Osmoprotection, cobalt, molybdenum and co-inoculation in soybean culture

Osmoproteção, cobalto, molibdênio e coinoculação na cultura da soja

Abstract

Inoculation is essential to supply nitrogen (N) to soybeans and includes live organisms, which may have reduced viability when coming into contact with seed treatment (ST) products. To evaluate the effectiveness of an osmoprotectant between co-inoculation and the application of Cobalt (Co) and Molybdenum (Mo) during ST and specify the best time to apply Co and Mo to soybeans. In the 2017/2018 and 2018/2019 harvests, four experiments were carried out, using the cultivars NS 5959 IPRO and TMG 7062 INTACTA. An experimental design was adopted in randomized blocks with four replications, in a factorial scheme (2 x 3) + 1 experiment, consisting of the use or not of osmoprotectant and application of Co and Mo to the seeds, foliar or without its application + control. The use of osmoprotectant combined with chemical treatment of seeds reduced the dry mass of nodules, the mass of a thousand grains, the number of pods and soybean productivity. Co and Mo in ST, in the absence of osmoprotectant, increased grain yield (GY) by an average of 12.6% for the cultivar NS 5959 IPRO. Annual seed co-inoculation promoted increases of up to 11.8% in grain productivity. Co-inoculation increases the crude protein content. Co-inoculation with application of Co and Mo increases soybean yield without harming the environment and at low cost. The use of Co and Mo at the seed in areas with co-inoculation allows obtaining more fertile areas and with greater potential for soybean productivity under intensive management.

Keywords:
biological fixation of nitrogen; micronutrients; protein content; co-inoculation; soybean productivity

Resumo

As sementes são agentes de introdução das tecnologias de proteção e nutrição de plantas, pois as inovações são aplicadas via tratamento de sementes (TS) pelo menor custo e facilidade de operações. Contudo, o processo de inoculação é primordial para fornecimento de nitrogênio (N) para a cultura e envolve organismos vivos, os quais em contato com produtos do TS podem ter sua viabilidade reduzida. Objetivou-se avaliar a utilização de um osmoprotetor entre a co-inoculação e a aplicação de Cobalto (Co) e Molibdênio (Mo) via TS, além da definição do modo de aplicação do Co e Mo na cultura da soja. Foram conduzidos dois experimentos na safra 2017/2018 e dois na 2018/2019 com as cultivares NS 5959 IPRO e TMG 7062 INTACTA. O delineamento experimental foi de blocos ao acaso com quatro repetições, envolvendo um bifatorial (2 x 3) + 1, constituído por utilização ou não de osmoprotetor e aplicação de Co e Mo nas sementes, foliar ou sem sua aplicação + testemunha. O uso de osmoprotetor juntamente ao fungicida, inseticida, Co e Mo nas sementes reduziu à massa seca de nódulos, massa de mil grãos, número de legumes e produtividade de grãos da soja. O Co e Mo aplicados no TS, na ausência de osmoprotetor incrementou em média 12,6% a produtividade de grãos (PG) em relação ao somente co-inoculado para a cultivar NS 5959 IPRO. A co-inoculação anual das sementes promoveu acréscimos de 11,8% e 2,4% na produtividade de grãos das cultivares NS 5959 IPRO e TMG 7062 INTACTA, respectivamente. A co-inoculação aumenta o teor de proteína bruta nos grãos da cultura da soja. A utilização da co-inoculação associado ao Co e Mo ampliam a produtividade da cultura da soja.

Palavras-chave:
fixação biológica de nitrogênio; micronutrientes; conteúdo de proteína; co-inoculação; produtividade de soja

1. Introduction

Soybean (Glycine max (L.) Merrill) plays an important role in the world economy. This crop is unique for its oleaginous quality and high nutritional value, as the grains are high in proteins, vitamins, minerals and fiber (Medic et al., 2014). As the protein content of the grain is around 40%, it is essential as food and feed. Besides, due to its large-scale production and high protein yield per area, the purchase cost is much less than that of the other protein. These factors have contributed to the expansion of its cultivated area, however, with an estimated population growth of 9.55 billion people by 2050, world food demand will more than double, requiring increased productivity in already cultivated areas (FAO, 2014).

Plant nutrition is fundamental to boost grain production and between the agricultural years 2007/2008 and 2015/2016 it was responsible for 27.82% of crop costs (CONAB, 2016). Nitrogen (N) ranks first among the nutrients essential for good crop performance, because of the high protein content in the soybean grains. The N is produced through the symbiotic process between the plant and bacteria belonging to genus Bradyrhizobium. Through the biological nitrogen fixation process (BNF) is produced 84% of the total N requirement for this cultivar, assuring good economic value and low environmental damage by avoiding the application of nitrogen fertilizers. In addition to the bacteria of the genus Bradyrhizobium, another group of bacteria have been excelling in their use along with these, are those belonging to the genus Azospirillum. These associative bacteria facilitate plant growth via the release of growth hormones (Giri et al., 2025), inducing resistance to diseases and environmental stresses (Müller et al., 2025; Souza et al., 2023), enabling phosphate solubilization (Tahir et al., 2013) and performing BNF (Sun et al., 2025) resulting in boosting the crop yield (Queiroz Rego et al., 2018).

However, some factors exert a negative effect on the BNF like micronutrient deficiency, including Cobalt (Co) and Molybdenum (Mo). The Mo in legumes is involved in plant nutrition and N uptake as part of enzymatic components that catalyze reactions and participate in electron transfer processes (Hille, 2013). The electron transfer from Mo to nitrite is observed for the reduction of NO^3- to NO^2-, the first step in the integration of N as NH₂ into protein. It is also an essential component of the enzyme nitrogenase as Fe-Mo protein, which receives the Fe-protein electrons as they bind to the N₂. Thus, when these electrons are present, the H arising from the respiration process via oxidative metabolism is incorporated with the ATP to cause the N₂ triple bond to break and release the NH₃ + H₂. Both processes, reduction and nitrogenase formation, facilitate the N₂ to be transformed into forms that the plant can assimilate (Hu and Ribbe, 2013).

Co is an element essential to the microorganisms involved in BNF, as it is part of the vitamin B12 structure and participates in building the cobalamide coenzyme, the precursor of legmoglobin, which controls the nodular activity (Mus et al., 2016).

In their study, Dourado Neto et al. (2012) and Galindo et al. (2017) reported a boost in the soybean yield when Co and Mo were used during inoculation with Bradyrhizobium or co-inoculation. However, others studies also demonstrated that when the Co and Mo were applied to the seeds, they could lower the survival of the inoculated bacteria because of the pH, salinity and bactericidal action of some of the products, thus causing a drop in the number of viable cells to nodulate the plant roots (Silva et al., 2011). As an alternative method, the Co and Mo could be sprayed to prevent direct contact with the bacteria-inoculated seed, while maintaining their survival.

However, as seeds are recommended as agents of plant protection and nutrition technologies because they are easy and cost effective in terms of operation, suitable options are being sought for the maintenance and viability of these seed treatment (ST) methods. One such possibility is by applying osmoprotectants onto the seeds. Osmoprotectants contain cellular protectors in their make-up which minimize the toxic effects of seed treatment, polymers which lower the influence exerted by the fluctuations on relative humidity, raise the inoculant adhesion onto the seeds, offer substrates that act as nutrients for the bacteria, and also provide signaling substances that enhance the bacteria-plant root communication (Deaker et al., 2007; Araújo et al., 2017; Stecca et al., 2019).

Considering that the bacteria used for co-inoculation do not form spores, they are more susceptible to deleterious factors that occur during the inoculation process and after in soil (O'Callaghan, 2016; Berninger et al., 2018), and that its use is indispensable for soybean cultivation, since they reduce the cost of production of the crop and do not cause damage to the environment, products and technologies that aim at the survival of inoculated bacteria are necessary, keeping the viable cells for occupy the rhizosphere in sufficient numbers. Therefore, objectified aimed at evaluating the relevance of the osmoprotectant application between co-inoculation and the addition of Co and Mo through ST, as well as to specify the best moment of applying the Co and Mo in soybean.

2. Material and Methods

The field experiments were performed on the 2017/2018 and 2018/2019 crops, in a specifically allotted area in Departmento de Fitotecnia da Universidade Federal de Santa Maria, (29° 43'2.81 "S, 53° 43'58.28" W, 116 m a.s.l). The climate in this region of Rio Grande do Sul, based on the Köeppen classification is Cfa (86.7%) and Cfb (13.3%) (Alvares et al., 2013).

The soil in the area is classified as an Ultisol (USDA, 2022), with the following chemical analysis (Table 1). The sowing experiments were performed on November 30, 2017 and November 6, 2018, on the remains of the earlier wheat cultivation. The NS 5959 IPRO and TMG 7062 INTACTA soybean cultivars were used, at sowing densities of 37 and 26 seeds/m², respectively, during both seasons.

Table 1
Physicochemical analyses of the soil from the first year (2017) and the second year (2018) of the experiments. UFSM, Santa Maria (RS).

	Year 2017
Layer (cm)	pH	MO	CLAY	P	K	H + Al	CTC pH 7	V
		-------%-------		---mg/dm3---		----cmolc/dm3----		---%---
0-10	5.7	2.0	21	35.9	53.9	3.9	11.8	67
	Year 2018
Layer (cm)	pH	MO	CLAY	P	K	H + Al	CTC pH 7	V
		-------%-------		---mg/dm3---		----cmolc/dm3----		---%---
0-10	5.5	2.2	26	11.8	64.12	3.9	12.2	67.9

• pH = Hydrogen Potential; MO = Organic matter; P = Phosphorus; K = Potassium; H+Al = Potential acidity; CTC pH 7 = Cation Exchange Capacity at pH 7; V = Base saturation.

Each experiment was conducted in a randomized complete block design, with four replications per treatment, involving a factorial model (2 x 3) + 1 constituted by the use or not of osmoprotectant and Co and Mo application via ST, leaf V3 or not application, totaling six treatments and the control. The treatments included T1: only seed treatment (Control); T2: only co-inoculated; T3: co-inoculated + Co and Mo in ST; T4: co-inoculated + Co and Mo in V3; T5: co-inoculated + osmoprotectant; T6: co-inoculated + Co and Mo and osmoprotectant in ST; T7: co-inoculated + osmoprotectant in ST and Co and Mo in V3. Each experimental unit was 7.75 m in length and 2.25 m in width, with five rows having 0.45 m spacing, for a total of 17.4 m² of total area and 6.75 m² of usable area.

From the chemical analysis of the soil was applied the fertilization of the area, where the macronutrients P and K were supplied in the furrow using the fertilizer sowing, for expectation of five tons of soybean yield per hectare, together with the sowing process. Pests, disease and weed management measures were implemented based on the technical recommendations, circumventing any interference by these factors during the crop development (Salvadori et al., 2016).

The chemical treatment of the seeds involved the addition of Pyraclostrobin 25 g/l i.a. + methyl thiophanate 225 g/l i.a. + Fipronil 250 g/l i.a. (Standak® Top) at a dose of 2 ml/kg of seeds. The seeds were covered with 20 ml/ha Mo and 2 ml/ha Co, and when applied via leaf in V3 the doses were doubled. The osmoprotectant 1 ml/kg of seeds (water, active metabolites of bacteria, sugar complex and biopolymers) were applied. Co-inoculation was done using a liquid inoculant for the bacterium Bradyrhizobium japonicum (7x10⁹ CFU/ml) at a dose of 2 ml/kg of seeds, and for Azospirillum brasilense (2x10⁸ CFU/ml) at a dose of 2 ml/kg of seeds.

At the phenological stage R2 of the based on the phenological scale of Fehr and Caviness (1977), the number of root nodules was noted. For the assessment, four plants were randomly picked from each experimental unit. All four plants were maintained in a pre-established soil volume of 0.008 m³, with dimensions of 0.2 (S1) x 0.2 (S2) x 0.2 (H) collected using a shovel. The number of nodules per plant was recorded by directly counting the nodules in the main and secondary roots (NNP, number/plant). Next, the nodules were washed and subsequently dried in drying oven at 65°C for 48 hours. The samples were then weighed by determining the nodule dry mass per plant (NDM, mg/plant). The aerial parts of these same plants were dried in a forced circulation oven at 65 °C for 48 hours and weighed to ascertain the plant dry mass (PDM, g/plant).

At the plant stage R2, the soil was taken to isolate and identify the Azospirillum using the techniques adopted by Döbereiner et al. (1995); Bradyrhizobium was similarly checked using the methodologies adopted by Hungria and Araújo (1994).

At the time for full crop maturation (R8), five plants were collected in sequence, from the cultivation row of the useful area of the experimental unit, and the number of pods per plant were evaluated (NPP, number/plant). Five plants were harvested from the three central rows of the experimental unit (6.75 m²). The samples were thrashed and cleaned to estimate and correct the grain moisture (base 13%) to calculate the grain yield (GY, kg/ha) and mass of one thousand grains (MTG, g). Next, the seed samples drawn from each treatment were transported to the União de Ensino do Sudoeste do Paraná (UNISEP) to evaluate the grain protein content (PC) using near infrared spectroscopy (NIRS). Protein data were evaluated according to the completely randomized design.

The data for both years in each experiment were subjected to a factorial analysis of variance and the means were submitted to complementary procedures based on the results presented by the interaction. The degrees of freedom versus the control by the Scheffé test were contrasted at 5% probability of error and significant interactions were submitted to the comparison test Scott-Knott averages at 5% probability of error using the Genes® statistical software.

3. Results

Tables 2 and 3 summarize the results of variance analyses of cultivars NS 5959 IPRO and TMG 7062 INTACTA. For the variables NNP and NDM, both revealed no significant difference between the factorial and its respective control, during both experimental years (P>0.05). In the cultivar NS 5959, the control showed values which fell below the average of the factorial for the PDM, NPP and GY, while the MTG and PC values were also lower in the 2017/2018 crop. In TMG 7062 the PC content was above in the 2018/2019 crop factorial. In the 2017/2018 crop the control PDM showed higher values than did the factorial and in the 2018/2019 this as well as the MTG gave lower values.

Table 2
Summary of the variance of analysis for the cultivars NS 5959 IPRO and TMG 7062 INTACTA for the 2017/2018 crop, represented by the mean squares of the explanatory variable: number of nodules (NNP, number/plant), nodule dry mass (NDM, mg/plant); shoot dry mass (PDM, g/plant), thousand-grain mass (MTG, g), number of pods (NPP, number/plant), grain yield (GY, kg/ha), protein content crude (PC).

SV	DF	NNP	NDM	PDM	MTG	NPP	GY	PC 1
NS 5959 IPRO
Blocks	3	155.75	600.14	0.69	15.59	12.24	7523.96	-
Treatments	6	349.24	3429.03*	2.75*	85.16	64.23*	451321.30*	1.28*
Factorial	5	394.4	4113.47*	2.28*	17.13	46.97*	232361.73*	0.69*
Contrast	1	123.43	6.88	5.11*	425.32*	150.48*	1546119.16*	4.22*
Error	18	409.78	917.89	0.78	34.08	12.43	7588	0.01
Control Treatment Average		109.75	220	9.17	180.53	50.08	3580.38	31.35
CoMo (D)	2	412.13	892.54	3.81*	31.58	46.63	254527.34	1.25
Osmoprotector (C)	1	16.67	4160.67	0.96	1.84	26.04	10973.07	0.05
DxC	2	565.54	7310.79*	1.41	10.32	57.79*	320890.45*	0.45*
Average		115.75	218.58	10.39	191.67	56.87	4251.92	32.64
CV (%)		17.49	13.9	8.53	3.04	6.19	2.05	0.34
TMG 7062 INTACTA
Blocks	3	141.75	1316	0.22	170.43	1.24	76645.58	-
Treatments	6	541.61*	5619.20*	19.69*	146.63	60.98*	242193.00*	0.84*
Factorial	5	649.90*	6203.84*	14.58*	167.74	70.18*	283330.70*	0.92*
Contrast	1	0.21	2696	45.24*	41.09	15.02	36504.49	0.43
Error	18	180.44	1140.47	0.54	73.4	16.25	20791.04	0.23
Control Treatment Average		93.25	272.75	15.42	209.85	55.67	3385.08	31.6
CoMo (D)	2	916.13	8177.54	2.32	6.64	39.31	134224.7	0.82
Osmoprotector (C)	1	560.67	12.04	33.18	12.11	0.05	162006.4	0.07
DxC	2	428.29	7326.04*	17.59*	406.66*	136.12*	493098.80*	1.44*
Average		93	244.71	11.78	213.31	57.76	3488.3	32.01
CV (%)		14.44	13.8	6.25	4.01	6.98	4.13	1.52

• ^*
  Significant by the F test (P<0.05).
• Source of variation (SV); Degree of freedom (DF); Coefficient of variation (CV%).
• ¹
  PC was evaluated in a completely randomized design with three replications; the DF error was 14.

Table 3
Summary of the variance of analyses for the cultivars NS 5959 IPRO and TMG 7062 INTACTA for the 2018/2019 crop, represented by the mean squares of the explanatory variable: number of nodules (NNP, number/plant), nodule dry mass (NDM, mg/plant); shoot dry mass (PDM, g/plant), thousand-grain mass (MTG, g), number of pods (NPP, number/plant), grain yield (GY, kg/ha), protein content crude (PC).

SV	DF	NNP	NDM	PDM	MTG	NPP	GY	PC 1
NS 5959 IPRO
Blocks	3	615.76	3805.67	22.52	30.33	14.23	190422.61	-
Treatments	6	2772.70*	8475.18	81.64*	785.32*	42.39*	198776.90*	0.18
Factorial	5	3325.85*	9917.54	69.19*	477.21*	49.45*	175103.97*	0.16
Contrast	1	6.96	1263.36	143.93*	2325.89*	7.08	317141.55*	0.27
Error	18	496.75	3707.87	14.29	95.22*	10.28	54645.31	0.14
Control Treatment Average		85.8	384.42	27.52	76.23	169.57	3572.17	31.96
CoMo (D)	2	1072.14	1254.76	55.41	576.3	0.79	59813.56	0.11
Osmoprotector (C)	1	13066.66	40319.4	190.97	186.48	196.65	359097.27	0.37
DxC	2	709.15	3379.39	22.07	523.48*	24.52	198397.73*	0.12
Average		84.37	403.62	34	102.27	171.01	3876.31	32.29
CV (%)		26.41	15.08	11.11	9.54	1.87	6.03	1.16
TMG 7062 INTACTA
Blocks	3	126.95	3465.71	10.14	71.75	20.53	20053.76	-
Treatments	6	911.39*	6456.16	9.84	155.07*	82.43*	180275.84*	0.33*
Factorial	5	1093.04*	7238.06*	0.98	186.07*	69.27*	213225.21*	0.37*
Contrast	1	3.14	2546.62	54.12*	0.05	148.22*	15528.99	0.12*
Error	18	317.09	2477.35	7.32	53.38	18.11	40231.76	0.01
Control Treatment Average		83	358	24.41	72	187.95	3479.8	32.05
CoMo (D)	2	676.79	3990.09	0.18	114	57.35	3115.27	0.39
Osmoprotector (C)	1	1426.04	16752.92	0.92	260.04	100.04	485254.65	0.04
DxC	2	1342.79*	5728.61	1.81	221.16*	65.81*	287320.44*	0.53*
Average		83.7	330.74	28.38	71.87	194.53	3547.1	32.27
CV (%)		21.27	15.05	9.53	10.16	2.18	5.65	0.38

• ^*
  Significant by the F test (P<0.05).
• Source of variation (SV); Degree of freedom (DF); Coefficient of variation (CV%).
• ¹
  PC was evaluated in a completely randomized design with three replications; the DF error was 14.

For cultivar NS 5959 the NPP and GY of the factorial were confirmed to be 11.9% and 15.8% higher, respectively, than the control in the 2017/2019 crop and 25% and 7.8% in the 2018/2019 harvest. In the TMG 7062, although the means showed no significance, the GY and PC were also greater, implying the benefits of re-inoculation in each season crop (Tables 2 and 3).

For the variables NPP, GY and PC, the factorial (Osmoprotectant X Co and Mo) demonstrated double interactions in cultivar NS 5959 in the 2017/2018 crop (P<0.05), and for the NPP and GY in the 2018/2019 harvest. For TMG 7062 double interaction was evident for PDM in the 2017/2018 crop, MTG an PC in the 2018/2019 (Tables 2 and 3).

The 2017/2018 crop for the cultivar TMG 7062 showed a decrease in the PDM when the osmoprotectant was applied in combination with the Co and Mo in ST, and without the application of Co and Mo. It is also observed that even though there was no reduction in TMG 7062 NDM, the treatment with osmoprotectant alone reduced the PDM (Table 4). When applied to the leaf, the Co and Mo, even together with the osmoprotectant application may have boosted the BNF efficiency of the bacteria that produced the nodules of these roots (Table 3). For the same variable PDM and in the same crop, in NS 5959, this effect was noteworthy for the Co and Mo factors, highlighting its application in ST (Table 5).

Table 4
Nodule dry mass (NDM, mg/plant), dry mass of plant shoot in R2 (PDM, g/plant), one thousand grain mass (MTG, g) as a function of the application of the osmoprotectant, as well as Co and Mo for the cultivar TMG 7062 INTACTA, in the 2017/2018 crop.

Interaction (Osmoproctetor X Co and Mo)
	Without Osmo	With Osmo		Without Osmo	With Osmo		Without Osmo	With Osmo
	NDM			PDM			MTG
Without CoMo	267 Aa*	295 Aa		13.7 Aa	10.9 Bb		212 Aa	217 Aa
ST CoMo	257 Aa	186 Bb		13.8 Aa	8.7 Bc		220 Aa	205 Bb
V3 CoMo	212 Aa	251 Aa		11.4 Aa	12.2 Aa		206 Ba	220 Aa

• ^*
  Means followed by the same letter, uppercase in the row and lowercase in the column, do not differ statistically from each other by the Scott-Knott test (P>0.05).

Table 5
Dry mass of the nodules in R2 (NDM, mg/plant), number of pods (NPP, number/plant), grain yield (GY, kg/ha), crude protein in the grain (PC, %) and dry mass of the plant shoot in R2 (PDM, g/plant) as a function of the osmoprotectant application and Co and Mo application for the cultivar NS 5959 IPRO, in the 2017/2018 crop.

					Interaction (Osmoproctetor X Co and Mo)										Main fator
		Without Osmo		With Osmo			Without Osmo	With Osmo		Without Osmo	With Osmo		WithoutOsmo	With Osmo
		NDM					NPP			GY			PC			PDM
Without CoMo		252 Aa*		178 Ba			53 Ab	56 Aa		3997 Bc	4178 Ab		32.98 Ba	33.24 Aa		10 b
ST CoMo		254 Aa		207 Ba			64 Aa	56 Ba		4694 Aa	4189 Bb		32.96 Aa	32.22 Bb		11 a
V3 CoMo		190 Ab		231 Aa			57 Ab	56 Aa		4129 Bb	4325 Aa		32.13 Ab	32.27 Ab		10 b

• ^*
  Means followed by the same letter, uppercase in the row and lowercase in the column, do not differ statistically from each other by the Scott-Knott test (P>0.05).

The variable NPP ranks among the principal constituents of the soybean crop yield. It was clear that when the Co and Mo were applied in combination with the osmoprotectant, plant development in the cultivar NS 5959 was reduced, but when the same were applied without the osmoprotectant in the ST, the NPP value exceeded that of the other treatments (Tables 5 and 6). The cultivar TMG 7062 in the 2018/2019 crop showed decreased MTG when the products were applied together (Table 6).

Table 6
Number of pods (NPP, number/plant), grain yield (GY, kg/ha) for cultivar NS 5959 IPRO, mass of one thousand grains (MTG, g) and crude protein content in the grain (PC, %) for TMG 7062 INTACTA after the application of the osmoprotectant and Co and Mo, in the 2018/2019 crop.

Interaction (Osmoproctetor X Co and Mo)
	NS 5959 IPRO								TMG 7062 INTACTA
		Without	With		Without	With			Without	With		Without	With
		Osmo	Osmo		Osmo	Osmo			Osmo	Osmo		Osmo	Osmo
		NPP			GY				MTG			CP
Without CoMo		89 Ac*	98 Aa		3830 Ab	3833 Aa			194.44 Aa	195.57 Aa		32.60 Aa	32.12 Ba
ST CoMo		122 Aa	99 Ba		4275 Aa	3676 Ba			196.76 Aa	186.53 Bb		32.67 Aa	32.27 Ba
V3 CoMo		104 Ab	101 Aa		3890 Ab	3752 Aa			198.51 Aa	195.36 Aa		31.69 Bb	32.28 Aa

• ^*
  Means followed by the same letter, upper case in the row and lower case in the column, do not differ statistically from each other by the Scott-Knott test (P>0.05).

In GY, a notable difference was evident (P<0.05) for the cultivar NS 5959 in response to the Co and Mo treatment, where higher yields were implied when they were applied in the ST without the osmoprotectant for both harvests, and in V3 when applied along with the osmoprotectant for the 2017/2018 crop (Tables 5 and 6). For the V3 the application of Co and Mo demonstrated a rise in the productivity in association these nutrients whith osmoprotectant. The PC content also declined when the Co, Mo and osmoprotectant were applied together (Tables 5 and 6).

The experiment incurred a monetary expenditure in the range of $ 754.5 to 775 per hectare, whereas costs involved for the other technologies are as mentioned: co-inoculation (US$ 3.52/ha), osmoprotectant (US$ 0.81/ha) and Co and Mo application (US$ 8.13/ha). When Co and Mo were applied in leaf, the total cost went up to US$ 8.13 excluding the cost of operation machinery/implements, as this application was normally done along with the herbicide application post-emergence (Table 7).

Table 7
Total production cost, grain yield (GY, kg/ha), gross revenue (GR, US$/ha), net revenue (NR, US$/ha) and net gain over control (NG, US$/ha) of the soybean crop as a function of the treatments.

		2017/2018 Harvest					2018/2019 Harvest
		NS 5959		TMG 7062			NS 5959			TMG 7062
Treatments		GY*	GR	GY	GR		GY	GR		GY	GR
Control Treatment		3600	930	3360	868		3600	930		3480	899
Co-inoculation		4020	1038	3360	868		3840	992		3720	961
Co-inoculation+ST CoMo		4680	1209	3840	992		4260	1100		3840	992
Co-inoculation+V3 CoMo		4140	1069	3480	899		3900	1007		3480	899
Osmo+Co-inoculation		4200	1085	3360	868		3840	992		3360	868
Osmo+Co-inoculation + ST CoMo		4200	1085	3180	821		3660	945		3180	821
Osmo +Co-inoculation + V3 CoMo		4320	1116	3720	961		3780	976		3600	930
Total Cost of Production ST CoMo		767					767
Total Cost of Production V3 CoMo		775					775
		NR	NG	NR	NG		NR	NG		NR	NG
Control Treatment	175		-	113	-	175		-	144		-
Co-inoculation	280		104.5	110	-3.5	234		58.5	203		58.5
Co-inoculation+ST CoMo	443		267.5	226	112.5	334		158.5	226		81.5
Co-inoculation+V3 CoMo	295		119.5	125	11.5	233		57.5	125		-19.5
Osmo+Co-inoculation	326		150.5	109	-4.5	233		57.5	109		-35.5
Osmo+Co-inoculation + ST CoMo	318		142.5	54	-59.5	178		2.5	54		-90.5
Osmo +Co-inoculation + V3 CoMo	341		165.5	186	72.5	201		25.5	155		10.5

• ^*
  Price per Metric Ton US$ 258.30. The value of US$ 3.69 considered for the dollar was the average quotation from October 2017 to April 2020 (Investing.com, 2025).

By Co-inoculation + the Co and Mo technologies in ST an average net gain was evident when compared with the control of US$ 213/ha for NS 5959 and US$ 97/ha for TMG 7062, affirming the efficiency of these and the benefits of low cost and high financial returns. The results also indicate profit for the producer when the Co and Mo are applied to the leaf, which offers the producer this alternative if he is unable to apply it during ST. However, the responses for both cultivars were different, and for TMG 7062 in some treatments there was a reduction in the profit to the producer when compared to the control.

4. Discussion

The use of treatment control without inoculation was performed in seeds treated only with fungicides and insecticides, technical broadly used to the Brazilian farmers. However, inoculation is a necessary practice, as one of the recommendations in the technical indications for soybean cultivation, because the Bradyrhizobium bacteria are not autochthonous (Salvadori et al., 2016), and in the regions where they have been in use over the long term, they occur in low populations having decreased FBN efficiency (Zerpa et al., 2013). This is the likely reason for the reduced GY in the control treatment. According to Hungria et al. (2013) annual inoculation can stimulate a rise in the grain yield by 8.4% and through co-inoculation by 16.1%.

As for Bradyrizobium, bacteria that usually survive in soil from previous inoculations no longer have the same efficiency as those inoculated, and because they are more environmentally daped friendly, they become highly competitive (Grönemeyer et al., 2014), and the challenge is fast adaptation of inoculated bacteria (Ouma et al., 2016). This efficiency loss is possibly because of the negative microbial interaction or incompatibility with the other symbionts in the soil (Nkot et al., 2015). According to the research by Ferreira et al. (2000), after 17 years of inoculation only 38% of the soil bacteria isolated came from the inoculated strains.

Considering the bacteria belonging to genus Azospirillum, a higher population is observed in the factorial, which suggests that the plants have a greater lengthening and higher number of lateral roots, resulting in larger the volume of soil explored which, in turn, raises the water and nutrient uptake capacity (AL-Tammar and Khalifa, 2022; Deak et al., 2019), thus impacting the crop development and grain output. These benefits were particularly significant principally for the 2017/2018 crop, because during the flowering and grain filling stages, there were days on which the crops received rainfall less than the average quantity (Figure 1). This. according to and Ku et al. (2013) and Mutava et al. (2015) may have been the principal reason for inhibiting the photosynthesis, thus lowering transpiration in the plant by stomatal closure which in turn limits the CO₂/O₂ ratio in the leaf. The plant while trying to cope with this water stress, and the changes in the root architecture, may have had other mechanisms being induced by these bacteria, like osmotic adjustment through the production of the proline (García et al., 2017).

Figure 1
Distribution of rainfall (mm) and average temperatures (°C) in ten years from November to December 2017 and January, February, March and April 2018 for the municipality of Santa Maria, Rio Grande do Sul. Sowing: 2017/2018 crop on 11/28/2017 and 2018/2019 crop on 11/06/2018 (SM), Flowering: 2017/2018 crop on 02/15/2018 and 2018/2019 crop on 01/16/2019 (FL) and harvest: 2017/2018 crop on 04/12/2018 and 2018/2019 crop on 03/25/2018 (CO).

Regarding the increase of CP in co-inoculated treatments (factorial), there is also a relationship with the introduction of new bacteria in the area with each crop season, which provide more efficient N for protein formation in the grains. This rise is particularly significant, especially for the industry producing animal feed. Besides, the utilization of soy protein is advantageous for its low cost, which has driven the food industry because of its growing usage and demand.

In the treatments where the joint application of the osmoprotectant, Co and Mo occurred in ST, the results can be explained by the incompatibility between the ST and bacteria, which caused the lowered MNP and thereby that of the BNF, resulting in the reduced N supply for the plant growth and development. Incompatibility is the outcome of the toxic effect of the seed treatment products (fungicide, insecticide, Co and Mo) which, with the application of the osmoprotectant, produces a more highly concentrated syrup, with greater adherence, which decreases the bacterial survival. However, to ensure greater establishment of the plants during the conditions unfavorable for germination, chemical ST becomes indispensable (Balardin et al., 2011). However, the study demonstrates that this incompatibility between the chemical treatments and the inoculated bacteria induces a decrease in the number of viable cells that can nodulate the plant roots (Costa et al., 2013; Silva et al., 2018) possibly caused by the pH, salinity and bactericidal properties of some of the products (Bárbaro et al., 2009; Silva et al., 2011).

When the osmoprotectant is used, the polymers that constitute it, according to Hartley et al. (2013), cause changes in the seed drying, thus inducing mortality in the inoculated rhizobia. In fact, Deaker et al. (2007) reported that water loss in the polymer treatments is associated with the different properties of moisture absorption, which alter the rates of cell rehydration and dehydration, reducing the oxygen flow to the bacteria, which affects their survival.

One more observation indicated that although the TMG 7062 NDM, showed no decrease after the application of the osmoprotectant alone, the MPA decreased, implying that the bacteria responsible for nodulating these roots had lowered efficiency (Table 4). This fact may be associated with the autochthonous bacteria possessing higher ability to compete against the inoculated bacteria, thereby inducing root infection (Grönemeyer et al., 2014), because the soil in this treatment revealed the highest CFU for Bradyrhizobium sp. (Table 8). Therefore, it is likely that these bacteria have low metabolic capacity and require other factors to be able to efficiently perform BNF. In addition, when nodulation occurs by indigenous bacteria, it is usually delayed and located one, two centimeters from the root crown resulting in less vigorous plants (Hungria and Mendes, 2015).

Table 8
The Bradyrhizobium sp. and Azospirillum sp. populations per gram of soil (CFU/g soil) in the area where the experiments were conducted, in 2018 and 2019.

Treatment	NS 5959 IPRO						TMG 7062 INTACTA
	Bradyrhizobium			Azospirillum			Bradyrhizobium			Azospirillum
	2018	2019		2018	2019		2018	2019		2018	2019
Control Treatment	3.2x106	2.6x106		9.5x102	1.5x105		3.4x106	7.0x105		4.0x103	1.1x106
Co-ino*	3.4x106	1.1x106		2.5x104	1.2x105		3.2x106	2.0x 105		2.5x104	3.0x104
Co-ino + ST CoMo	3.8x106	2.5x106		9.5x103	3.0x105		3.0x106	1.2x106		2.0x103	1.5x105
Co-ino + V3 CoMo	3.1x106	1.5x106		9.5x102	1.5x105		3.4x106	1.8x106		2.0x104	2.0x105
Osmo + Co-ino	2.6x106	2.9x106		9.5x102	2.0x105		3.6x106	1.2x106		9.5x103	4.0x104
Osmo + Co-ino + ST CoMo	3.5x106	7.7x106		2.0x103	3.0x105		3.1x106	7.5x105		3.0x103	1.1x106
Osmo + Co-ino + V3 CoMo	2.7x106	1.8x106		2.5x103	2.0x105		2.9x106	1.1x106		4.5x103	3.0x105

• ^*
  Osmo abbreviation for osmoprotectant.
• Co-ino short for co-inoculation.

When foliar application of the Co and Mo is done, even in combination with the osmoprotectant, the BNF efficiency of the bacteria that formed the nodules of these roots may be increased (Table 3), because these micronutrients are essential in the BNF process. The Mo strongly participates in the formation of the enzyme nitrogenase, which controls the breakdown of N2 into plant-assimilable forms; the Co plays a key role in the legmoglobin synthesis, which determines the nodule activity (Mus et al., 2016). For the same variable PDM, and the same crop, in cultivar NS 5959, the influence exerted by the Co and Mo applied in the ST was remarkable. This was because the application method ensured a more uniform application in the field and the quantities provided were appropriate to ensure plant growth. Another likely reason is because the Co and Mo had already been applied at the commencement of plant development, they could raise the BNF even more efficiently in advance, because based on Welch and Shuman (1995) when the Co is applied via the leaf it requires more time to reach the plant roots, as it is partially mobile, and differs from that applied via the seed which is mobile. Besides, when foliar application had been performed, straw was present in the area which may have acted as a barrier and delayed its contact with the plant.

The differences for the variables NPP and MTG may have had as main influence the nutrient N, that is, the ability of the bacteria that infected the roots of the plants to perform BNF. Significantly, in the phenological stage of the full flowering of the crop (R2) the plants show high N accumulation in the leaves, purposed first for the non-abscission of flowers, followed by the vegetables. Already, when grain filling commences another accumulation of N is obvious resulting from the increased photosynthesis, due to the vigorous remobilization and translocation of the photoassimilates to the seeds (Câmara, 2014). Therefore, grain filling has direct links to the photorespiratory and photosynthetic activities of the plant (Ainsworth et al., 2012).

Regarding the GY in the cultivation of the NS 5959 variety using the treatment involving the Co and Mo application without the osmoprotectant, the utilization of these micronutrients becomes fundamental to the acquisition of greater crop yields. However, the osmoprotectant together with these in the seeds ended up damaging the NPP directly influencing the grain yield, as previously explained. In the application of Co and Mo in V3, it is important to note that the osmoprotectant may have provided the inoculated bacteria with longer survival time and also greater competition ability, since in the constitution of the osmoprotectants there are substrates that serve to protect these bacteria (Araújo et al., 2017), signaling substances that benefit the communication between the bacteria and the plant roots are present as well as substrates that serve as nutritive substrate. Root infection by inoculated bacteria, which are more efficient, combined with the application of Co and Mo also needed in the FBN process may have been primordial in the construction of this grain yield.

The combined application of the Co, Mo and osmoprotectant also reduced the PC content (Tables 5 and 6). This decrease may also be attributed to the fewer numbers of bacteria which had infected these plants, because protein formation depends upon the amount of N available to the plant. Sadly, throughout the harvests the PC has been declining, which hinders the countries exporting it. A big impediment in terms of raising the grain content of PC is the dearth of incentives to the producer to invest in technologies that can induce such gains. A good option would be to give the producer bonuses for improving the grain quality, specifically with regards to the PC.

Re-inoculation is evidenced with each soybean crop, because even with the high population of Bradyrhizobium in the control treatment (Table 3), yield increases and protein content in grains were found, being the important process to ensure persistence elite strains of bacteria, which have greater efficiency in the process of BNF (Fulaneti et al., 2025; Mendes et al., 2004). One must also consider the cultivars used owing to plant-bacterial interaction, because differences were seen in the parameters assessed. The necessity for the micronutrients Co and Mo, in plant development and growth were notable as they induced improvements in the crop yield constituents, and were thus responsible for the increased grain yield and higher protein content. We acknowledge the crucial the need for more research on the compatibility between the additives, polymers and osmoprotectants, among others substances and the inoculated bacteria employed in seed treatment.

Regarding the investment for technology implementation, the profitability for the producer is phenomenal, with results that concur with those of Galindo et al. (2017). However, both cultivars gave different responses, and for TMG 7062 in some treatments a decrease in producer profit was observed when compared to the control. Therefore, further studies are required, with the focus on the interaction with the bacteria and cultivars.

5. Conclusions

The osmoprotectant associated with the chemical seed treatment (fungicide, insecticide, Co and Mo) negatively affected the constituents of the yield assessed was therefore responsible for the decreased yield of the soybean grain.

The Co and Mo used in seed treatment, in the absence of osmoprotectant, increases en 22% the number of pods in plants and 12.6% yield of soybean grains in relation to the use of co-inoculation for cultivar NS 5959 IPRO.

The annual co-inoculation of soybean seeds promotes average increase of 11.8% and 2.4% in grain yield of cultivars NS 5959 IPRO and TMG 7062 INTACTA, respectively.

The annual co-inoculation was observed to boost the content of crude protein in the soybean grains.

Co-inoculation together with the application of Co and Mo is the most suitable process that enhances the soybean grain yield without damaging the environment and involving comparatively low production costs.

Acknowledgements

Funding for this research was provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Data Availability Statement

The data that support this study will be shared upon reasonable request to the corresponding author.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  11 May 2026
• Date of issue
  2026

History

• Received
  20 Oct 2025
• Accepted
  11 Mar 2026