Organomineral Fertilization Associated with Inoculation of Rhizobium tropici and Co-Inoculation of Azospirillum brasilense in Common Bean

Abstract

The use of organomineral fertilizers can serve as a nutritional source for crops, ensuring sustainability in the production system. Another alternative approach is through the inoculation and co-inoculation of microorganisms. The objective of this study was to evaluate the development, nutrition, and agronomic yield of common beans through fertilization with the organomineral formula “OMF”, derived from sewage sludge biosolids, combined with Rhizobium tropici inoculation and co-inoculation with Azospirillum brasilense techniques. Three bean cultivars from different commercial groups, Pérola, BRS Esteio, and BRS Pitanga, were tested. They underwent OMF application; OMF application combined with seed inoculation with Rhizobium tropici and re-inoculation; seed co-inoculation with Rhizobium tropici + Azospirillum brasilense prior to sowing; and foliar re-inoculation with Rhizobium tropici + Azospirillum brasilense. The results indicated that seed inoculation, combined with leaf re-inoculation at the V4 stage, resulted in greater bean productivity. The combination of Rhizobium tropici with co-inoculation with Azospirillum brasilense generally led to increased productivity compared to mineral nitrogen fertilization. The exclusive use of OMF enabled development and productivity gains. The Pérola bean cultivar showed better adaptation to the applied treatments. In conclusion, this research suggests that there are benefits to using OMF with symbiotic bacteria and growth promoters.

1. Introduction

The bean plant, like most crops, requires prepared soil to provide all the necessary nutrients for development. The key macronutrients for this purpose are nitrogen (N), phosphorus (P), and potassium (K). As the most-needed nutrient for plants, nitrogen is particularly crucial, and tropical soils often have limited availability due to environmental dynamics.

Nitrogen fertilizers, primarily urea, are extensively used in agriculture. However, these fertilizers come with a high production cost and substantial energy consumption, constituting significant greenhouse gas (CO₂) emissions. This is because the mineral nitrogen compounds derived from ammonia, obtained through the Haber–Bosch process, rely on nitrogen gas (N₂) from the air and hydrogen (H) from burning fossil fuels [1], posing a considerable environmental threat.

Moreover, Brazil relies heavily on importing over 80% of the fertilizers (NPK) used in the country. The use of these products directly influences national agricultural expansion, prompting the development of new technologies and the adoption of biological fertilizers to invigorate the lucrative fertilizer market. Responding to this demand, Brazil implemented the National Fertilizer Plan through Decree no. 10,991 of 2022, aiming to boost national production, reduce imports, and enhance the competitiveness of the national agribusiness sector.

In the search for greater sustainability, different sources of nitrogen fertilization are essential for the current scenario. One alternative is the use of sewage sludge generated in the treatment process of this affluent, called biosolid, which, after being decontaminated and enriched with small doses of mineral fertilizers, is called Organomineral Formulated (OMF). This by-product has proven to be a viable source for replacing traditional fertilizer, in addition to being a way of sustainably reusing waste [2].

Another alternative to address the demand for nitrogen (N) in crop nutrition is biological nitrogen fixation (BNF), a method predominantly used with Fabaceae, including the common bean. This process occurs through the inoculation of bacteria capable of promoting plant benefits by establishing symbiosis. For satisfactory BNF in bean plants, it is necessary to consider soil fertility, pH, adequate humidity and temperature, and precautions with respect to the use of fungicides and insecticides during seed handling and product storage [3].

One of the primary species capable of nodulating the common bean is Rhizobium tropici [4,5]. This species can fix atmospheric nitrogen through inoculation and make it available to the plant [6]. The use of inoculation and re-inoculation with Rhizobium tropici as a nitrogen source for the bean cultivar BRS Valente [7] resulted in a grain yield of 2827 kg ha⁻¹. Despite enabling the replacement of nitrogenous chemical products and offering economic, social, and environmental advantages for producers, consumers, and the environment, bean producers exhibit some resistance to inoculation due to limited knowledge about the subject, making the adoption of this technique challenging.

An alternative widely studied to enhance its efficiency is the combination of BNF with bacteria that promote root system growth, such as Azospirillum brasilense, through a co-inoculation process. Studies involving the co-inoculation of common beans [8,9,10], in general, demonstrate the efficiency of combining inoculation and co-inoculation techniques compared to mineral nitrogen fertilization alone. However, investigative studies involving the combination of inoculation or co-inoculation with the application of OMF in bean crop nutrition are non-existent.

Considering the aforementioned problems of using mineral fertilizers, the aim of the present study was to evaluate the performance of the inoculation of Rhizobium tropici with the co-inoculation of Azospirillum brasilense using OMF derived from sewage sludge as fertilization in the nutrition, development, and yield of common bean crops.

2. Materials and Methods

2.1. Study Area Description

The experiment was conducted in the 2021 winter season at an experimental area of the Goiás State Agency for Technical Assistance, Rural Extension, and Agricultural Research—EMATER, located in Anápolis-GO, Brazil, the geographic coordinates of which are 16°20′44.27″ S, 48°52′44.67″ W, with an average altitude of 1032 m [11]. The climate in the region is humid tropical, Aw-type, and is characterized by dry winters and rainy summers, with an average annual precipitation of 1600 mm, according to the Köppen classification.

The soil in the area is classified as dystrophic Red Oxisol, and sampling was carried out in the 0–20 cm layer. Initially acidic, it was corrected with liming of 1.5 tons per hectare before planting the beans.

2.2. Origin and Characterization of Organic Waste

The residue used as an organic base in the OMF formulation was derived from sanitized sewage sludge. The initial composition of the sewage sludge was 70% moisture and 30% solids. This material underwent chemical processes, incorporating 30% hydrated lime into the solid part present in the centrifuge, while still wet, and mixed using a concrete mixer. Subsequently, the material was packed into rectangular containers made of galvanized zinc (30 × 30 × 100 cm), covered with a transparent canvas with a thickness of 200 microns, and exposed to sunlight and ultraviolet rays for 15 days for thermal treatment. After removing the canvas, the material underwent a drying process in the open air for 30 days, stabilizing at 20% humidity.

2.3. Formulation of the Organomineral Fertilizer

The OMF was prepared based on the chemical composition of the sewage sludge and to meet the needs of the common bean crop for a yield of 2500 to 3000 kg ha⁻¹. The OMF preparation aimed to fulfill the requirements for 10 kg ha⁻¹ of nitrogen, 90 kg ha⁻¹ of phosphorus, and 50 kg ha⁻¹ of potassium at planting, along with an organic base that guaranteed a minimum of 8% organic carbon in the composition of the OMF.

To meet these specifications, the resulting OMF had a composition of 02–18–10. The quantities required for the formulation were: 3.7 kg of urea as a nitrogen source; 38.4 kg of MAP as a phosphorus source; 17.2 kg of KCl as a potassium source, and 40.7 kg of treated sewage sludge. The MAP added at planting contained 10–12%, ensuring robust seedling vigor from emergence until the establishment of the inoculated bacteria population in the soil, effectively facilitating biological nitrogen fixation for the bean plants.

2.4. Experimental Design and Treatments

A randomized block design was employed, with treatments arranged in a 3 × 4 × 3 factorial scheme and four replications. The treatments involving the use of OMF at sowing included three distinct bean cultivars based on grain colors: Pérola (carioca bean group), BRS Esteio (black bean group), and BRS Pitanga (purple bean group). These cultivars underwent fertilization with OMF without inoculation and co-inoculation techniques; fertilization with OMF involving seed inoculation with Rhizobium tropici before sowing and re-inoculation during topdressing; co-inoculation of seeds with Rhizobium tropici + Azospirillum brasilense before sowing and re-inoculation during topdressing of Rhizobium tropici + Azospirillum brasilense; a control treatment without any fertilization, inoculation, or co-inoculation; and an additional treatment meeting the nutritional needs of the three cultivars exclusively via mineral fertilization.

The quantity applied in the sowing fertilization of the OMF formulation 02-18-10 (600 kg ha⁻¹) for the cultivars was calculated to meet the nutritional requirements for achieving a grain yield of 2500–3000 kg ha⁻¹ based on the results of a soil analysis. A liquid inoculant based on Rhizobium tropici was utilized at a rate of 150 mL 50 kg⁻¹ of seeds and 150 L ha⁻¹ in the topdressing at the V4 stage (initiated when the third trifoliate leaf was fully open). The root growth regulator Azospirillum brasilense was applied in the seed treatment at a rate of 100 mL 50 kg⁻¹ of seeds and 100 mL ha⁻¹ in topdressing at the V4 stage. The control group did not receive any inoculant or fertilizer. The treatments using OMF as fertilization did not receive topdressing fertilization due to the combination of inoculation and co-inoculation techniques. It is essential to note that the application of OMF was exclusively performed before the sowing stage. The dose of 400 kg ha⁻¹ of mineral formula 5-25-15 and the topdressing fertilization of 50 kg ha⁻¹ of N at stage V3, using urea as a source in the additional treatment, was calculated to achieve the same grain yield of 2500–3000 kg ha⁻¹, as was used in the treatments where OMF was used as fertilization.

The inoculation and co-inoculation of the seeds in treatments that received them were carried out just before sowing, with no seed treatment being performed to avoid the death of the inoculated bacteria. Reinoculation during topdressing was executed with a backpack sprayer with a capacity of 20 L, using a fan-type nozzle, with a jet directed toward the soil at the base of the plants. This procedure was consistently carried out in the late afternoon, aiming at better inoculation efficiency. The inoculants were diluted according to the manufacturer’s recommendations in an aqueous solution to deliver a final volume of 150 L ha⁻¹.

2.5. Experimental Unit and Crop Treatments

The experimental area consisted of plots with four rows of 5 m each, spaced 0.5 m apart. The two central rows were used for evaluations in each experimental plot. The beginnings and ends (0.5 m) of the plot were used to conduct plant development analyses at the R1 stage—full flowering—and the rest were reserved for the evaluation of the yield and its components. The sowing of the cultivars was carried out using 12 plants per meter, dispensing with thinning for all cultivars.

Irrigation was performed by sprinkling on alternate days in the morning to meet the crop’s needs. The crop treatments employed were those commonly applied to common beans through monitoring the crop, using the boom sprayer, and recommended products.

2.6. Development Analysis

Five plants were randomly collected from the central rows (0.5 m at each end) with a hoe for the development analyses. After the applications of the treatments for reinoculation in the topdressing, ten plants were collected to conduct the following evaluations: leaf area (LA), leaf area index (LAI), plant height (PH), root length (RL), shoot dry mass (SDM), and root dry mass (RDM).

The LA was obtained by removing the leaves from the plants and passing them through the portable meter model CI-202 Portable Leaf Area Meter from CID Bio-Science. The leaf area index (LAI) was obtained by determining the ratio between the sum of the leaf area of a plant and the soil surface it occupies as shown in Equation (1):

LAI = (LA ∗ FS)/10,000

(1)

wherein: LAI = leaf area index; LA = average leaf area (m²) and FS = final stand of plants per hectare.

The plant height (PH) was obtained with the aid of a tape measure used to measure between the base of the plant and the apical end of the main stem of the plant in cm. The root length (RL) was also obtained with a tape measure by measuring the length of the main root in cm. The shoot dry mass (SDM) and root dry mass (RDM) were obtained by separating the organs of the plants into roots, stem, leaves, and pods, packed in kraft paper bags, and then placed in the greenhouse at 75 °C until they reached a constant weight. Afterward, they were weighed on a precision scale (0.01 g) to obtain the dry mass [12].

2.7. Analysis of Yield and Its Components

The yield components were analyzed based on data from ten plants collected at bat each experimental unit at the time of harvest, evaluating the number of pods per plant (NPP), an average obtained based on counting the number of pods obtained from the ten plants collected. The average number of grains per pod (NGP) is based on the number of grains obtained from the total pods of the ten plants. The 100-grain weight (W100) was obtained using random samples of 100 grains from the ten plants. Grain yield (YIELD) was obtained by harvesting and threshing the remaining plants collected in the plot, weighing their grains with 13% moisture on a 0.01g precision scale; the result was extrapolated to kg ha⁻¹.

2.8. Statistical Analysis

Data were initially subjected to tests of homogeneity of variances and normality of residuals by the Levene and Shapiro–Wilk tests, respectively. Then, the analysis of variance for the data was conducted, and the means of the factorial were compared using Tukey’s test, and the additional treatment was evaluated using the Dunnett test at a 5% probability. R software (R Core Team, version 3.6.0) was used in the analyses.

3. Results and Discussion

3.1. Development Analysis

The results of the analysis of variance (

Table 1. Results of analysis of variance performed on bean cultivars subjected to different forms of inoculation with Rhizobium tropici and co-inoculation with Azospirillum brasilense.

Mean Squares
SV	DF	LA	LAI	PH	RL
Blocks	3	281,614.34	0.3455	4.2232	22.9099
Cultivars (A)	2	201,460.8 NS	0.3008 NS	3.6618 **	12.80083 *
Treatments (B)	3	1024,133.1 **	2.8318 **	0.9285 NS	5.6335 NS
A × B	6	64,204.18 NS	0.8425 *	3.5602 **	3.4325 NS
Addi. Treatment	2	14,252.64 NS	0.3025 NS	1.1633 NS	1.5925 NS
Treatments × addi. Treatment	1	1,149,486.1 NS	0.2470 NS	2.6250 NS	9.8820 NS
Residue	42	229,949.04	0.3547	0.6950	3.6582
CV (%)		56.4	21.8	11.9	13.1
SV	DF	SDM	RDM
Blocks	3	0.1063	0.6461
Cultivars (A)	2	3.7731 **	0.9058 **
Treatments(B)	3	10.6535 **	0.9568 **
A × B	6	0.6097 **	1.2058 **
Addi. Treatment	2	0.97 **	0.2158 **
Treatments × addi. Treatment	1	6.8343 **	0.1760 *
Residue	42	0.0774	0.3413
CV (%)		11.9	12.1

** significant at 1% probability by the F test. * significant at 5% probability by the F test. ^NS not significant by the F test. LA—leaf area; LAI—leaf area index; PH—plant height; RL—root length, SDM—shoot dry mass, and RDM—root dry mass.

) show the variables influenced by the tested treatments. Good experimental precision was observed for most variables, according to the coefficient of variation (C.V) values obtained when compared to the values cited by [13]: low when less than 10%; medium between 10 and 20%; high between 20 and 30%; and very high when greater than 30%. In other words, the higher the C.V value, the lower the experimental precision. Despite the high variability observed in the results of the LA analysis, it was possible to detect the difference between the treatments for LA and LAI.

Comparing the responses among the treatments for LA, it was observed that treatment with the inoculation of Rhizobium tropici presented a higher mean (1232.71 m²) compared to treatments with the co-inoculation of Rhizobium tropici + Azospirillum brasilense (994.76 m²) and with only fertilization with OMF at the base (920.98 m²); however, these did not differ statistically (Figure 1).

Reinoculation provided greater development for LA, which may be related to the symbiosis promoted by the inoculants added to the crop. When studying different combinations of inoculants in the common bean crop [14], an approximate value of 1380 m² for LA inoculation with Rhizobium tropici was found, a result similar to that found in this study, where Rhizobium tropici provided 1232.71 m² of LA.

The relationship between treatments and cultivars for LAI showed no significant difference for the Pérola and Pitanga cultivars. The results for the BRS Esteio cultivar (Figure 2) showed positive responses to the treatments, which did not present a significant difference except for the control treatment. The highest average, in this case, was represented by the co-inoculation treatment of Rhizobium tropici + Azospirillum brasilense (3.6).

The response of the LAI variable depends on the number of trifoliate leaves per plant, which are formed by increasing the leaf area, allowing the plant to take better advantage of the incidence of light. This whole process is only possible if the plant is well-nourished, and nitrogen is an essential nutrient in vegetative development [15]. In general, there was an increase in the LAI after reinoculation; this result was also observed by [16].

There was no significant difference in plant height (PH) for BRS Esteio and BRS Pitanga cultivars. The cultivar Pérola, using the OMF treatment without any inoculation, had a better PH, with 30% more than the control treatment (Figure 3).

The use of an OMF based on biosolids in the bean crop also promoted an increase in plant height according to [17]. These authors observed a 67% increase in PH in relation to the control. This type of fertilizer, composed of organic matter, is rich in humic substances, allowing for the release of negative charges capable of controlling the adsorption of phosphorus and other cationic nutrients by iron and aluminum oxides, thus providing nutrients to plants.

Phosphorus, in addition to being a component of several biochemical reactions in plants, is also part of the composition of the ATP molecule, which is of paramount importance for the execution of photosynthetic activity [18]. The predominant humic substances in the OMF may be related to the PH additions verified in the Pérola cultivar.

The root length (RL) differed among the studied cultivars, with the highest average observed in the Pérola cultivar (Figure 4). Ref. [19] investigated the genetic variation response of the bean plant using fertilizers containing ammonium polyphosphate, single superphosphate, biostimulants, and micronutrients. In their results, it was possible to perceive the influence of genetic variability on the response to the analyzed parameters, including root length. This allowed us to conclude that the Pérola and Pitanga cultivars demonstrated better adaptation in relation to RL for the applied treatments.

The results of shoot dry mass (SDM) for the cultivar Pérola (Figure 5a) were influenced by the treatments, and the highest mean was observed in the Rhizobium tropici treatment (4.08 g plant⁻¹). For the cultivar BRS Esteio (Figure 5b), it was verified that there was a significant difference only for the control treatment. BRS Pitanga (Figure 5c) showed a similar result to that of the Pérola cultivar, emphasizing that the Rhizobium tropici treatment (3.38 g plant⁻¹) provided a greater accumulation of SDM.

The root dry mass (RDM) differed significantly for all cultivars. The Pérola cultivar obtained the best response to the isolated addition of the OMF (2.1 g plant⁻¹) (Figure 5d); BRS Esteio showed no statistical difference in this parameter for the treatment with inoculation of Rhizobium tropici (Figure 5e). Co-inoculation with Azospirillum brasilense resulted in a mean value lower than the other treatments for BRS Pitanga (1.48 g plant⁻¹) (Figure 5f).

Differences were detected by [20] among bean cultivars when evaluating biomass accumulation due to seed inoculation with two strains of Rhizobium. They observed that the use of the inoculant influenced biomass accumulation, concluding that the use of inoculated strains provided greater nitrogen availability, with an increase ranging from 15 to 20% in SDM.

This same percentage was found when comparing the inoculation for the Pérola cultivar, in which SDM was increased by 20% compared to the isolated use of OMF. In a study developed by [21], the amount of SDM was influenced by the reinoculation of Rhizobium tropici, corroborating the results found in this study. The increase in bean plant SDM through inoculation was also noted by [22]. Therefore, it can be observed that the use of inoculants can assist in plant development.

The accumulation of RDM may be related to the genetic trait of the cultivar and the morphological growth characteristics of different genotypes. An evaluation of OMF with an application of Rhizobium tropici and co-inoculated with the Bradyrhizobium strain for beans did not find significant differences for these treatments, according to [23]. The result was similar to that of the Pérola and BRS Pitanga cultivars. The response variations between cultivars may indicate that the response of OMF to the root system varies according to the cultivar used.

A significant difference in SDM was observed for all additional factors. Mineral fertilization for the Pérola cultivar (Figure 6a) showed a significant difference. The highest average for the Pérola cultivar was associated with the exclusive use of OMF (2.1 g plant⁻¹), which provided an increase of 30% in dry mass compared to the use of mineral fertilizer (1.48 g plant⁻¹). A comparison with the additional treatment for BRS Pitanga (Figure 6b) also showed a lower result than the other treatments, except for the controls.

For the RDM variable, the additional treatment of the Pérola cultivar showed a significant difference (1.48 g plant⁻¹) compared to the Pérola-OMF treatments (2.1 g plant⁻¹), which increased the RDM by 30% (Figure 6c). The additional factor, BRS Pitanga (Figure 6d), differed statistically, with a higher average (1.9 g plant⁻¹) from the BRS cultivar treatment Pitanga-Rhizobium tropici + Azospirillum brasilense (1.48 g plant⁻¹).

The SDM showed, in general, a better response to treatments with the inoculation of Rhizobium tropici and co-inoculation of Azospirillum brasilense, as identified in the results of [24], in which the response of inoculation and co-inoculation of Rhizobium tropici and Azospirillum brasilense for bean cultivation was analyzed; better results were also obtained with a combination of seed inoculation with Rhizobium tropici and foliar application of two doses of Azospirillum brasilense. This favorable outcome was also observed in a study involving soybeans. Ref. [25] observed an increase in SDM for a common bean crop when using the co-inoculation of Rhizobium tropici and Azospirillum brasilense, corroborating the results found in this study. This occurs due to the increased efficiency of Rhizobium when using Azospirillum, which produces plant growth-stimulating hormones and improves nitrogen availability through symbiotic nitrogen fixation, among other factors.

An analysis of the results showed that, for RDM, the most significant treatment results were obtained with OMF, which were similar to, or superior to, mineral fertilization. Obtaining the agronomic response of using OMF compared to mineral fertilizer in bean cultivation, ref. [26] observed that the best results for RDM were obtained at dosages of between 100% and 150% of OMF compared to the tested mineral fertilizer. Likewise, in a study with beans, ref. [27] also obtained better RDM results for the use of the OMF compared to the NPK source used as a reference. These studies corroborate the results found in this research, proving that the use of the OMF can be sufficient to increase RDM.

3.2. Components and Grain Yield

The analysis of variance demonstrated significance for all variables (

Table 2. Results of analysis of variance, its components, and yield in common bean cultivars subjected to different forms of inoculation with Rhizobium tropici and co-inoculation with Azospirillum brasilense.

Mean Squares
SV	DF	NPP	NGP	W100	YIELD
Blocks	3	1.0388	2.5333	55.0666	62,777
Cultivars (A)	2	63.8125 **	0.8125 NS	85.5625 **	2,374,793.58 **
Treatments (B)	3	87.5833 **	0.2222 NS	21.3611 **	2,536,109.13 **
A × B	6	1.3958 NS	0.0347 NS	8.8402 NS	182,579.44 *
Addi. Treatment	2	12.2500 NS	0.2500 NS	36.0833 **	1,213,846.58 **
Treatments × Addi. treatment	1	54.1500 NS	0 NS	40.0166 NS	434,435.51 **
Residue	42	8.2508	0.2714	4.79286	56,048.44
CV (%)		33.2	17.3	7.6	22.6

** significant at 1% probability by the F test. * significant at 5% probability by the F test. ^NS not significant by the F test. NPP—number of pods per plant; NGP—number of grains per pod; W100—100-grain weight; YIELD—yield.

). As for experimental precision, it can be verified that, in general, good precision in obtaining the data of the variables studied, except for the number of pods per plant (NPP), which presented a value superior to 30% when compared to the values considered by [14]: low, when less than 10%; medium, between 10 and 20%; high, between 20 and 30%; and very high if greater than 30%. Despite this, it was possible to detect a significant difference between treatments for NPP.

The number of grains per pod (NGP) did not present a significant difference, a result also found by [28], who, studying the efficiency of inoculation and co-inoculation of Rhizobium tropici and Azospirillum brasilense in common bean, did not reach significance for that variable. According to the authors, this fact is linked to the high genetic heritability of the cultivars. On the other hand, the 100-grain weight (W100), despite being a variable predominantly influenced by the genetic part and the NGP results, differed depending on the treatments.

The Pérola cultivar produced the highest NPP (11.38), differing from the other studied cultivars (Figure 7a). Comparing the development of two bean cultivars with four nitrogen doses, ref. [29] also observed the influence of the cultivar factor on NPP and the interaction between cultivars and nitrogen doses. Also, for this study, the cultivar that presented the best performance was the IAC Imperador, with an average of 13 pods per plant; however, it did not show a response to the fertilizer dosages. Although lower than that found in this research, this result shows an influence of treatments for the studied cultivars.

The NPP differed only from the control treatment regarding applied treatments (Figure 7b). The OMF, Rhizobium tropici + Azospirillum brasilense, and Rhizobium tropici treatments provided increases of 54%, 51%, and 47%, respectively, for this variable.

This result is related to the fact that the symbiosis between the inoculant bacteria and the plant is capable of increasing the yield components [6]. This is due to the symbiosis between the inoculating bacteria and the plant being able to increase the yield components. In a study evaluating the inoculation of different strains in common bean seeds, ref. [30] obtained superior results for NPP and NGP components when the seeds were inoculated with Rhizobium tropici and in combination with other strains. Ref. [17] observed that the use of OMF was able to increase the bean yield components. It is notable that the most significant result for NPP was fertilization with OMF at 200 kg ha⁻¹. In this study, it was possible to notice the influence of treatments compared to the control, which increased NPP.

A comparison of the cultivars showed a significant difference between the cultivars for W100. The Pérola cultivar had the highest average (31.19 g), and the BRS Esteio cultivar had the lowest average (26.56 g) (Figure 8a). A comparison between treatments showed that the use of OMF (30.08 g) and the use of Rhizobium tropici + Azospirillum brasilense (29.67 g) did not differ from each other, and the lowest result was verified in the control treatment (27.08 g) (Figure 8b).

Analyzing the results found for W100 in the comparison between cultivars, it is possible to verify that there was a difference in their adaptation, either due to the type of treatment applied or the influence of seasonality and soil type; the genetic differences between the bean plants also influence results. Ref. [31] showed that some bean cultivars that are better adapted to rhizobial inoculation, in the same way that there are also soils that are not adapted to this practice. Considering this information, it is possible to observe that there is a difference between commercial bean cultivars; that is, cultivars considered more efficient for FBN may respond to native or exogenous rhizobia, while others may adapt only to nitrogen fertilization. Therefore, it is likely that the BRS Esteio cultivar did not adapt to the inoculated treatments and presented inferior results.

The application of treatments significantly influenced grain weight, a result also observed by [32], in which a statistical difference for W100 of the beans using different combinations of Rhizobium tropici and Azospirillum brasilense were found. Similarly, ref. [33], while testing the effects of Rhizobium tropici reinoculation, also found a statistical difference for this variable. Additionally, according to [18], W100 may be related to the type of management used and environmental conditions, which can directly influence the outcome of the variables, even with the high heritability conferred upon it.

The yield (YIELD) differed between the genotypes and the treatments studied. Notably, the Pérola cultivar yield averages with the addition of OMF, Rhizobium tropici + Azospirillum brasilense, and Rhizobium tropici, were respectively 68%, 61%, and 58% higher than that of the control (Figure 9a). Among the BRS Esteio (Figure 9b) and BRS Pitanga (Figure 9c) cultivars, differences were also detected between the treatments, with the Rhizobium tropici, OMF, and Rhizobium tropici + Azospirillum brasilense treatments providing increases in grain yield of 76%, 73%, and 70%, respectively; in BRS Pitanga, grain yields of 71%, 76%, and 60%, respectively, were obtained compared with the control.

It is also noteworthy that the highest yield averages were obtained in the Pérola and BRS Esteio cultivars, with averages higher than the national average obtained in the 2020/2021 harvest, around 1.0 ton. ha⁻¹ [32]. On the other hand, the lowest yield was verified in the cultivar BRS Pitanga in response to the treatments. Additionally, the response to the addition of OMF alone or as a base in the treatments that received the microorganisms Rhizobium tropici seed and Rhizobium tropici + Azospirillum brasilense in the topdressing were significant.

Comparing different dosages of OMF with either biosolids or filter cake for bean yield, ref. [17] obtained an increase of 46% for the treatment with biosolids at a dose of 50%, compared to the filter cake. In the present study, the use of OMF increased the yield by 68% compared to the control. The positive result of this material, as indicated by the findings in this research, may be related to the availability of N, P, and K, which promote the mineralization and decomposition of organic matter [17].

In this study, it was observed that inoculation with Rhizobium tropici did not differ statistically from co-inoculation with Azospirillum brasilense. Both treatments differed from the control and provided higher yields. This same response was found in [24]. While studying different inoculant doses in common bean plants, it was concluded that both combinations of Rhizobium tropici and Azospirillum brasilense, and the combination of Azospirillum brasilense inoculated and sprayed, resulted in significantly higher grain yields compared to single inoculations. Similar results were obtained for the cultivars BRS Esteio and BRS Pitanga.

The yield with additional treatments for the cultivar Pérola (Figure 10a) showed a statistical difference compared to the control treatment. The treatment with mineral fertilizer showed the best results (1466 kg ha⁻¹). Even though there was no difference, the Pérola-OMF treatment (1931 kg ha⁻¹) obtained a grain yield 24% higher than that of mineral fertilization.

A comparison of treatments for the cultivar BRS Esteio (Figure 10b) showed that there was no significant difference between the treatments, except for the control treatment, which presented a lower yield. Although not significantly different, grain yield increased by 12% with Rhizobium tropici. Additional treatments for the cultivar BRS Pitanga (Figure 10c) differed from treatments of cultivar Pitanga with OMF and Rhizobium tropici + Azospirillum brasilense, presenting lower yields than mineral fertilization. No difference was seen for cultivar BRS Pitanga treated with Rhizobium tropici. This result shows that the treatments could not provide the same yield as the mineral fertilizer for this cultivar. It indicates that the BRS Pitanga cultivar is less resistant to pests and diseases and, at the same time, is less compatible with inoculation.

Observing the influence of OMF use on bean production in comparison to mineral fertilizer, ref. [27] demonstrated higher grain productivity for the OMF treatment. This led to the conclusion that there is possibly greater nutritional availability for this type of agricultural fertilizer, in addition to providing better mineralization of organic matter and the best C:N ratio, resulting in positive outcomes.

In a study comparing OMF with mineral fertilizers in bean cultivation, ref. [34] obtained higher yields with the use of OMF, which presented higher productive performance than other treatments. These results are partially consistent with those obtained in this research regarding the increase in yields of bean cultivars generated by fertilization with OMF based on sewage sludge combined with seed inoculation with Rhizobium tropici and reinoculation with co-inoculation using Rhizobium tropici + Azospirillum brasilense in the topdressing at the R4 stage.

The differences in productivity among the cultivars are notable, with the most significant being that of the Carioca cultivar. Results obtained in [35] showed that the Carioca cultivar had the most satisfactory response to inoculation, resulting in higher productivity. This is in accordance with the results found in this study.

4. Conclusions

Inoculation applied via seed, combined with re-inoculation in the topdressing at the V4 stage, made it possible to obtain the highest grain yield of the common bean crop and can replace the use of mineral nitrogen fertilizer.

The combination of Rhizobium tropici with the co-inoculation of Azospirillum brasilense, in general, provides yield increases compared to mineral fertilization. The isolated use of OMF allows for gains in the development of common beans, in addition to grain yield and its components, making it an alternative source for fertilization.

The bean cultivar Pérola showed a higher grain yield with OMF combined with inoculation with Rhizobium tropici before sowing and co-inoculation with Azospirillum brasilense in the topdressing at the V4 stage, compared to the response capacity of the cultivars BRS Esteio and BRS Pitanga.

Considering the benefits of using alternative nutrition techniques for mineral fertilization, the use of OMF with symbiotic bacteria and growth promoters can be of benefit in agricultural practices, and help to ensure sustainable development.