Reinoculation in Topdressing of Rhizobium tropici, Azospirillum brasilense, and the Micronutrients Mo/Co in Common Bean

Abstract

Biological nitrogen fixation (BNF) can provide the necessary nitrogen for bean crops; however, for this to occur, important limitations involving the inoculant application technology need to be overcome.The use of co-inoculation is a management technique used to obtain benefits and increase the potential of N₂ fixation from the association between bacteria from the rhizobia group, such as R. tropici, and bacteria that promote plant growth, such as A. brasilense, in association with the addition of nutrients that allow greater efficiency of bacteria fixing atmospheric N₂. This study aimed to evaluate the bean response to the reinoculation of R. tropici in co-inoculation with A. brasilense in a mixture with the micronutrients Co/Mo, in the winter season of 2021, in Anápolis-GO, Brazil. A randomized block design was used, with four replications, and the following treatments (TRs) were studied: TR1—reinoculation with R. tropici; TR2—reinoculation with co-inoculation of R. tropici + A. brasilense; TR3—reinoculation of R. tropici + Mo/Co micronutrients; TR4—reinoculation with co-inoculation R. tropici + A. brasilense + Mo/Co micronutrients; TR5—inoculation via seed, without reinoculation; TR6—mineral N fertilization in the sowing furrow and topdressing; TR7—control, without any N source. At stage R6, nodulation characteristics (number and dry mass of nodules) and the morphophysiological parameters of the plants (main root length, root dry mass, plant height, shoot dry mass, leaf area, and leaf N content in the shoot) were evaluated. At harvest, the final plant stand and components (number of pods per plant, number of grains per pod, and average weight of one hundred grains) were determined, in addition to grain yield. It was concluded that inoculation followed by reinoculation in topdressing with R. tropici in co-inoculation with A. brasilense plus Mo/Co, compared to mineral nitrogen fertilization, improves the efficiency of the nodulation process and the morphophysiological characteristics of the common bean crop. Seed inoculation and topdressing application with R. tropici, associated with co-inoculation with A. brasilense + Mo and Co, have the potential to completely replace mineral nitrogen fertilization in common bean crops.

1. Introduction

Common bean (Phaseolus vulgaris L.) belongs to the Fabaceae family andis the most cultivated species of bean worldwide [1], feeding more than 300 million people across the globe, especially in Latin America, Africa, and Asia, with Brazil being the world’s largest producer of the species [2,3]. The total area of common bean production globallyin 2020 was 29 million hectares, with a production of 27 million tons [4], the nutritional issue being one of the factors that corroborates this low level of yield, with emphasis on nitrogen (N₂), the nutrient most demanded by the crop [5].

The recommended dose of N for the common bean crop varies from 40 to 100 kg ha⁻¹ in Brazil, depending on the technological level applied [6]. This demand can be supplied by N from the soil, nitrogen fertilization, and N₂ fixation, which is the first technique commonly used to supply the N demand for the common bean crop [7,8]. Biological nitrogen fixation by legumes such as the common bean ranges from 20 to 200 kg N fixed ha⁻¹ yr⁻¹ [9,10], fully meeting the needs of the crop. However, the use of biological inputs to replace industrialized chemical inputs has been increasingly frequent in the research field, being considered as an attractive N supply alternative from an economically and ecologically sustainable point of view since it reduces the use of nitrogen fertilizers and thus contributes to avoiding contamination of aquifers, lakes, and rivers caused by the leaching of this nutrient from the soil [11,12].

Nitrogen-fixing bacteria play an important ecological role, acting in the conversion of atmospheric nitrogen (N₂), which is not usable by plants and other groups of organisms, into ammonia (NH₄+), a form that can be assimilated by plants [13]. Bacteria of the Rhizobium group can fix nitrogen from the atmospheric air and supply it to the plant. The plant receives nitrogen fixed by the microsymbiont rhizobium in ammoniacal form, assimilating it into nitrogenous compounds that can be translocated to its different parts [14]. The leguminous plants develop by symbiotic association in the roots with the rhizobial bacteria, and when these bacteria are present in the soil, naturally or via inoculation, they recognize and infect the roots of the host plant, causing the formation of nodules where the biological fixation of N₂ occurs [15,16].

Currently, the strains Semia 4077, Semia 4088, and Semia 4080, belonging to the species of Rhizobium tropici, make up the commercial inoculants intended for use in the common bean crop [17,18], notably in Brazil, the world’s largest producer of common beans. These strains are resistant to high soil temperatures, acidity conditions, and greater competitiveness compared to natural rhizobia populations in the soil, making them more adapted to cultivation in tropical soils [19]. These characteristics have practically changed the paradigm of the subject concerning common bean since numerous investigative studies prove the ability of N₂ fixation to supply the N₂ demanded by the crop under field conditions, provided that correct inoculation practices are employed [8,20].

The co-inoculation technique is beginning to be explored in the species to improve the performance of rhizobia and, consequently, the efficiency of N₂ fixation in common bean plants. Co-inoculation in common bean is a management technique used to obtain benefits and increase the potential of N₂ fixation from the association between bacteria from the rhizobium group and bacteria that promote plant growth, such as A. brasilense. This alternative is represented by a group of associative bacteria capable of promoting plant growth through physiological changes due to an increase in root growth [21,22].

Several studies have found that co-inoculation consists of combinations of different microorganisms, which produce a multiplier effect that surpasses the results produced in isolation, promoting increases in the yield of crops [23,24,25]. Due to the positive interaction between bacteria, co-inoculation has been suggested to enhance nodulation, stimulate plant growth, and benefit the biological process of nitrogen fixation. The bacterium A. brasilense promotes plant growth due to its ability to fix nitrogen, produces hormones such as auxins, cytokinins, gibberellins, and ethylene, and increases nitrate reductase activity [26,27]. The presence of bacteria of the genus Azospirillum can release phytohormones that induce the formation of root hairs in the bean, making it possible to increase the production of dry mass and the number of nodules per plant [28].

Adding nutrients in co-inoculation can also improve N₂ fixation efficiency in soybean (Glycine max), especially the micronutrients molybdenum and cobalt [29]. Molybdenum is indispensable for the metabolism of N₂, as it is part of the enzymes nitrate reductase and nitrogenase, essential for plant growth and development [30]. Due to the high mobility of molybdenum in the plant, topdressing fertilization can provide good results as long as it is carried out at the beginning of the period of vegetative development of the crop [31]. Cobalt is useful in the Fabaceae family because it participates in the structure of vitamin B12, which regulates oxygen concentration in the nodules, preventing the inactivation of the enzyme nitrogenase [32].

The application of rhizobian cells, associated with A. brasilense, is commonly performed via seed before sowing [33,34]; however, the initial formation of nodules is critical for the common bean in the first 15 days after sowing [35], and only a set of fully functional nodules can supply the appropriate amount of N to the crop, which will happen after this period. Premature aging of the population of nodulating bacteria associated with environmental stress can cause nodular senescence and cessation of N₂ fixation of legumes generally coinciding with the flowering and filling stage of grains [36,37], phases in which precisely the demand for nutrients, water, and photoassimilates by the plant is greater. In addition, nodules that appear late with the advancement of the crop cycle can contribute in a restricted way to crop nutrition and increase yield [38,39]. In this way, the reinoculation in topdressing made directly in the soil can renew the rhizobian population effectively in incorporating N₂ [39,40], associating the application of A. brasilense [41] and the Mo/Co micronutrients, exactly in a phase in which the bean crop has a greater nutritional demand. However, the effect of the association of these products applied to bean cultivation is little investigated by scientific research.

Agricultural production can be improved by using microorganisms, which are presented as alternatives for sustainable nutritional management in the current scenario. Thus, the joint action of the two microorganisms associated with adding Mo/Co micronutrients constituted an excellent solution in searching for higher yields of the common bean crop based on nutrition with sustainable resources.Thus, this study aims to evaluate the nodulation process, morphophysiology, and yield of bean plants in response to reinoculation with R. tropici, the co-inoculation of A. brasilense, and the application of micronutrients Co/Mo in topdressing at the V4 stage.The hypotheses raised in the research are as follows: (i) the association of R. tropici + A. brasilense and the micronutrients Mo/Co can increase bean nodulation; (ii) bean morphophysiology is influenced by the reinoculation of rhizobial cells with the growth regulator azospirillum and the micronutrients Mo/Co; and (iii) bean reinoculation involving the addition of R. tropici associated with the addition of azospirillum and the micronutrients Mo/Co presents results equivalent to nitrogen mineral fertilization in terms of grain yield.

2. Materials and Methods

2.1. Characterization of the Study Area

The experiment was conducted under field conditions in the winter season of 2021 in the experimental area of the Goiás State Agency for Technical Assistance, Rural Extension, and Agricultural Research—EMATER, located in Anápolis-GO, Brazil, ingeographical location of 16°20′12.13″ S, 48°53′15.96″ W, and an average altitude of 1058 m [42]. According to the Köppen classification, the climate of the region is tropical humid, AW-type, with a dry season in the autumn–winter period (May–October) and a rainy season in the spring–summer period (November–April), and the average annual temperature is 22 °C, with average annual precipitation of 1677 mm [43]. During the experiment period, the average temperature was around 25 °C, with no rainfall occurring.

Samples of soil as Yellow Red Latosol, previously cultivated with corn (Zea mays), were taken from the 0–20 cm layer and sent to soil laboratory for physical and chemical analysis; the results were as follows: pH (CaCl₂) = 4.8; P (mg dm⁻³) = 4.2; K (cmol_c dm⁻³) = 0.21; Ca (cmol_c dm⁻³) = 1.4; Mg (cmol_c dm⁻³) = 0.6; Al (cmol_c dm⁻³) = 0.2; H+Al (cmol_c dm⁻³) = 2.4; base saturation (%) = 48; B (mg dm⁻³) = 0.21; Cu (mg dm⁻³) = 2.8; Fe (mg dm⁻³) = 33.8; Mn (mg dm⁻³) = 17.6; Zn (mg dm⁻³) = 6.3; organic matter (g dm⁻³) = 20.5; sand (g kg⁻¹) = 415; silt (g kg⁻¹) = 105, and clay (g kg⁻¹) = 480.

Liming was conducted using limestone filler at a dose of 1.5 ton ha⁻¹ of limestone, thus aiming to reach base saturation of 70%, waiting two months for the reaction of the corrective in the soil area where the sowing was carried out.

2.2. Experimental Design and Treatments

The experiment was conducted in a randomized block design (RBD), with four replications, with the following treatments (TRs) being tested: TR1—reinoculation with R. tropici; TR2—reinoculation with co-inoculation of R. tropici + A. brasilense; TR3—reinoculation of R. tropici + Mo/Co micronutrients; TR4—reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co micronutrients; TR5—inoculation via seed, without reinoculation; TR6—nineral N fertilization at the sowing furrow and in topdressing; TR7—control treatment, without any source of N.

According to the recommendations of the manufacturer, 150 mL per hectare was used for reinoculation. The inoculant used for inoculation and reinoculation was Biomax premium liquid (bean inoculant), based on R. tropici (Semia 4077, Semia 4080, and Semia 4088) with a guarantee of 2 × 10⁹ CFU mL⁻¹. The inoculant based on A. brasilense belonging to the Azotrop brand was used at a dose of 100 mL ha⁻¹. The micronutrients molybdenum and cobalt were used from the Biocross brand with a recommended dose equivalent to 30 and 3 mL ha⁻¹, respectively. The source of mineral nitrogen fertilizer used at the sowing and topdressing in TR6 was urea at doses of 20 and 40 kg ha⁻¹ of N, respectively. All treatments that previously received reinoculation in topdressing received inoculation with R. tropici + A. brasilense + Mo/Co micronutrients before sowing, with the same products and their respective doses mentioned above, but readjusting the doses for seed treatments.

The seeds used in the experiment belong to a common bean cultivar BRS Estilo that belongs to the pinto bean group, registration number 25,746 at the “Ministério da Agricultura, Pecuária e Abastecimento—MAPA” of Brazil, and has an erect plant architecture, type II indeterminate growth, and an average cycle of 92 days, adapted to direct mechanical harvesting. This cultivar also has a high yield potential and stability, with clear grains and excellent commercial quality. Regarding diseases, the cultivar is resistant to Bean common mosaic virus, Colletotrichumlindemuthianum, and Uromycesappendiculatus.

2.3. Implementation and Conduction

The experimental area consisted of plots of four rows 5 m in length each, with a spacing between rows of 0.5 m, taking the two central rows as useful areas. The beginning and end of the rows (0.5 m) were used for development analysis, and the central part was intended for analysis of grain yield and its components.

After the corrective reaction period (two months), the soil was prepared conventionally, plowing once and harrowing twice. Then, the sowing fertilization was carried out in the treatments, using 400 kg ha⁻¹ of the fertilizer formulated 00-20-20. The TR6 treatment, with mineral fertilization, received the 05-25-20 formula at the sowing and 60 kg ha⁻¹ of N at the R4 stage, using urea as the source. The sowing density was 12 seeds per meter. Treatments with mineral N fertilization were applied at the sowing furrow and topdressing (TR6), while the control treatment (CRT) only with 400 kg ha⁻¹ of fertilizer formulated 00-20-20 was applied at sowing, without any N source. Treatments were inoculated and sown immediately in the soil.

Reinoculationwas performed at the V4 stage, with the aid of a backpack sprayer with a capacity of 20 L, with a fan-type nozzle, with a jet directed toward the ground at the base of the plants, always performed in the late afternoon, aiming at better inoculation efficiency. The inoculants were diluted according to the recommendations of the manufacturerin an aqueous solution to deliver a final volume of 150 L ha⁻¹.

The crop treatments employed were those commonly applied to common bean through monitoring the crop, using the boom sprayer and recommended products, which included weed control, through the chemical method, withthe association of Flex^® (Syngenta, São Paulo, Brazil) at a dose of 0.06 L ha⁻¹ and Fusilade^® (Syngenta, West Yorkshire, UK) at a dose of 0.08 L ha⁻¹; to control bugs, Engeopleno^® (AlzChem Group AG, Trostberg, Germany) was used at a dose of 0.01 L ha⁻¹, and to control white flies, the insecticide Sperto^® (GSP Crop Science Private Ltda, Gujarat, India) 0.01 g ha⁻¹ was used. During the entire experiment, sprinkler irrigation was carried out on alternate days and always in the morning to meet the crop needs.

2.4. Evaluated Characteristics

At R6 stage, five plants were collected in the first and last 0.5 m of the two useful lines of each plot, containing the common bean root system (Figure 1A), with the aid of a straight shovel, removing the roots in the 0–30 cm layer, where the root system is concentrated, to evaluate the nodulation characteristics (number and dry mass of nodules) and morphophysiological parameters of the plants (main root length, root dry mass, plant height, shoot dry mass, leaf area, and leaf N content in the shoot).

The roots were separated from the aerial part using a stylus and then washed in running water to remove the adhering soil (Figure 1B), for later analysis of thetotal nodules (TN), and the number of nodules found was counted. For counting nodules, nodules with 2 mm or more in diameter were considered, and the result was expressed in units per plant. After evaluation, the nodules were detached, packed in kraft paper bags, and taken to a forced air circulation oven for approximately 48 h at +/− 65 °C, with the dry material being weighed on a precision scale (0.01 g) to determine the dry mass of nodules per plant (NDM). Based on these results, the ratio between the dry mass and the number of nodules per plant was calculated to obtain the dry mass per nodule in grams [44].

With the aid of a graduated ruler, the length of the main root (RL) was evaluated, measuring the distance between the base of the plant and the end of the root (cap) and the height of the plant (PH) and measuring the region from the base of the plant to the apex of the main stem; the results were expressed in cm. The leaf area index (LAI) was obtained using the CI-202 (CID Bioscience, Inc., Camas, WA, USA) leaf measurer with a scanner. To obtain the dry mass, the plant organs were separated into roots (RDM) and shoots (SDM), the samples were placed in kraft paper bags and placed in an oven for approximately 48 h at +/− 65 °C until they reachedconstant weight, and subsequently, the dry mass of the separated plant organs was quantified on a precision scale (0.01 g), with the results expressed in grams per plant [45]. In the dry mass of the leaves, the nitrogen content (NC) was determined by the Kjeldahl method, as described by [46].

At the time of harvest, when the plants were falling off their leaves and pods with a straw yellow color (stage R9), the final stand (FS), the components (number of pods per plant—NPP, number of grains per pod—NGP, and 100-grain weight—W100), and its grain yield (YIELD) were evaluated. The final stand was obtained by computing the final number of plants harvested in the useful area of the plot. Data for the NPP, NGP, and W100 were obtained by counting 10 plants collected in the useful area of each plot, and the YIELD was obtained by collecting the rest of the plants in the useful area of the central rows. The NPP was obtained based on the average number of pods per plant, expressed in n^o. plant⁻¹. The NGP was obtained by averaging the number of grains obtained from the total number of pods harvested, and the result was expressed in n^o. pod⁻¹. The W100 was obtained using random samples of 100 grains, and the result was expressed in grams. The YIELD was obtained by weighing the mass of the grains from all the plants collected in the useful area on a precision scale of 0.01 g, the results obtained were extrapolated to kg ha⁻¹, and the water content was corrected to 13%.

2.5. Statistical Analysis

The data obtained were subjected to analysis of variance (p ≤ 0.05) and, when significant, were submitted to the Tukey test at 5% probability. Statistical analysis was obtained using the Sisvar 5.6 software [47].

3. Results

3.1. Nodular Analyses

The highest number of nodules (TN) with 2 mm or more in diameter, present in the common bean root system, was found in the treatment with reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4), with 20.4 unit plant⁻¹, but not statistically different from the other treatments that received inoculation in the seed and reinoculation (Figure 2A). On the other hand, the treatment that received nitrogen fertilization in the roots (TR6) followed by the control treatment (TR7) presented the lowest average values of nodules per plant, with respective average values of 6.3 and 5.0 nodules per plant⁻¹, respectively.

The TR4 treatment provided the highest NDM average (15.35 g plant⁻¹), followed by the other treatments in which reinoculationwas used, except for the TR3 treatment, which differed from the others. The lowest mean values for NDM were detected in the TR6 treatments with nitrogen fertilization and in control TR7, without any source of inoculation and nitrogen fertilizer (Figure 2B).

3.2. Morphological Analyses

The highest RL (25.8 cm) was found in the treatment with mineral fertilizer (TR6), followed by the treatment with reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4) that presented an RL of 24.6 cm. The treatments in which reinoculation with R. tropici (TR1), reinoculation with co-inoculation of R. tropici + A. brasilense (TR2), reinoculation of R. tropici + Mo/Co (TR3), and inoculation via seed (TR5) were performed obtained RLs of 23.2, 21.5, 19.3, and 22.4 cm, respectively. On the other hand, the control treatment (TR7) had the lowest average for RL—6.33 cm (Figure 3A).

A comparison of the means of the PH variable showed that treatment with mineral nitrogen fertilization (TR6) provided the highest mean (58.1 cm), followed by treatments with reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4), with an average of 54.3 cm; reinoculation of R. tropici (TR1), with an average of 51.9 cm; inoculation via seed, without reinoculation (TR5), with an average of 45.9 cm; and reinoculation with co-inoculation of R. tropici + A. brasiliense (TR2), measuring 42.3 cm. The treatments that presented the lowest averages for PH were the control, without any N source (TR7), and the reinoculation of R. tropici + Mo/Co (TR3), with 19.7 and 39.1 cm, respectively, differing statistically from the other treatments (Figure 3B).

The mean LAI values showed a significant difference between treatments, with treatments with mineral N fertilization at the sowing and topdressing (TR6) and reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4) showing the best averages with 2.5 and 2.3, respectively, followed by the reinoculation treatment with R. tropici (TR1), which differed statistically from the other treatments (Figure 4A).

The highest mean values of RDM were verified in treatments involving reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4), mineral N fertilization (TR6), inoculation via seed, without reinoculation (TR5), and reinoculationof R. tropici (TR1), with the respective mean values of 14.2, 13.9, 11.7, and 9.7 g plant⁻¹, differing from the other inoculated treatments, reinoculation with co-inoculation of R. tropici + A. brasilense (TR2) and reinoculation of R. tropici + Mo/Co (TR3),which presented mean values for RDM of 8.2 and 7.4 g plant⁻¹ (Figure 4B).

The highest average values of SDM were verified in treatments with mineral N fertilization at the sowing and topdressing (TR6) and reinoculation with co-inoculation R. tropici + A. brasilense + Mo/Co (TR4), with 332.2 and 328.8 g plant⁻¹, not statistically different from inoculation treatments via seed, without reinoculation (TR5), reinoculation of R. tropici (TR1), and reinoculation with co-inoculation of R. tropici + A. brasilense (TR2), whose SDM values were 273.3, 266.7, and 230.4 g plant⁻¹, respectively. Treatment with reinoculation of R. tropici + Mo/Co (TR3) showed an average value of 217.89 g plant⁻¹. The control treatment, without any source of N, presented the lowest average to the other treatments for the analyzed variable, with 49.8 g plant⁻¹ (Figure 4C).

Among the treatments studied, it can be seen that the average values of NC showed a significant difference only for the control treatment, without any source of N (TR7), whose foliar N content was 21 g kg⁻¹, differing from the treatments inoculated via seed and reinoculated in topdressing and with mineral nitrogen fertilization, which presented average values of NC between 45 and 55 g kg⁻¹ (Figure 4D).

3.3. Agronomic Analyses

The average values of FS differed statistically between the treatments, and the control, without any N source (TR7), presented an average value of 7.0 plants per meter, while in the other treatments tested, including the inoculation of seeds and reinoculation in the topdressing of R. tropici + A. brasilense and the Mo/Co micronutrients, the FS ranged from 10 to 12 plants per meter, demonstrating that there was a compromise in the FS in the control treatment (Figure 5A).

For the number of pods per plant (NPP) (Figure 5B), it is observed that there was a significant difference between the treatments, in which the mineral fertilization (TR6) presented the highest average value (19.9 unit plant⁻¹), followed by the treatments that received inoculation via seed and reinoculation in topdressing, not statistically different from each other. On the other hand, the lowest NPP was verified in the control treatment, without any source of N with 7.2 pods plant⁻¹, confirming that the restriction of N to the plants compromised the NPP component of beans.

The number of grains per pod (NGP) showed similar behavior to NPP, with treatments involving seed inoculation and topdressing reinoculation differing statistically only from the control (Figure 5C), which had the lowest mean (3.9), without any source of N. The results of the treatments reinoculation with co-inoculation of R. tropici + A. brasilense + Mo/Co (TR4) and inoculation via seed, without reinoculation (TR5), with 5.4 pods plant⁻¹ and the treatment with reinoculation stand out from R. tropici (TR1) with 5.3 pods plant⁻¹, followed by treatments with mineral N fertilization at the sowing and topdressing (TR6), reinoculation with co-inoculation of R. tropici + A. brasilense (TR2), and the reinoculation treatment of R. tropici + Mo/Co (TR3) which did not differ statistically from each other and presented means of 5.0, 4.6, and 4.6 pods per plant, respectively.

The 100-grain weight (W100) differed significantly between treatments. The treatments that showed the highest means were mineral N fertilization at the sowing and topdressing (TR6) and reinoculation with co-inoculation R. tropici + A. brasilense + Mo/Co (TR4) with respective means of 27.2 and 26.9 g, followed by reinoculation of R. tropici (TR1) and seed inoculation without reinoculation (TR5). In contrast, the lowest W100 values were observed in treatments with reinoculation of R. tropici + Mo/Co (TR3) with 19.9 g, reinoculation with co-inoculation of R. tropici + A. brasilense(TR2) with 19.6 g, and the control treatment, without any source of N (TR7), with 19.2 g (Figure 5D).

The grain yield values differed statistically between the tested treatments, with emphasis on mineral N fertilization at the sowing and topdressing (TR6) and reinoculation with co-inoculation R. tropici + A. brasilense + Mo/Co (TR4), with a grain yield of 2952 and 2865 kg ha⁻¹, respectively, statistically differing from treatments with reinoculation of R. tropici (TR1) and inoculation via seed, without reinoculation (TR5), whose respective means of grain yield were 2426 and 2298 kg ha⁻¹, in addition to the treatments reinoculated with R. tropici + Mo/Co (TR3) and reinoculated with co-inoculation of R. tropici + A. brasilense (TR2) with 1555 and 1477 kg ha⁻¹, and the control treatment, without any source of N (TR7), with only 707 kg ha⁻¹ (Figure 5E).

4. Discussion

4.1. Nodular Analyzes

The greater number of nodules produced by bean plants in the treatment that involved inoculation with R. tropici, A. brasilense, and Mo/Co strains with the other reinoculated treatments and treatments with inoculation via seed, can be attributed to the fact that the nodulation process is a complex and distinct event according to [48] since the efficiency is not determined by the variety of the plant nor by the strain of rhizobia, but by the interactions between the symbiotic.

The decrease in the number of nodules in the common bean root system in the treatment with mineral nitrogen fertilizer is related to the supply of readily assimilable N2 to the bacterial population, thus inhibiting the process of N2 fixation in the atmospheric air. These results are consistent with [49] when they observed that nitrogen fertilization decreases nodule activity in bean roots. It is also noteworthy that the population of native fixative bacteria found in the soil was not efficient in promoting nodulation in common bean because it had the lowest number of nodules per plant, thus demonstrating the importance of the inoculation and reinoculation processes in topdressing to meet the requirement of the bean plant regarding the need for nitrogen.

The results referring to the TN and NDM are in agreement regarding the effects of the treatments tested, with emphasis on the T4 treatment in which the association R. tropici, A. brasilense, and the Mo/Co micronutrients were used. The addition of A. brasilense promotes an increase in root growth, while Mo/Co improves N metabolism and the activity of the bacterial population [30] and consequently improves the efficiency of the R. tropici strain population in the common bean N2 fixation.

In a study carried out by [25], a positive effect of Mo on N2 fixation was verified since there was more N accumulated per root and nodule mass, that is, greater radicular and nodular efficiency in N absorption, corroborating in part with the results obtained in the present study. Similarly, in a study conducted by [5], it was noted that the increase in the dose of mineral nitrogen fertilizer provided a reduction in N2 fixation, thus corroborating the increase in the concentration of mineral N in the soil solution, thus disfavoring the symbiosis between plant and the rhizobia. The N concentration in the soil solution is due to the accumulation of nitrite and nitrate at the root level, which can reduce the availability of the bacteroid.

4.2. Morphological Analyzes

The availability of N readily assimilable by the plants through the use of mineral nitrogen fertilizer, from the emergence phase onwards, can be pointed out as responsible for the greater root growth of bean plants presented in the TR6 treatment. However, the treatments that received inoculation via seed and reinoculation in topdressing, with the association of R. tropici and A. brasilense, presented root length values very close to the plants that received mineral fertilization, thus confirming the effective action of A. brasilense in promoting growth associated with increased N-fixing activity of R. tropici, as pointed out by [33,34].

The smallest bean PH was verified in control (TR7) among all the treatments tested, probably due to the omission of nitrogen supply in the mentioned treatment since it presents a positive correlation with the photosynthetic rates, with a consequent influence on the development of the plants. In research carried out by [50], significant differences were observed for bean plant height, so treatments with inoculation using only R. tropici and co-inoculation with R. tropici + A. brasilense showed greater height compared to the control.

The treatments with the lowest mean values were with inoculation via seed, without reinoculation (TR5)—0.9 and control, without any N source (TR7) (0.4), demonstrating in both treatments that only the inoculation of R. tropici via seed and the population of native fixing bacteria were unable to promote an increase in leaf area, probably due to the reduced amount of N supplied to the plants.

The control treatment (TR7) presented the lowest mean value for RDM (3.4 g plant⁻¹), confirming that the lack of nitrogen fertilization and/or inoculation via seed/reinoculation in topdressing with R. tropici + A. brasilense + micronutrients Mo/Co impaired the development of the common bean root system.

Oliveira et al. [20] observed that root dry mass and molybdenum doses promoted an increase in bean root mass in the treatment without inoculation and decreased when inoculated. These results are contrary to those obtained in the present research, a difference that can be attributed to the increasing doses of Mo investigated in that study, and the highest doses (150 g ha⁻¹), above all, impaired the action of the inoculated rhizobian bacteria. It is also noteworthy that the association of A. brasilense with Mo/Co micronutrients in co-inoculation with R. tropici may have contributed to greater efficiency of symbiotic N fixation, reducing the plant’s need to invest in the production of roots to increase the efficiency of N absorption from the soil.

The results obtained for SDM and PH showed similar behavior in terms of significance of the tested treatments, demonstrating a strong relationship between these variables.

It is noteworthy that the control was the only treatment that showed symptoms of generalized chlorosis in the bean foliage leading to the detection of deficiency due to the lower amount of N available to the plants (21 g kg⁻¹), and confirmed by information in the literature [51,52], whose value found is below the range of adequate N content present in the bean leaf at flowering, between 30 and 50 g kg⁻¹. On the other hand, the values of foliar N contents between 45 and 55 g kg⁻¹ detected in the treatments inoculated via seed and reinoculated in topdressing and with mineral nitrogen fertilization are within the values mentioned in the literature.

The application of nitrogen fertilizers, assimilated more quickly by the plant, can clarify the results obtained in the treatments with mineral fertilizer, but the treatments with inoculation via seed and reinoculated in topdressing showed similar foliar levels of N, except for the reinoculation treatment with co-inoculation of R. tropici + A. brasilense + micronutrients Mo/Co (TR4), which can be explained by the “dilution factor” of N in the plant according to [30] since this one generally stood out from the others with the additions of the nodulation process and the morphological characteristics analyzed.

N is directly linked to the increase in plant biomass, thus, an efficient N2 fixation makes more N available to plants and increases plant biomass [25]. A study by [15] found that the N content accumulated by inoculated plants is higher than that of non-inoculated plants due to N2 fixation. Such results were confirmed in the present research. It is also noteworthy that the N content or nitrogen utilization index is directly proportional to the above ground part biomass and the nitrogen content in the plant [53], and thus, it can be considered that the greater accumulation of N in bean leaves is associated with high rates of nitrogenase enzyme activity, thus justifying the greater efficiency of N2 fixation, equivalent to treatments with mineral nitrogen fertilization, corroborating the reports by [25] on the subject.

4.3. Agronomic Analyzes

Possibly with a direct reflection on the components and the bean grain yield since the desirable FS for high grain yield of beans is around 12 plants per meter [54].

Research carried out by [55] found that NPP depends on the strain of R. tropici used, as these strains have different host specificity and, therefore, can demonstrate differential behavior and different values for nitrogen fixation and accumulation in plants, as found in this research. The efficiency of inoculation with R. tropici associated with A. brasilenseis attributed to the physiological changes caused in the plants due to the release of hormones such as auxins and cytokinins that increase root growth, and with that, the plant tends to absorb water and nutrients [21], like Mo and Co.

In the research carried out by [20], it was found that the NGP did not differ depending on the inoculation and doses of Mo applied via the foliar, justifying the lack of a high genetic heritability response, however, these results are divergent from this study, which can be attributed to differences in the management conditions applied to the plant sand the genotype used.

Although the W100 is a variable influenced predominantly by the genetics of the plant [40], there was an influence of the treatments on it, like the NGP, due to the same factors mentioned above.

When comparing the average grain yield obtained from the common bean crop in Brazil in the last harvest (2020/2021), 990 kg ha⁻¹ [56], to the grain yield verified in this study, specifically in the treatment with inoculation and reinoculation in topdressing with R. tropici + A. brasilense and the micronutrients Mo/Co, whose average was 2865 kg ha⁻¹, it appears that this value is almost three times the national average, and only 3% lower than the average obtained in the treatment with mineral nitrogen fertilization, confirming the capacity of the inoculant R. tropici in association with the application of A. brasilense plus Mo/Co to meet the demand for N by the common bean crop. In addition, these results confirm that seed inoculation alone is not enough to meet the common bean demand for nitrogen, requiring the reinoculation in topdressing at the R4 stage and the inability of soil-native fixing bacteria to provide the N need for the crop in question.

Recently, some research results have been unanimous in stating that the reinoculation procedure with R. tropici in the common bean crop, conducted close to flowering, promotes the reinvigoration of the fixative activity of the population of bacteria inoculated at sowing [39,40,41,49]. In this study, it can be noted that the morphological and agronomic analyzes obtained similar results, but with the inclusion of other products in the application, such as A. brasilense in association with Mo/Co, which ensured the achievement of high levels of grain yield, equivalent to the use of mineral nitrogen source.

Thus, it is evident that the inoculation of R. tropici through N2 fixation in association with A. brasilense plus micronutrients added to the seeds and later in topdressing can supplement or even replace nitrogen fertilization in the bean crop, allowing to reduce doses of nitrogen fertilizers without causing a reduction in grain yield, reducing costs and alleviating environmental problems.

5. Conclusions

The nodulation process and the morpho-physiological characteristics of common bean are improved by using an inoculant in combination with A. brasilense plus Mo/Co micronutrients for mineral nitrogen fertilization.

The use of mineral nitrogen fertilizer can increase the nitrogen content in the shoot; however, it reduces the number of nodules and the efficiency of nitrogen use.

Using the mixture inoculant + A. brasilense + Mo/Co micronutrients applied to the seed and reinoculated in topdressing at the R4 stage allows obtaining a bean yield of up to 2865 kg ha⁻¹, statistically equivalent to the yield obtained in the treatment with mineral nitrogen fertilizer, 2952 kg ha⁻¹, equivalent to a difference of just 3% between the yields obtained in both treatments.

Inoculation of seeds with R. tropici in topdressing, associated with co-inoculation with A. brasilense + Mo/Co, can completely replace mineral nitrogen fertilization in common bean.