Agronomic efficiency of inoculation with Bacillus consortium - B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05 - in corn and soybean crops under different soil and climate conditions in BrazilABSTRACT
Sustainable corn (Zea mays L.) and soybean (Glycine max L.) production in tropical soils is challenged by high phosphate fertilizer dependence and low phosphorus (P) use efficiency. This study assessed the agronomic efficiency of BTP 010-19 (B. subtilis + B. amyloliquefaciens + B. pumilus) in compensating for a 25% reduction in recommended phosphate fertilization under diverse field conditions. Multi-location field trials were conducted across four Brazilian edaphoclimatic regions using a randomized complete block design. Treatments included inoculation factors (non-inoculated control, BTP 010-19 via seed treatment and in-furrow, and a positive control with Pseudomonas fluorescens and Azospirillum brasilense) and P doses (75% and 100% of recommended). The results demonstrated that BTP 010-19 application, particularly in-furrow, significantly enhanced grain yield in both crops. In corn, BTP 010-19-FURROW achieved the highest mean yield (8,264.25 kg ha-¹), representing a 7.1% increase over the non-inoculated control. Crucially, at 75% of the recommended P rate, this inoculation strategy boosted corn yield by 9.2% and soybean yield by 13.9%. These impressive gains were associated with improved root architecture, evidenced by a 6.6% increase in corn root length and a 5.0% increase in soybean root length, alongside significant shoot (8.4%) and root (19.4%) biomass gains in corn. The stability of 1000-grain weight and foliar nutrient concentrations indicated that growth promotion was primarily driven by enhanced nutrient use efficiency rather than increased uptake. These findings validate the Bacillus consortium’s practical potential to effectively offset substantial phosphate reductions, fostering the sustainable intensification of corn and soybean production in tropical soils.
Index terms:
Sustainable agriculture; microbial inoculation; plant growth promotion; tropical soils
RESUMO
A produção sustentável de milho (Zea mays L.) e soja (Glycine max L.) em solos tropicais é desafiada pela dependência de fertilizantes fosfatados e baixa eficiência no uso do fósforo (P). Este estudo avaliou a eficiência agronômica do BTP010-19 (B. subtilis + B. amyloliquefaciens + B. pumilus) em compensar uma redução de 25% na fertilização fosfatada recomendada sob condição de campo, em quatro regiões edafoclimáticas brasileiras. Os tratamentos incluíram fatores de inoculação (controle não inoculado (NI), BTP010-19 via tratamento de sementes e sulco, e um controle positivo com Pseudomonas fluorescens+Azospirillum brasilense) e doses de P (75% e 100% do recomendado). Os resultados demonstraram que BTP010-19, em sulco, aumentou significativamente a produtividade de grãos em ambas as culturas. No milho, alcançou a maior produtividade média (8.264,25 kg ha-¹), com aumento de 7,1% em relação ao NI. Com 75% de P, esta estratégia de inoculação elevou a produtividade do milho (9,2%) e da soja (13,9%). Esses ganhos associam-se à melhoria da arquitetura radicular, evidenciada pelo aumento no comprimento da raiz do milho (6,6%) na soja (5,0%), juntamente com ganhos de biomassa da parte aérea (8,4%) e da raiz (19,4%) no milho. A estabilidade do peso de 1,000 grãos e das concentrações de nutrientes foliares indicou que a promoção do crescimento foi impulsionada principalmente pela eficiência no uso de nutrientes, e não pelo aumento da absorção. Essas descobertas validam o potencial do consórcio de Bacillus para compensar reduções substanciais de P, promovendo uma produção, de milho e soja, mais sustentável em solos tropicais.
Termos para indexação:
Agricultura sustentável; inoculação microbiana; promoção do crescimento vegetal; solos tropicais
Introduction
Corn (Zea mays L.) and soybean (Glycine max L.) are fundamental crops for global food security and the Brazilian economy. However, their sustainable production faces major challenges due to the high dependence on phosphate fertilizers in tropical soils. These highly weathered soils are dominated by 1:1 clay minerals (mainly kaolinite) and iron and aluminum oxides, which confer low cation exchange capacity and high phosphorus fixation capacity (Johan et al., 2021; Ramaroson et al., 2023; Schaefer et al., 2025). As a result, excessive phosphate application leads to rapid adsorption and precipitation of P, resulting in very low use efficiency, accumulation of legacy phosphorus in the soil profile, and substantial increases in production costs (Silva et al., 2024; Pavinato et al., 2020; Rodrigues et al., 2016). Although P leaching is generally limited in these soils, the continuous buildup of phosphorus increases the risk of losses by surface runoff and potential contribution to eutrophication, besides high greenhouse gas emissions associated with fertilizer production and transport (Johan et al., 2021; Pavinato et al., 2020; Rodrigues et al., 2016). Reducing reliance on high P rates is therefore essential to improve both the economic viability and the environmental sustainability of Brazilian agribusiness.
Phosphate fertilization is essential for key plant metabolic processes, including ATP synthesis, root development, and grain yield (Calvo et al., 2014). However, in Eutrophic Cambisol, Dystrophic Red Latosol, and Red Latosol soils, phosphorus availability is naturally limited due to strong adsorption and fixation in clay minerals and iron and aluminum oxides (Pavinato et al., 2020; Rodrigues et al., 2023). This low natural availability commonly demands high rates of phosphate fertilizers, substantially increasing production costs for soybean and corn growers (Gotz et al., 2023). In a scenario of rising fertilizer prices and strong dependence on mineral inputs, strategies that allow a safe reduction in phosphate application rates without compromising yield are of great economic importance (Valdes, Gillespie, & Dohlman, 2023). Such approaches can enhance the profitability of the production system, reduce the producer’s exposure to input price volatility, and contribute to more sustainable agriculture.
Plant growth-promoting rhizobacteria (PGPRs) offer a promising pathway to enhance nutrient use efficiency and crop yield (Bashan et al., 2014; Meyer & Giachini, 2024). Among PGPRs, Bacillus species have garnered significant attention for their ability to solubilize phosphorus through the production of organic acids and enzymes (e.g., phosphatases and phytases), thereby increasing P availability in the rhizosphere (Alori et al., 2017; Sharma et al., 2013). Additionally, Bacillus strains synthesize phytohormones, siderophores, and antimicrobial compounds. These mechanisms stimulate root growth, improve stress tolerance, and increase grain yields by 10-40% in various crops, including soybean and corn (Calvo et al., 2014; Vejan et al., 2016).
Recent studies have demonstrated the potential of Bacillus inoculation in corn and co-inoculation strategies in soybean (Araújo et al., 2021; Oliveira-Paiva et al., 2024; Marchão et al., 2025). For instance, strains of Bacillus subtilis and B. megaterium have been shown to improve P acquisition and increase corn yield under reduced phosphate fertilization (Sousa et al., 2021; Ibarra-Galeana et al., 2017). In soybean, inoculation with Bacillus consortia resulted in yield increases of up to 18-25%, achieved through improvements in root architecture, biofilm formation, and endophytic colonization (Bai, Zhou, & Smith, 2003; Marchão et al., 2025).
Despite these advances, a significant knowledge gap remains regarding the efficacy of specific Bacillus consortia in compensating for substantial reductions in phosphate fertilization under diverse soil and climate conditions in Brazil (Mosela et al., 2025; Vasconcelos et al., 2025). The edaphoclimatic variability across the country - particularly in regions with highly weathered soils such as Oxisols (Latosols) and Cambisols, under precipitation regimes ranging from 800 to 2,000 mm year⁻¹ - reinforces the need for robust multi-location validation (Pavinato et al., 2020; Rodrigues et al., 2016). Multicenter field studies comparing different application methods (seed treatment vs. in-furrow) and reduced P doses are still scarce. Such comprehensive validation is essential for the development of effective microbial strategies that can be widely adopted across different Brazilian edaphoclimatic regions (Vey et al., 2025).
Therefore, this study aimed to evaluate the agronomic efficiency of a Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) in corn and soybean crops at four distinct locations in Brazil. The guiding hypothesis of this study was that inoculation with this specific consortium compensates for a 25% reduction in recommended P₂O₅ fertilization through rhizospheric P mobilization and vegetative promotion induced by phytohormones, thereby contributing to more sustainable agroecosystems.
Material and Methods
Field experiments were conducted to validate the efficiency and viability of the bacterial consortium containing different Bacillus strains (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05), in accordance with the protocols established by the Ministry of Agriculture, Livestock, and Supply (MAPA) for the registration of products based on plant growth-promoting microorganisms. The trials followed SDA Instructions No. 13, dated March 25, 2011 (Brasil, 2011), No. 25, dated July 28, 2009 (Brasil, 2009), and No. 53, dated October 24, 2013 (Brasil, 2013).
Four locations with distinct edaphoclimatic characteristics in Brazil were selected (Tables 1 and 2). The experiments were carried out during the 2024/2025 crop season.
Description of the experimental sites for evaluating the agronomic efficiency of inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) in soybean (Glycine max L.) and corn (Zea mays L.) crops across four distinct edaphoclimatic regions in Brazil.
Physical and chemical soil characterization prior to trial establishment for evaluating the agronomic efficiency of inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) in soybean (Glycine max L.) and corn (Zea mays L.) crops across four distinct edaphoclimatic regions in Brazil.
The experiments were conducted in a randomized complete block design (RCBD) with eight treatments and four replications. The description of the treatments, the active ingredients present in the tested biological products, as well as the guaranteed concentration and application method used in the treatments, are described in Table 3. All Bacillus isolates from the BTP010-19 treatment were collected in the municipality of Lapa, in the state of Paraná, and were deposited at Embrapa Londrina (CCTB04 and CCTB05 in September 2019, and CCTB09 in January 2020).
Description of the treatments, active ingredients, guaranteed concentration, dose, and application method for evaluating the agronomic efficiency of inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) in soybean (Glycine max L.) and corn (Zea mays L.) crops across four distinct edaphoclimatic regions in Brazil.
For soybean and corn crops, in seed application, a slurry volume of 500 mL per 100 kg of seeds was used for Bacillus-based treatments and the positive control with the Biofree® product. For in-furrow application at planting, 60 L ha⁻¹ of application slurry was used.
Each experimental unit consisted of seven rows 7.0 m long by 3.0 m wide, with a spacing of 0.45 m between rows, totaling 22.05 m² of working area. The description of the crop calendar for soybean and corn, the cover fertilization performed, and the products used for seed treatment and post-emergence are described in Table 4.
Description of management practices (crop calendar, cover fertilization, seed treatment, and post-emergence applications) for evaluating the agronomic efficiency of inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) in soybean (Glycine max L.) and corn (Zea mays L.) crops across four distinct edaphoclimatic regions in Brazil.
In accordance with SDA Instructions No. 13 and 53, the minimum agronomic parameters for registration of plant growth promoters were evaluated. For soybean, the following were assessed: yield (kg ha⁻¹), 1,000-grain mass (g), grain protein concentration (g kg⁻¹), nitrogen (N) concentration in grains (g kg⁻¹), foliar N, phosphorus (P), and potassium (K) concentrations (g kg⁻¹), nodule number, nodule dry mass (g), root dry mass (g), shoot dry mass (g), root length (cm), and plant height (cm). For corn, the following were evaluated: yield (kg ha⁻¹), 1,000-grain mass (g), grain protein concentration (g kg⁻¹), N concentration in grains (g kg⁻¹), foliar N, P, and K concentrations (g kg⁻¹), root dry mass (g), shoot dry mass (g), root length (cm), and plant height (cm). The chemical analyses were performed as described in Silva (2009).
For plant height, root length, and shoot and root dry mass determination, 10 plants per plot were collected. For dry mass analyses, plants were dried in a forced-air circulation oven at 65 °C until constant mass was reached. For foliar analysis, at least 20 plants per plot were collected, selecting the most developed but non-senescent leaves, totaling a 100 g sample at 35 days after emergence (DAE). For grain yield, moisture correction to 13% was performed, and harvest was manual, with grain threshing using a mechanical thresher.
The data were submitted to analysis of variance (ANOVA) for randomized complete block design in a factorial scheme (2x4), considering the inoculation and phosphorus dose factors. Subsequently, simple effects were unfolded, and the LSD (Least Significant Difference) mean comparison test was applied at 5% probability. The analysis was performed using the R environment (version 4.5.0, R Core Team, 2023) the “emmeans” package.
Results and Discussion
Productive response of corn and soybean to inoculation with PGPRs
Inoculation with the Bacillus consortium (BTP010-19) in the forms of in-furrow application at planting (BTP010-19-FURROW) and seed treatment (BTP010-19-ST), combined with phosphate fertilization doses of 75% and 100% of the recommended rate, promoted consistent increases in grain yield for corn and soybean during the 2024/2025 crop season. This validates its efficiency in compensating for a 25% reduction in the recommended phosphate fertilization (Tables 5 and 6). The observed yield increases may be attributed to yield components such as the number of pods per plant and the number of grains per pod, as no statistically significant differences were detected for the 1000-grain weight.
Evaluation of grain yield and 1000-grain weight in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in corn (Zea mays L.) across four distinct edaphoclimatic regions in Brazil.
Evaluation of grain yield and 1000-grain weight in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in soybean (Glycine max L.) across four distinct edaphoclimatic regions in Brazil.
For corn grain yield, inoculation and P doses were significant at the 5% probability level in three of the four locations (except Ponta Grossa-PR for inoculation, and Chapadão do Sul-MS for P dose, p ≤ 0.10), and the interaction between inoculation and P dose was significant at the 5% probability level in one of the four locations (Piracicaba-SP) (Table 4). The BTP010-19-FURROW treatment presented the highest mean yield (8,264.25 kg ha⁻¹), corresponding to a mean increase of 5.8% compared to the non-inoculated control (7,811.91 kg ha⁻¹). At the reduced dose of 75% P, BTP010-19-FURROW statistically differed from the other treatments in all locations, showing an increase of 9.2% (8,138.71 kg ha⁻¹) relative to the control. Inoculation with Bacillus strains in corn increased yield by 12 to 21% under reduced doses of 25 to 50% of the phosphate fertilization required for corn (Alori et al., 2017; Oliveira-Paiva et al., 2024; Sousa et al., 2021), corroborating the results of this study. This yield increase can be attributed, at least in part, to the mineralization of organic phosphorus by phytases and phosphatases produced by Bacillus strains, such as B. megaterium and B. subtilis, together with the action of siderophores capable of chelating Fe-bound P. Biofilm formation and exopolysaccharide (EPS) production may have additionally favored root colonization, thereby enhancing nutrient acquisition efficiency through microbial enzymatic activity (Sousa et al., 2021), Table 5.
It is noteworthy that, despite the expressive gains in yield, the 1000-grain weight (TGW) was not significantly influenced by the treatments, P doses, or the interaction between inoculation and P dose.
In soybean, the effects of inoculation and P dose were significant, although without interaction (Table 6). The BTP010-19-FURROW treatment proved superior, achieving yield increases of 13.9% and a mean yield of 3,872.92 kg ha⁻¹ under the 75% P dose relative to the control at the same dose, but did not differ from the other inoculated treatments. At the 100% P dose, BTP010-19-FURROW presented the highest mean yield (4,114.70 kg ha⁻¹) but did not statistically differ from the control and other treatments in the evaluated locations. In studies by Bai, Zhou and Smith (2003) and Marchão et al. (2025), inoculation with Bacillus strains resulted in yield increases of up to 25% in soybean grain yield, corroborating the performance of the BTP010-19 consortium. These data corroborate the grain yield increase in the Bacillus strain consortium (BTP010-19) and may be linked to the species’ capacity for solubilization and mineralization of insoluble P, hormonal promotion through IAA synthesis, root architecture, stable biofilm formation, and endophytic colonization, ensuring continuous delivery of beneficial metabolites and protection against nutritional stress (Bai, Zhou, & Smith, 2003; Marchão et al., 2025).
In a manner consistent with that observed in corn, soybean TGW remained stable, showing no statistical difference among treatments and P doses.
Plant architecture and vegetative development of corn and soybean in response to PGPR inoculation
Vegetative development in corn and soybean responded positively to inoculation with the Bacillus consortium (BTP010-19), with significant improvements in root architecture and aerial growth (Tables 7 and 8). These morphophysiological gains provide the mechanistic basis for the observed yield increases, indicating plant investment in resource capture structures.
Evaluation of plant architecture and vegetative development (root length, plant height, shoot and root dry mass) in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in corn (Zea mays L.) across four distinct edaphoclimatic regions in Brazil.
In corn, the BTP010-19-FURROW treatment promoted consistent increases in root length (23.5 cm) and plant height (108.8 cm) relative to the control, with mean increases of 6.6% and 4.7%, respectively. These improvements in plant architecture were accompanied by substantial biomass increases, raising root and shoot dry mass by an average of 19.4% (10.5 g plant-1) and 8.4% (73.0 g plant-1), respectively. Such results are corroborated by other studies, where inoculation with Bacillus velezensis, B. amyloliquefaciens, and B. subtilis under reduced phosphate fertilization resulted in 15 to 22% increases in root biomass, attributed to rhizosphere acidification and bacterial auxin production that stimulate root system development (Oliveira-Paiva et al., 2024; Ercole et al., 2023).
Corn biomass responded differently in significance to inoculation and P doses across variables within the tested locations (Table 8). Shoot dry mass (SDM) in corn was minimally responsive to inoculation, P dose, and the interaction between inoculation and P dose, showing no statistical difference among treatments within P doses. Root dry mass (RDM), on the other hand, showed no statistical difference only in one of the four evaluated locations (Ponta Grossa-PR for SDM and Lavras-MG for RDM, p ≤ 0.10).
Evaluation of plant architecture and vegetative development (root length, plant height, shoot and root dry mass, nodule number and dry mass) in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in soybean (Glycine max L.) across four distinct edaphoclimatic regions in Brazil.
Overall, the BTP010-19-FURROW treatment presented the highest means for P doses of 100% and 75%, for shoot dry mass (71.24 g plant-1 and 74.75 g plant-1, respectively) and root dry mass (10.0 g plant-1 and 11.1 g plant-1, respectively). Under the reduced P dose, increases of 9.0% in SDM and 19.9% in RDM were observed for the BTP010-19-FURROW treatment, although these differences were not statistically significant. In the study by Araújo et al. (2023), the use of a consortium of Bacillus strains inoculated into maize increased root and shoot dry matter by 5.5% and 7.8%, corroborating the performance of BTP010-19. However, Ibarra-Galeana et al. (2017) and Mosela et al. (2025) found yields exceeding 35% for RDM and SDM with inoculation of B. siamensis, B. aryabhattai, and B. subtilis under reduced P doses. These results can be explained based on the capacity of Bacillus species to utilize mechanisms such as chelation by siderophores and IAA production (12.5 µg mL⁻¹), which optimize root and shoot architecture (Ibarra-Galeana et al., 2017).
In soybean, vegetative development also responded positively to BTP010-19 inoculation, with significant increases in root length (5.0%) and shoot dry mass (8.4%) relative to the control, particularly in-furrow application (Table 8). These effects were more pronounced at the reduced P dose, where BTP010-19-FURROW increased root dry mass by 19.4% and nodule number by 12%, without significantly altering nodule dry mass. Similar studies indicate that Bacillus consortia improve rhizobial symbiosis in soybean under nutritional stress, enhancing nodulation and N fixation by up to 15% (Ferguson et al., 2019; Kaschuk et al., 2022) Table 7.
In soybean, the plant height and root length were significant for inoculation and P dose in two of the four locations evaluated, and the interaction between inoculation and P dose was not significant. The BTP 010-19-FURROW treatment stood out, promoting the greatest increases in plant height (9.3% - 39.1 cm) and root length (5.0% - 17.1 cm) at the reduced P dose (Table 9).
Number and dry mass of nodules in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in soybean (Glycine max L.) across four distinct edaphoclimatic regions in Brazil.
The number of nodules in soybean roots was influenced by inoculation and P dose in only one of the four locations evaluated. The BTP 010-19-FURROW treatment presented the highest means in both P doses tested, 75% and 100%, of 21.85 and 24.65 nodules/plant, respectively, without differing from the other treatments (Table 8). This can be explained by the plant’s homeostatic regulation of nodulation, mediated by autoregulation of nodulation (AON), suppressing additional responses when the primary inoculation with Bradyrhizobium was already efficient. And by rhizospheric competition for adhesion sites in root hairs and low production of flavonoid signalers by Bacillus preventing synergy in nodulation factor (NF) induction, limiting the effect to general vegetative growth (Ferguson et al., 2019; Kaschuk et al., 2022; Reid et al., 2011).
The root dry mass and nodule dry mass were little influenced by the inoculation factor and the interaction between inoculation and P dose. The BTP 010-19-FURROW treatment presented the highest means of soybean root dry mass, 1.22 and 1.29 g/plant at 75% and 100% P doses, respectively. While in nodule dry mass variable, this treatment presented the lowest means in the 25% reduced P fertilization (0.14 g/plant). The aerial part of the plant showed a significant effect of inoculation in three of the four locations. The BTP 010-19-FURROW treatment showed itself superior to the others, with means of 6.52 and 7.44 g/plant at 75% and 100% P doses, respectively. The increase in SDM mainly derives from PGPR actions of Bacillus spp., mediated by organic acids, such as gluconic acid, reducing nutritional deficiency in acidic soils, stimulating enzyme activity like rubisco and improving photosynthetic rate by 15-20%, resulting in greater aerial biomass accumulation, i.e., favoring carbon allocation for aerial structures (Dhole, Shelat, & Vyas, 2023). Thus, inoculation selectivity favors SDM by PGPR mechanisms, while RDM and NDM are endogenously regulated to avoid metabolic overload.
Foliar and grain nutrient concentrations
In contrast with the positive responses in yield and vegetative development, inoculation with the Bacillus consortium (BTP010-19) did not promote consistent increases in nutrient concentrations in foliar tissues and grains of corn and soybean (Tables 10 and 11). This apparent dissociation between growth and nutrient concentration offers valuable insights into the indirect growth promotion mechanisms involved.
Determination of foliar N, P, and K concentrations, and grain N and protein concentrations in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in corn (Zea mays L.) across four distinct edaphoclimatic regions in Brazil.
Determination of foliar N, P, and K concentrations, and grain N and protein concentrations in response to inoculation with Bacillus consortium (B. subtilis CCTB04 + B. amyloliquefaciens CCTB09 + B. pumilus CCTB05) combined with phosphate fertilization doses (75% and 100% P) in soybean (Glycine max L.) across four distinct edaphoclimatic regions in Brazil.
In corn, distinct patterns emerged. Leaf K concentration was the most responsive parameter. Significant effects of inoculation were observed in three locations, Ponta Grossa, Piracicaba, and Chapadão do Sul. This indicates that the BTP010-19 consortium consistently enhances K uptake across multiple locations. This response is likely associated with organic acid production, which facilitates K release from soil minerals. Modulation of root ion channels involved in K acquisition may also contribute (Etesami, Emami, & Alikhani, 2017; Awoniyi et al., 2025).
The P dose factor and the inoculation by P dose interaction were significant for leaf P and N concentrations in only one location or none. The same was observed for grain N and protein contents. Despite the absence of statistically significant differences in the present study, previous research has demonstrated positive effects. Tropical Bacillus isolates (B. subtilis and B. amyloliquefaciens) secrete organic acids (gluconate and citrate) and phytases. These compounds elevate rhizospheric P availability. They also stimulate nitrate reductase and glutamine synthetase activity. This resulted in grain N concentrations (14.5-16.8 g kg⁻¹) higher than the control (12.8-13.5 g kg⁻¹). Nitrogen use efficiency reached 55-62 kg grain kg⁻¹ N in a Ferralsol under 50% P (Sousa et al., 2021). Additionally, IAA production by these isolates induces ZmNRT2.1 expression, a high-affinity nitrate transporter. It also induces genes encoding storage proteins (zeins). This promotes translocation of vegetative N to grains, achieving protein concentrations of 9.8-10.5% (Sousa et al., 2021). In the present study, however, such effects were not consistently observed across locations. This underscores the influence of edaphic conditions and microbial community structure on the expression of these mechanisms (Rossmann et al., 2020).
In soybean, a diferente pattern emerged. Leaf P concentration was the most responsive parameter. Significant effects of inoculation were observed in three locations (Ponta Grossa, Piracicaba, and Chapadão do Sul). A significant inoculation by P dose interaction was detected in Piracicaba and Chapadão do Sul (Table S2). This pattern indicates that P uptake promotion by Bacillus spp. depends on soil P availability, acting preferentially under conditions of lower P supply. This action occurs through phosphate solubilization and mineralization, mediated by enzymes such as phosphatases and phytases (Sharma et al., 2013; Alori et al., 2017).
Beyond these location-specific responses, the integrated interpretation of the obtained results reveals a consistent pattern, where inoculation with the Bacillus consortium (BTP010-19), particularly in-furrow application at planting, acts as a systemic modulator of plant development, promoting gains through a reconfiguration of plant physiology that prioritizes efficiency over accumulation. The success of in-furrow application is a critical starting point, as this practice ensures high viable cell density of the inoculum in a protected microenvironment, facilitating early and uniform rhizospheric colonization along the developing root system (Bashan et al., 2014). This robust bacterial establishment is a prerequisite for the efficacy of subsequent mechanisms. In contrast, conventional seed treatment (ST) systems expose soybean and corn seeds to a high quantity of chemical products, which may negatively impact the survival and efficiency of biological inoculants (Sartori et al., 2023).
Once established in the rhizosphere, Bacillus strains, especially B. subtilis and B. amyloliquefaciens, initiate a profound morphological remodeling of the root system. Through the production of phytohormones, with emphasis on auxins such as indole-3-acetic acid (IAA), and various other signaling metabolites, these bacteria orchestrate root development. In addition to auxins, cytokinin production promotes cell division and lateral root branching, acting in synergy with auxins (Arkhipova et al., 2007).
An indirect but crucial mechanism is the synthesis of the enzyme ACC deaminase, produced particularly by B. subtilis, which degrades the ethylene precursor (ACC), reducing levels of this root elongation-inhibiting hormone in the rhizosphere and allowing deeper growth of the primary root (Glick, 2014). Additionally, bacterial volatile organic compounds (VOCs), such as 2,3-butanediol, can act as long-distance signals, modulating plant gene expression to favor growth (Ryu et al., 2003).
Polyamine production (e.g., spermidine) by Bacillus spp. stimulates plant cell division via regulation of biosynthetic pathways and membrane stabilization, while biofilm formation creates a favorable rhizospheric microhabitat, with an exopolysaccharide matrix that retains moisture/nutrients and potentiates PGPR interactions (Al-Ali et al., 2018; Chieb & Gachomo, 2023; Ganchev, 2021; Vejan et al., 2016). Collectively, these metabolites stimulate cellular elongation, branching, and root system density.
The increase in aerial biomass and the corresponding elevation in foliar N, P, and K concentrations can be explained by the “carbon dilution” phenomenon, in which rapid accumulation of carbohydrates and structural biomass exceeds the nutrient accumulation rate in tissues, resulting in stable or even reduced concentrations (Jarrell & Beverly, 1981). However, the most significant mechanism is the enhancement of internal nutrient use efficiency. Bacillus acts as systemic biostimulants that optimize internal physiological processes, redirecting nutrient flux and utilization. For P, the action is predominantly rhizospheric and biochemical. Exudation of low-molecular-weight organic acids, such as gluconate and citrate, acidifies the rhizosphere microenvironment, chelating cations (Ca²⁺, Al³⁺, Fe³⁺) and releasing phosphate (PO₄³⁻) from insoluble compounds. Concomitantly, production of acid and alkaline phosphatases by the bacteria hydrolyzes organic P forms (such as phytate), mineralizing it and making it bioavailable (Sharma et al., 2013). This solubilized P is rapidly absorbed and, instead of being stored, is immediately allocated to the biosynthesis of critical molecules such as ATP, nucleic acids, and membrane phospholipids, which are demanded to sustain rapid cell growth and division induced by bacterial hormones.
For nitrogen, evidence indicates that Bacillus spp. can modulate the expression of nitrate transporter genes and nitrogen metabolism enzymes, improving N assimilation and translocation (Calvo et al., 2019). Studies with Arabidopsis thaliana and corn have shown that Bacillus spp. inoculation can up-regulate the expression of high-affinity nitrate transporter genes (such as NRT2.1), facilitating nutrient uptake (Calvo et al., 2019). Additionally, there is induction of key nitrogen metabolism enzyme activity, such as nitrate reductase (NR) and glutamine synthetase (GS). NR catalyzes the rate-limiting step of N assimilation (reduction of NO₃⁻ to NO₂⁻), while GS incorporates ammonium into carbon skeletons for amino acid synthesis. By optimizing this assimilation “pipeline,” the plant converts inorganic N into organic forms more efficiently, directing amino acids to protein synthesis necessary for building new tissues (aerial parts and grains) at the expense of temporary accumulation in leaves (Calvo et al., 2019; Souza et al., 2013). Thus, nutrients (N and P) are not merely absorbed and stored; they are cyclically mobilized and metabolically channeled. The solubilized P and assimilated N with greater efficiency are promptly used as building blocks and energy sources for anabolic processes that sustain vegetative and reproductive growth. This continuous and directed flow explains why nutrients do not accumulate statically in foliar tissue-the “intermediate port”-but rather flow dynamically to the final “sinks”: growth organs and, mainly, forming grains, resulting in the final yield increase (Jarrell & Beverly, 1981; Calvo et al., 2019).
The fact that 1000-grain weight (TGW) and grain N and protein concentrations showed no differences relative to the negative control, in contrast to the yield increase, demonstrates that the action of Bacillus strains (BTP010-19) focuses on strengthening the reproductive “sink”-notably through increased grains per plant-without altering individual filling potential or the intrinsic biochemical composition of each grain. In this scenario, resources, mainly photoassimilates, N, and P, are distributed across a greater number of grains, keeping the average allocation per unit constant and, consequently, the TGW. Simultaneously, the classic physiological trade-off between yield and protein concentration is evident, where total available nitrogen is diluted by the greater total grain biomass produced, maintaining its final concentration unaltered, as Bacillus does not reprogram the metabolic pathways governing the carbon-nitrogen balance during grain filling (Sadras & Lawson, 2011).
In summary, the integration of results demonstrates that the 25% reduction in phosphate fertilization combined with yield gains, dissociated from elevations in foliar nutrient concentrations or alteration of established symbiosis, points to a central mechanism: increased nutrient use efficiency. Plants inoculated with the Bacillus consortium (BTP010-19), notably in-furrow, did not simply absorb more nutrients but became significantly more efficient in their utilization, optimizing processes such as translocation and remobilization of P and N from vegetative tissues to forming grains (Alori et al., 2017). This enhanced efficiency is the key point of the described systemic reprogramming, which encompasses root morphological remodeling, physiological modulation, and strategic resource allocation. Thus, this reprogramming may have allowed corn and soybean crops to maintain the yield of the fully fertilized control treatment, even under a 25% reduction in phosphate fertilizer application. Therefore, Bacillus-mediated productivity increments represent a manifestation of more efficient biological agriculture, where productivity is potentiated by the optimization of natural processes, validating this technology as a robust tool to reduce reliance on external inputs and confer greater economic-environmental resilience and sustainability to agribusiness.
Conclusions
Inoculation with Bacillus consortium (BTP 010-19) via in-furrow application enables a 25% reduction in phosphate fertilization for corn and soybean. At 75% of recommended P, yields increased by 9.2% and 13.9%, respectively, matching 100% P results. These gains reflect improved root architecture and nutrient use efficiency without changing foliar concentrations. This innovative strategy reduces mineral fertilizer dependence, mitigating leaching and emissions while enhancing resilience to Brazilian edaphoclimatic variability. It validates the consortium as a vital tool for sustainable agriculture.
Acknowledgements
The authors thank Biotrop Soluções Biológicas for providing the microbial inoculants (Biofree® and the BTP010-19 consortium) and technical support during field trials.
Data Availability Statement
Data available upon request to authors.
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Editor de seção:
Renato Paivahttp://orcid.org/0000-0001-5107-0285
