, Fe, Mn, and Zn = 4 and 4; 0.26 and 0.21; 0.4 and 0.5; 12 and 12; 9.3 and 9.8; 0.7 and 0.5 mg dm⁻³, respectively; and 67 and 57% base saturation, respectively.

The granulometric analyses were performed via the pipette method according to the methodology described [31], whose values were as follows: 471, 439, and 90 g kg⁻¹ of clay, sand, and silt in the clayey soil and 105, 822, and 73 g kg⁻¹ of clay, sand, and silt in the sandy soil, respectively.

2.2. Treatments and Experimental Design

The experimental design used was randomized blocks arranged in a 4 × 4 factorial scheme with four replications. The treatments consisted of four types of co-inoculation (1- control, inoculation only with Rhizobium tropici (control), 2- R. tropici + Azospirillum brasilense, 3- R. tropici + Bacillus subtilis, and 4- R. tropici + Trichoderma harzianum) associated with the application of 0%, 33%, 66%, and 100% of the amount of base fertilizer with N-P₂O₅-K₂O recommended for common beans at sowing, respectively (Figure 3). Mineral fertilization was carried out in the common bean sowing furrow on the basis of the analysis of initial soil fertility and crop requirements, following the recommendation [14]; considering the respective previously established treatments, a physical mixture of commercial N-P₂O₅-K₂O (NPK) (Aduborg, Aduborg Fertilizantes, Itapetininga, São Paulo, Brasil) formula 08-28-6 was used. NPK fertilization was carried out in doses of (zero) as a control treatment, followed by 33%, 66%, and 100%, in which the 100% dose was selected on the basis of the results of the soil analysis, and the expected grain yield ranged from 4000 to 5000 kg ha⁻¹ for each location studied. In the seeding fertilization for the locality of Selvíria, MS, the recommended doses of 100%, 66%, and 33% NPK (N-P₂O₅-K₂O) in the clay soil were 235, 155, and 78 kg ha⁻¹, respectively. In the locality of Dracena, SP, the recommended doses of 100%, 66%, and 33% NPK in sandy soil were 357, 235, and 118 kg ha⁻¹, respectively.

The experimental plots consisted of six rows five meters in length, with the four central rows considered useful areas, disregarding 0.5 m at both ends of each row. The spacing between rows was 0.45 m, with a seed distribution to obtain 10 plants per meter of row.

Before sowing, the weeds were desiccated via the herbicides glyphosate (1800 g ha⁻¹ of active ingredient (a.i.)) (Bayer, Wuppertal, Renânia do Norte-Vestfália, Germany) and 2,4-D (670 g ha⁻¹ of a.i.), 15 days before sowing (DAS), followed by preparation of the area with a mechanical disintegrator of plant residues (Triton).

2.3. Installation and Conduct of the Experiment

The experiment was carried out with the common bean cultivar IAC 1850 (Instituto Agronômico de Campinas, State of São Paulo, Brazil), with indeterminate growth habit type II and grains of the carioca type, which are recommended and adapted to both regions. The seeds were previously treated with insecticides and fungicides (Standak Top, Basf, Ludwigshafen, Reno, Germany), such as pyraclostrobin (2.5 g ha⁻¹ a.i.), thiophanate methyl (22.5 g ha⁻¹ a.i.), and fipronil (25 g ha⁻¹ a.i.), on the basis of the manufacturer’s recommendations for the crop. Following the drying of the seeds treated with insecticide and fungicide, the common bean seeds were inoculated with Rhizobium tropici (SEMIA 4077) (Nitro 1000, Cascavel, State of Parana, Brazil) as a peat inoculant at a dose of 200 g for 100 kg of seeds with a guaranteed concentration of 2 × 10⁹ CFU g⁻¹. A 10% sugar solution was added to the seeds for the adhesion of the bacteria to the seeds. The co-inoculations of the seeds inoculated with the beneficial microorganisms were carried out according to the manufacturer’s recommendation for the formulation used. For co-inoculation with Azospirillum brasilense (strains Ab-V5 and Ab-V6) (Azotrop, Biotrop, Vinhedo, State of São Paulo, Brazil), a dose of 100 mL of liquid inoculant was used for 100 kg of seeds (guarantee of 2 × 10⁸) CFU mL⁻¹); inoculation with Bacillus subtilis (strain CCTB04) (Panta Premium, Geoclean, Araraquara, State of São Paulo, Brazil) occurred at a dose of 100 mL for 100 kg of seeds (guarantee of 1 × 10⁸ CFU mL⁻¹). The inoculation with Trichoderma harzianum (strain T-22) (Tritter, NOOA Brasil, Indaiatuba, State of São Paulo, Brasil) was performed at a dose of 100 g of inoculant (wettable powder) per 100 kg of seeds (guarantees of 1 × 10⁸ CFU mL⁻¹). The inoculations and co-inoculations of the seeds were carried out in the shade two hours before sowing the common bean. The first experiment was sown on 6 May 2023 in Selvíria/MS via the direct planting system. The second experiment in Dracena, SP, was sown on 5 June 2023, via direct seeding, without soil preparation before installation. Both experiments were sown mechanically, using a tractor and a six-row pneumatic seeder-fertilizer, with a setting of 15 seeds per meter of furrow. All preparations followed the same standard in both soil types, and the management aimed to maintain the same cultural practices to avoid influencing the final response in both locations.

Nitrogen topdressing was applied between the common bean rows, at a dose of 45 kg ha⁻¹ of nitrogen, in the form of urea, in all the treatments, 30 days after plant emergence (DAE) in Selvíria, MS. In the experiment in the Dracena area, SP, owing to the sandy texture of the soil, was used, and the application of nitrogen fertilization was divided into topdressing doses, 50% of the dose when the plants reached the V3 stage and 50% at the V4–5 stage, as recommended [14].

The area located in Selvíria, MS, was irrigated by a central pivot, with an average depth of 14 mm every three days or when necessary. The second experimental area located in Dracena, SP, was irrigated by a conventional sprinkler with 80% efficiency, whose sprinkler used was the Agropolo, model NY25 with 2.8 × 2.5 mm nozzles, composed of two lines with eight sprinklers in each, with a spacing of 12 m between the sprinklers, to obtain good application uniformity with a net depth of 4.9 mm h⁻¹ at a service pressure of 2.0 kgf cm⁻². Irrigation was carried out at an average depth of 14 mm every four days or when necessary.

At full flowering, when the plants reached 50% open flowers, the roots of five plants from each plot were collected with the aid of a hoe. The roots were detached from the entire plant, and the nodules were removed, washed for counting, and subsequently dried in a forced ventilation oven at 65 °C for 72 h, followed by weighing. The relative leaf chlorophyll content was also measured indirectly via readings from the portable chlorophyll meter ClorofiLOG^®, model CFL 1030 (Falker Automação Agrícola^®), which uses a third fully expanded trifoliate from the apex, in five plants in the useful area of each plot.

At the time of full flowering, five plants were collected at a predetermined location in the useful area of the plots, placed in paper bags, duly identified and subjected to drying in a forced ventilation oven at an average temperature of 65 °C for 72 h. Subsequently, the samples were weighed, and the values were converted to g plant⁻¹.

At harvest, five plants were collected from the useful area of the plots to evaluate the number of pods per plant, which was determined by the ratio of the total number of pods/number of plants; the number of grains per plant, which was determined by the ratio of the total number of grains/number of plants; and the weight of one hundred grains, which was obtained via random collection and weighing of a sample of one hundred grains per plot.

The plants from the useful area of the plots were harvested and left to dry in full sun. After drying, they were subjected to mechanical threshing, the grains were weighed, and the data were transformed into kg ha⁻¹ on a 13% wet basis.

2.4. Statistical Analysis of Data

The data were subjected to analysis of variance (F test) after normality and homoscedasticity. The means were compared via the Tukey test at 5% significance for the inoculation of R. tropici and co-inoculation of beneficial microorganisms.

When a significant interaction between the sources of variation was found, unfolding was performed, and the means were compared via the Tukey test, adopting 5% significance [32]. The R program version 4.3.2 [33] was used with the ExpDes.pt package.

The doses of NPK fertilizer used during sowing were analyzed via polynomial regression, adjusted to the models of significant linear and quadratic equations via the F test, and plotted in graphs with the aid of the SigmaPlot^® [34].

3. Results

3.1. Analysis for Unidirectional Statistics in Clayey and Sandy Soils

The emergence of common bean plants at both cultivation sites occurred uniformly on the sixth day after sowing (DAS). Full flowering of common bean occurred 40 and 42 days after plant emergence (DAE), respectively, in clayey and sandy soils. Common bean were harvested at 96 DAE in the Red Latosol and 94 DAE in the Red–Yellow Argisol.

There were significant interactions of NPK level × co-inoculation (CI) for plant dry mass, number of nodules per plant, number of pods per plant, and common bean grain yield (

Table 1. Average values of relative leaf chlorophyll content (ICF), plant dry mass (MSP), number of nodules per plant (NP), nodule dry mass (NDM), number of grains per plant (NGP), number of pods per plant (NPP), hundred-grain mass (M100G) and grain yield (GP), of common bean co-inoculated with beneficial microorganisms associated with reduced sowing fertilization NPK, in clayey oxisol, Selvíria, Mato Grosso do Sul, Brazil, 2023 harvest.

	ICF	MSP	NP	NDM	NGP	NPP	M100G	GP
		g plant−1		g plant−1			g	kg ha−1
F values
CI	0.000 *	0.633 ns	0.247 ns	0.642 ns	0.004 *	0.000 *	0.447 ns	0.079 ns
NPK	0.000 *	0.601 ns	0.000 *	0.927 ns	0.000 *	0.002 *	0.000 *	0.000 *
CI × NPK	0.000 *	0.006 *	0.032 *	0.134 ns	0.000 *	0.001 *	0.294 ns	0.000 *
Least Significant Difference (LSD 5%)
CI	5.09	2.58	5.96	0.08	16.50	2.94	0.85	310.22
CV (%)	15.12	23.50	35.80	31.65	17.06	24.29	3.74	11.70
Overall average	35.75	11.64	17.64	0.16	102.64	12.86	24.17	2.810
Standard error	1.35	0.68	1.57	0.02	4.37	0.78	0.22	82.20
Co-inoculation of microrganisms (CI)
R. tropici	41.3	12.4	16.3	0.16	110.6	15.4	24.1	2.900
R. tropici + A. brasilense	33.8	11.5	18.1	0.16	92.2	10.7	23.9	2.789
R. tropici + B. subtilis	34.6	11.3	16.0	0.14	96.6	11.8	24.5	2.914
R. tropici + T. harzianum	33.2	11.4	20.1	1.18	111.2	13.5	24.1	2.638
NPK doses (%)
Zero	41.1	11.4	12.3	0.17	87.3	12.5	24.8 (1)	2.725
33%	37.8	11.4	14.7	0.15	112.1	14.6	24.3	2.887
66%	28.2	11.8	22.6	0.16	100.2	10.4	23.9	2.893
100%	35.7	12.1	21.0	0.16	110.8	13.9	23.6	2.736

Note: CI: Co-inoculation; *—Significant at 5% significance; ^ns—not significant by the F test. Significant for quadratic regression ⁽¹⁾ $y=0.33x^2-1.55x+24.81$(R² = 50.97%).

and

Table 2. Average values of relative leaf chlorophyll content (ICF), plant dry mass (MSP), number of nodules per plant (NP), nodule dry mass (NDM), number of grains per plant (NGP), number of pods per plant (NPP), hundred-grain mass (M100G) and grain yield (GP), of common bean co-inoculated with beneficial microorganisms associated with reduced sowing fertilization NPK, in sandy ultisol, Dracena, São Paulo, Brazil, 2023 harvest.

	ICF	MSP	NP	NDM	NGP	NPP	M100G	GP
		g plant−1		g plant−1			g	kg ha−1
F values
CI	0.983 ns	0.425 ns	0.240 ns	0.401 ns	0.032 *	0.045 *	0.865 ns	0.909 ns
NPK	0.000 *	0.009 *	0.014 *	0.402 ns	0.000 *	0.006 *	0.000 *	0.268 ns
CI × NPK	0.165 ns	0.039 *	0.033 *	0.454 ns	0.432 ns	0.030 *	0.106 ns	0.000 *
Least Significant Difference (LSD 5%)
CI	4.44	1.66	3.39	0.07	14.40	2.39	1.22	541.12
CV (%)	10.12	25.42	34.30	37.30	31.25	21.84	5.23	24.55
Overall average	46.54	6.92	8.12	0.80	48.90	11.63	24.85	2.336
Standard error	1.17	0.44	0.89	7.36	3.81	0.63	0.32	143.38
Co-inoculation of microrganisms (CI)
R. tropici	46.4	7.4	7.8	0.80	40.6 b	12.8	24.6	2.315
R. tropici + A. brasilense	46.7	7.1	7.3	0.78	46.2 ab	12.2	24.9	2.293
R. tropici + B. subtilis	46.2	6.4	7.6	0.79	55.2 a	11.2	24.9	2.426
R. tropici + T. harzianum	46.8	6.7	9.7	0.80	53.6 a	10.3	24.9	2.310
NPK doses (%)
Zero	57.9 (2)	5.6	6.5	0.80	53.4 (3)	10.1	23.4 (4)	2.109
33%	48.0	7.3	10.3	0.80	57.2	11.0	25.3	2.380
66%	41.6	7.2	8.7	0.81	50.6	12.0	25.8	2.473
100%	38.6	7.5	6.9	0.77	34.4	13.3	24.9	2.383

Note: CI: Co-inoculation. *—Significant at 5% significance; ^ns—not significant by the F test. Means followed by the same letter, within co-inoculation, do not differ statistically at 5% significance. Significant for quadratic regression ⁽²⁾ $y=15.68x^2-34.95x+57.87\left(R^2=100\%\right);$ ⁽³⁾ $y=-44.78x^2+25.61x+53.44\left(R^2=99.95\%\right)$; ⁽⁴⁾ y = −6.14x² + 7.63x + 23.42 (R² = 100%).

) at both cultivation sites (Selvíria-MS and Dracena-SP). In addition, there were significant interactions between the relative leaf chlorophyll content and the number of grains per plant according to Selvíria-MS.

An analysis of the effects of the co-inoculation of microorganisms on the dry mass of common bean plants in clayey soil (clayey oxisol) revealed that the greatest increases in dry mass occurred at doses of 0% and 33% NPK, but only with inoculation with R. tropici. These two doses of NPK presented the highest averages, in the absence of NPK fertilization (zero), at 14.3 g plant⁻¹ and at 33% of the recommended dose of 13.4 g plant⁻¹ (

Table 3. Breakdown of the significant interaction of the analysis of variance for plant dry mass and relative leaf chlorophyll content of common bean co-inoculated with beneficial microorganisms associated with reduced sowing fertilization NPK, in clayey oxisol (Selvíria, Mato Grosso do Sul) and sandy ultisol (Dracena, São Paulo).

Dry mass of plants (g planta−1)—Selvíria, Mato Grosso do Sul
Clayey Oxisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	14.3 a	13.4 a	11.2	10.8	n.s
R. tropici + A. brasilense	11.5 ab	12.8 ab	9.0	12.8	n.s
R. tropici + B. subtilis	11.9 ab	7.7 b	12.9	12.6	QR (1)
R. tropici + T. harzianum	7.8 b	77.8 ab	12.3	13.6	QR (2)
LSD (5%) Co-inoculation within NPK-5.16
Relative chlorophyll content—Selvíria, Mato Grosso do Sul
Clayey Oxisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	58.9 a	40.1	33.6	32.4 ab	QR (3)
R. tropici + A. brasilense	30.8 b	38.3	27.0	38.9 ab	QR (4)
R. tropici + B. subtilis	39.1 b	34.1	24.1	41.3 a	QR (5)
R. tropici + T. harzianum	35.7 b	38.8	28.1	30.3 b	QR (6)
LSD (5%) Co-inoculation within NPK-10.19
Dry mass of plants (g planta−1)—Dracena, São Paulo
Sandy Ultisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	5.4	8.6	9.1	6.5	QR (7)
R. tropici + A. brasilense	6.5	7.3	5.9	8.9	n.s
R. tropici + B. subtilis	6.5	5.6	6.7	6.9	n.s
R. tropici + T. harzianum	4.1	7.9	7.1	7.8	QR (8)
LSD (5%) Co-inoculation within NPK—3.32

Note: Averages followed by the same letter, lowercase in the columns, do not differ statistically from each other by the Tukey’s test at 5% significance. LSD: least significant difference; n.s: not significant for quadratic or linear regression; QR: significant for quadratic regression; ⁽¹⁾ $y=8.80x^2-6.67x+11.21 \left(R^2=36.13\%\right)$; ⁽²⁾ y = −5.96x² + 11.35x + 8.02 (R² = 94.79%); ⁽³⁾ y = 40.12x² − 65.89x + 58.56 (R² = 99.47%); ⁽⁴⁾ y = 10.20x² − 6.19x + 32.92 (R² = 13.92%); ⁽⁵⁾ y = 50.24x² − 51.15x + 40.69 (R² = 71.11%); ⁽⁶⁾ y = −1.49x² − 6.55x + 37.06 (R² = 99.93%); ⁽⁷⁾ y = −13.27x² + 14.34x + 5.40 (R² = 99.93%); ⁽⁸⁾ y = −7.07x² + 10.17x + 4.38 (R² = 81.57%).

). In addition, there was an increasing quadratic adjustment of the NPK doses for the co-inoculation of R. tropici + T. harzianum with the maximum yield of dry mass of plants at the optimal dose of 94.58% of the recommended dose of NPK, whose increase in dry mass increased when more NPK was offered in clayey soil (Figure 4A). In sandy soil (sandy ultisol), the treatments did not significantly affect the plant dry mass, as observed in the breakdown of the means (Table 3). There was a quadratic fit for the inoculation of only R. tropici and R. tropici + T. harzianum, with maximum dry mass yield related to the optimal doses of 55.15% and 72.57% NPK, respectively (Figure 4A,B). Notably, the dry mass yield of common bean plants was greater when there was an increase in NPK dose for both types of co-inoculation, a result similar to that which also occurred in clayey soil (Figure 5A,B).

3.2. Evaluation of the Outcomes of Experiments Conducted in Clayey and Sandy Soils

With respect to the relative leaf chlorophyll content evaluated at the time of full flowering of the plants, a significant effect was observed for the co-inoculation of microorganisms, for the NPK doses and for the interaction of the co-inoculation of microorganisms with the NPK doses in clayey soil (Table 1). It is possible to observe that only the inoculation with R. tropici at the zero-dose resulted in a relatively high leaf chlorophyll content, with a value of 58.9, a result similar to that found at the 100% NPK dose in the co-inoculation of R. tropici + B. subtilis, with a value of 41.3 (Table 3). Analyzing the performance of microorganisms through quadratic adjustments, the inoculation of R. tropici tended to decrease, with the highest concentration of leaf chlorophyll occurring at the optimal dose of 0.82% and decreasing as the NPK dose increased (Figure 4B).

The co-inoculation of R. tropici + T. Harzianum showed positive increases in nodulation in clayey soil (clayey oxisol), in which the highest number of nodules was observed at the 100% NPK dose, with an average of 28.5 nodules per plant (

Table 4. Breakdown of the significant interaction of the analysis of variance for the number of nodules per plant of common beans co-inoculated with beneficial microorganisms associated with reduced sowing fertilization NPK, in clayey oxisol, Selvíria, Mato Grosso do Sul, and sandy ultisol, Dracena, São Paulo, 2023 harvest.

Number of nodules per plant—Selvíria, Mato Grosso do Sul
Clayey Oxisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	12.0	17.1	17.8	18.2 ab	n.s
R. tropici + A. brasilense	12.7	10.7	25.9	23.2 ab	QR (9)
R. tropici + B. subtilis	10.6	11.7	27.7	14.0 b	QR (10)
R. tropici + T. harzianum	13.8	19.2	18.8	28.5 a	QR(11)
MSD (5%) Co-inoculation within NPK-11.92
Number of nodules per plant—Dracena, São Paulo
Sandy Ultisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	5.8	9.2 b	10.4	6.0	n.s
R. tropici + A. brasilense	6.0	6.8 b	8.0	8.3	n.s
R. tropici + B. subtilis	6.7	7.9 b	8.8	7.0	n.s
R. tropici + T. harzianum	7.5	17.5 a	7.6	6.2	QR (12)
MSD (5%) Co-inoculation within NPK-6.78

Note: Averages followed by the same letter, lowercase in the columns, do not differ statistically from each other by the Tukey’s test at 5% significance. MSD: minimum significant difference; n.s: not significant for quadratic or linear regression; QR: significant for quadratic regression. ⁽⁹⁾ $y=-2.23x^2+16.16x+110.97\left(R^2=63.61\%\right)$; ⁽¹⁰⁾ $y=-33.48x^2+41.69x+8.34\left(R^2=47.88\%\right)$; ⁽¹¹⁾ $y=9.28x^2+3.85x+14.59\left(R^2=89.08\%\right)$; ⁽¹²⁾ $y=-25.09x^2+20.90x+9.00\left(R^2=50.05\%\right).$

). For this co-inoculation, the data were adjusted to a quadratic model, in which there was an increase in nodulation in common bean plants as the NPK dose increased, with the highest number of nodules being related to the 100% NPK dose in clayey soil (clayey oxisol) (Figure 4C).

With respect to the nodulation of common bean plants in sandy soil, there was a significant effect of the NPK dose and the interaction of the co-inoculation of microorganisms with the NPK dose (Table 2). Similar to the results reported in clayey soil, the co-inoculation of R. tropici + T. harzianum increased the number of nodules at the 33% NPK dose, with an average of 17.5 nodules per plant (Table 4).

An analysis of the effects of the average NPK dose within the co-inoculation of microorganisms on the number of common bean grains per plant when grown in clayey soil (clayey oxisol) revealed that the greatest highlight was at the dose of 33% NPK associated with the inoculation of R. tropici, with an average of 153.2 grains per plant (

Table 5. Breakdown of the significant interaction of the analysis of variance for the number of pods per plant and number of grains per plant of common beans co-inoculated with beneficial microorganisms associated with reduced sowing fertilization NPK, in clayey oxisol, Selvíria, Mato Grosso do Sul and sandy ultisol, Dracena, São Paulo, Brazil, 2023 harvest.

Number of pods per plants—Selvíria, Mato Grosso do Sul
Clayey Oxisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	15.7	17.3	16.3 a	12.5 ab	n.s
R. tropici + A. brasilense	10.0	13.1	4.1 c	15.6 ab	QR (13)
R. tropici + B. subtilis	13.6	13.4	9.6 bc	10.5 b	n.s
R. tropici + T. harzianum	10.8	14.6	11.6 ab	16.9 a	QR (14)
MSD (5%) Co-inoculation within NPK—5.89
Number of pods per plants—Dracena, São Paulo
Sandy Ultisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	12.2 a	13.5 a	13.1	12.2	n.s
R. tropici + A. brasilense	12.4 a	10.8 ab	10.3	15.1	QR (15)
R. tropici + B. subtilis	6.9 ab	8.6 b	13.0	13.5	QR (16)
R. tropici + T. harzianum	6.3 b	10.9 ab	11.8	12.3	QR (17)
MSD (5%) Co-inoculation within NPK—4.80
Number of grains per plant—Selvíria, Mato Grosso do Sul g planta−1
Clayey Oxisol	NPK Dose
Co-inoculation	0%	33%	66%	100%	Regression analysis
R. tropici	85.2	153.2 a	111.5	92.5	QR (18)
R. tropici + A. brasilense	88.7	85.0 b	85.5	109.5	n.s
R. tropici + B. subtilis	78.2	102.7 b	88.2	116.5	QR (19)
R. tropici + T. harzianum	96.7	107.5 b	115.7	124.7	n.s
MSD (5%) Co-inoculation within NPK—33.04

Note: Averages followed by the same letter, lowercase in the columns, do not differ statistically from each other by the Tukey’s test at 5% significance. MSD: Minimum significant difference; n.s: not significant for quadratic or linear regression; QR: significant for quadratic regression  ⁽¹³⁾ $Y=18.96x^2-16.62x+11.66\left(R^2=28.62\%\right)$; ⁽¹⁴⁾$y=-9.88x^2+8.96x+8.81\left(R^2=70.94\%\right)$; ⁽¹⁵⁾$y=14.35x^2-12.05x+12.65\left(R^2=94.19\%\right)$; ⁽¹⁶⁾$y=3.27x^2-1.56x+9.16\left(R^2=74.91\%\right)$; ⁽¹⁷⁾$y=-9.40x^2+15.06x+6.47\left(R^2=97.32\%\right)$; ⁽¹⁸⁾$y=-193.80x^2+187.72x+92.06\left(R^2=67.59\%\right)$; ⁽¹⁹⁾ $y=9.67x^2+20.05x+82.84\left(R^2=60.24\%\right)$.

).

By increasing the NPK dose within the co-inoculation of microorganisms (CI), it was observed that the co-inoculation of R. tropici in clayey soil at a dose of 66% NPK increased the number of pods per plant, with an average of 16.3 pods per plant (Table 5). At a dose of 100% NPK, co-inoculation with R. tropici + T. harzianum presented the highest average, with 16.9 pods per plant (Table 5). In sandy soil (sandy ultisol), the inoculation of R. tropici and the co-inoculation of R. tropici + A. brasilense in the absence of NPK (zero), the number of pods per plant increased, with averages of 12.2 and 12.4 pods per plant (Table 5). Similar to the results found in clayey soil, the inoculation of R. tropici in sandy soil resulted in the highest average number of pods per plant; however, this occurred at a dose of 33% NPK (Table 5).

In sandy soil (sandy ultisol), there was a quadratic adjustment for the co-inoculation of R. tropici + A. brasilense and R. tropici + T. harzianum; these co-inoculations increased, with the highest number of pods per plant being observed at a dose of 100% NPK (Figure 5C,D).

In the absence of sowing fertilization (zero) of NPK, only the inoculation of R. tropici was sufficient to show superior performance to the other co-inoculations, with an average grain yield of 3218 kg ha⁻¹ (Figure 6A). When the NPK dose was 66%, positive effects on grain yield of 3178 kg ha⁻¹ were observed only through the inoculation of R. tropici (Figure 6A). At 33% of the NPK dose, the best performance of the co-inoculation of R. tropici + A. brasilense was observed in relation to all the other doses tested, with an average yield of 3722 kg ha⁻¹ (Figure 6B). When the NPK dose was 100%, the co-inoculation of R. tropici + B. subtilis resulted in a greater increase in yield, with a value of 3448 kg ha⁻¹ (Figure 6D).

In sandy soil, there was a significant interaction effect between the co-inoculation of microorganisms and the NPK dose on grain yield. The breakdown of the means showed a significant difference only for the doses of 33% and 100% NPK (Figure 7B,D). At 33%, only R. tropici inoculation resulted in the highest mean yield, at 2932 kg ha⁻¹, followed by the co-inoculation of R. tropici + A. brasilense with 2619 kg ha⁻¹ and R. tropici + T. harzianum with 2765 kg ha⁻¹. The 100% NPK dose with the co-inoculation of R. tropici + B. subtilis had the highest mean grain yield of all the doses and co-inoculations tested, with a mean of 3245 kg ha⁻¹ (Figure 7D).

There was a quadratic adjustment for the inoculation of R. tropici; the maximum yield of common bean grains occurred at the optimal dose of 40.29% NPK for the inoculation of R. tropici, and the grain yield decreased as the dose of NPK increased (Figure 8A). The inoculation of R. tropici was adjusted to a decreasing quadratic model, and it was noted that there was greater grain yield at the optimal dose than at the 0.25% NPK dose in clayey soil (clayey oxisol) (Figure 8B).

The high grain yield observed in this study with the use of R. tropici + A. brasilense is due to the mechanism of action of the bacteria, as they assist in biological nitrogen fixation (BNF), and A. brasilense acts as a growth promoter, improving root exploration in the soil. In general, what was noted in the study is that the response to fertilization and the use of microorganisms in both soils follows a similar standard, which may be influenced by texture, but here it was also affected by the length of management, which is why the standard response was observed at lower doses. This can be explained by the time the soils have been managed under a no-tillage system, which has maintained good organic matter (OM) content and fertility in both locations. Due to conservation practices, the system has improved, influencing the standard response to fertilization and helping to maintain the microbial flora. For this study, it is proven that, in addition to the use of microorganisms to help reduce NPK doses for common bean cultivation, it is essential that both clayey and sandy soils maintain the pillars of the no-tillage system active for the construction of the system. Only then can we ensure effective and, most importantly, sustainable agriculture.

4. Discussion

The increase in the dry mass of common bean plants observed in clayey soil under R. tropici inoculation at lower doses of NPK (0% and 33%) is certainly related to the stimulation of biological nitrogen fixation caused by R. tropici. N is the main nutrient responsible for the increase in leaf area, and, as a consequence, it increases the photosynthetic rate of the plant, which is reflected in the increase in plant dry matter [35]. Biological nitrogen fixation (BNF) is highly influenced by soil acidity, temperature, fertility, and moisture. Clayey soils tend to be more prone to BNF than sandy soils, due to their ability to retain charges and higher concentrations of organic matter (OM), which can help regulate temperature, as well as nutrient availability, especially in tropical soils [15,23].

At relatively high doses of NPK (66% and 100%), no stimulation of biological nitrogen fixation was detected in the dry mass of the plants. This finding may be related to the excess N and the nutritional imbalance triggered by R. tropici. However, in sandy soil, the same effects could not be observed, possibly related to the difference in cation exchange capacity (CEC) and soil organic matter, which benefit the interaction of the clayey soil microbiota. In addition to the effects of losses due to leaching and volatilization in sandy soil, similar results were reported by Liliana et al. (2017) [36] in an area with higher organic matter content, where greater increases in the dry mass of the aerial parts of common bean plants inoculated with R. tropici associated with a dose of 40 kg ha⁻¹ of N in topdressing were observed.

Compared with the control treatment, the co-inoculation of R. tropici + T. harzianum had positive effects on common bean biomass, increasing the number of nodules in (36.15%) clayey soil and (47.43%) sandy soil, in addition to increasing the number of pods per plant in clayey soil at a dose of 100% NPK. It also positively affected grain yield at a dose of 33% NPK in sandy soil. The results of Liliana et al. (2017) [36] demonstrated that with the inoculation of T. harzianum in P. vulgaris with the application of only 50% of the dose of nitrogen fertilizers, a positive effect on the phenology of plant biomass and yield components was obtained, which the authors probably attributed to the conversion of seed and root exudates into the conversion of growth-promoting substances by the fungus Trichoderma sp. In the absence of NPK (zero), only the inoculation of R. tropici in clay soil increased the relative content of leaf chlorophyll.

The 33% NPK dose increased the number of grains per plant, whereas the 66% NPK dose increased the number of pods per plant. The 33% dose in sandy soil also increased the number of pods per plant. These effects can be explained by the increased efficiency of NPK fertilization when co-inoculated with R. tropici, as highlighted by Yadegari et al. (2014) [37], who reported that the number of pods per plant and the number of grains per pod increased in common bean inoculated with R. tropici, as did those inoculated with other strains of beneficial bacteria. The grain yield increased when R. tropici was inoculated without NPK fertilization and with only 33% of the recommended dose for clayey soil. Additionally, in cultivated sandy soil, the inoculation of R. tropici, associated with the 33% dose, increased the grain yield. Thus, it is evident that yield gains can be obtained in common bean crops through inoculation and the supply of nutrients at sowing, even if in a reduced form. A greater supply of N to plants in response to co-inoculation can increase chlorophyll synthesis [38], photosynthetic activity, and the vegetative growth of common bean plants and consequently increase the final yield [39].

Compared with all the other NPK doses tested, the co-inoculation of R. tropici and A. brasilense with the application of 33% of the recommended NPK dose resulted in the greatest increase in grain yield during cultivation in clayey soil (18.22%) compared with that in the control treatment and the best performance in sandy soil. There is evidence, according to Chibeba et al. (2015) [40], that one of the causes for the increase in yield in this co-inoculation is early nodulation since Azospirillum spp. colonizes the roots before Rhizobium spp. and produces flavonoids that attract this rhizobacterium. Only the inoculation of R. tropici in clayey soil in the absence of NPK fertilization was sufficient to achieve high grain yield in relation to the co-inoculation of R. tropici + B. subtilis, where a difference of 405.80 kg ha⁻¹ can be observed. Additionally, in another treatment, there was a notable difference in yield, with the use of 66% NPK in the sowing fertilization treatment, with only the inoculation of R. tropici, which presented a difference of 534.70 kg ha⁻¹ more than the co-inoculation of R. tropici + B. subtilis. When 33% of the recommended NPK dose was used and R. tropici + A. brasilense was co-inoculated, the grain yield was 678.0 kg ha⁻¹ greater in clayey soil. In contrast, in sandy soil, this same co-inoculation resulted in a difference of 312.4 kg ha⁻¹ from the inoculation of only R. tropici, corroborating the results of Hungria et al. (2013) [23], who reported that inoculation with R. tropici alone increased common bean grain yield by 98 kg ha⁻¹ (8.3%), and when R. tropici and A. brasilense were co-inoculated, yield increased by 285 kg ha⁻¹ (19.6%).

Sandy soils present a high management complexity, as their composition consists of sand (quartz), which has no charges and is known for being poor in nutrients. Therefore, these soils can deplete quickly due to their low cation exchange capacity (CEC), high sand content, and low organic matter (OM) content. Unlike sandy soils, clayey soils have a higher CEC, which facilitates charge retention, reduces leaching, and, if well managed, makes them easier to handle in agriculture and more prone to good yield [41,42]. Given the constant need for and general dependence on mineral nutrients, this research showed that combining microorganisms like R. tropici or the co-inoculation of R. tropici + A. brasilense with 33% of the recommended NPK dose, for both sandy and clayey soils, can achieve good yield for winter common bean. Since mineral nutrients are finite, reducing external use and dependence through sustainable techniques, such as the use of bacteria, can be an alternative to support the global food demand and improve the microbiology of these soils.

A fertile soil is capable of maintaining its system balanced, and management under a no-tillage system can be a practical alternative to assist in soil fertility. Especially in sandy soils, the need for OM is essential to help build the soil profile. Thus, in addition to combining microbiological techniques that assist in management, as observed in this study to balance the system and maintain high fertility levels, it is crucial to maintain the pillars of the no-tillage system. Doing so can help improve the microbiological health of the soil, which may reduce the future need for these microorganisms [21,23,43]. However, further studies on the dynamics of these microorganisms in soil and their resistance to stresses in tropical soils should be the subject of future research. In sustainable terms, combining the pillars of the no-tillage system, which improve soil fertility attributes, with the use of microorganisms has proven to be a great ally in reducing the use of mineral fertilizers, as well as contributing to the agricultural system by improving soil health and assisting in building a fertile soil profile.

Studies in the field confirm that the co-inoculation of R. tropici + A. brasilense via furrow or combined application can result in considerable yield gains, reaching up to 540 kg ha⁻¹ for common bean cultivation, similar to those observed here [44]. Furthermore, the use of R. tropici is seen as beneficial for common bean crops, as, due to its genetic characteristics, it has a lower capacity for BNF [45]. Other studies have also found that the use of R. tropici combined with insecticides does not affect grain yield and helps in the production of nodules, improving BNF, which justifies the use of microorganisms in the crop [46].

The positive results found here for T. harzianum still require further investigation. Since this species can be used in seed treatment to increase germination rates, root growth and length, seedling dry weight, and tolerance to physiological, saline, osmotic, and water stress, more research is still needed regarding its role in metabolism. However, recent studies confirm the fungus’s role in the synthesis of indole-3-acetic acid (IAA) and nutrient solubilization, such as phosphate [47,48].

Given the results obtained for common bean grain yield, greater synergy between inoculation and co-inoculation in clayey soil is evident. This fact can be attributed to the soil texture, organic matter content, porosity, and already consolidated direct planting system, in which the area where the experiment was carried out was clayey soil. This area has good soil coverage and a relatively high organic matter content, which favors moisture retention and greater soil fertility. With respect to prospects, inoculation and co-inoculation of beneficial microorganisms has proven to be an economically viable and sustainable alternative associated with reduced use of NPK during sowing, resulting in positive increases in productive components and, ultimately, grain yield. However, studies are still needed on the main effects of the synthetic machinery of plants and stimuli for biological nitrogen fixation in common bean crops.

5. Conclusions

The co-inoculation of R. tropici + A. brasilense was associated with a 66% reduction in the recommended dose of NPK at sowing, and the co-inoculation of R. tropici + B. subtilis was associated with a 100% dose of NPK fertilization, which increased the grain yield of common beans in clayey and sandy soils. The inclusion of beneficial microorganisms associated with the use of 33% mineral fertilizer (NPK) at the total recommended dose has greater efficiency in clayey and sandy soils. Therefore, we recommend the use of R. tropici inoculation and the co-inoculation of R. tropici + A. brasilense with a reduced dose of 33% NPK to cultivate common beans in winter to increase grain yield.

Recommending bacteria can help reduce the use of mineral fertilizers, lowering costs and consequently contributing to agricultural sustainability. However, further research is needed to better elucidate the dynamics of these bacteria in legumes and their behavior in tropical soils, whether or not managed under a no-tillage system. What is evident from the research is that maintaining soil management under conservationist systems and combining it with microorganisms is a key point for the development of efficient agriculture, which can reduce costs while increasing yield. The preliminary results are promising, as they demonstrate that the use of bacteria, specifically the co-inoculation of R. tropici + A. brasilense, can help reduce the use of mineral fertilizers, cutting costs and decreasing the external dependence of producing countries. Furthermore, in sustainable terms, this reduction can contribute to the development of regenerative agriculture and soil health.