Sustainable bioformulation of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis enhances agricultural productivity in different Brazilian statesABSTRACT
The use of plant growth-promoting microorganisms (PGPM) is a promising strategy to enhance crop productivity while improving soil functionality. This study evaluated the efficacy of fungal-bacterial consortium of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis, for promoting the growth of soybean and maize cultivated under distinct edaphoclimatic conditions across Brazil. Field trials were conducted in five locations within Rio Grande do Sul, Santa Catarina, São Paulo, and Minas Gerais. Treatments consisted of a fungal-bacterial consortium (200 g ha-¹, in-furrow at planting) combined with 50% or 100% of the recommended nitrogen rate. Shoot dry biomass, foliar nitrogen (N) and phosphorus (P) concentrations, grain yield, and soil microbial activity determined by fluorescein diacetate hydrolysis were assessed. The fungal-bacterial consortium significantly improved all variables in both crops. In soybean, shoot biomass increased by 10.7-13.4% and grain yield by 9.2-9.9%, while foliar N and P rose by 10.2-12.4%, and soil enzymatic activity increased up to 11.0%. In maize, biomass increased by 10.4-11.8% and grain yield by 13.1-13.9%, with foliar N and P increasing by 9.5-14.1% and soil enzymatic activity by up to 12.5%. Notably, positive responses were maintained under 50% nitrogen fertilization. These findings demonstrate that fungal-bacterial consortium enhances nutrient acquisition and soil microbial activity, improving crop performance under variable environmental conditions and reduced N input, supporting its potential as a biological tool for sustainable nutrient management in soybean and maize systems.
Index terms:
Agronomic efficiency; biological soil conditioner; corn crop; soybean crop; plant growth promotion microorganisms (PGPM).
RESUMO
A utilização de microrganismos promotores de crescimento de plantas (PGPM) é uma estratégia promissora para aumentar a produtividade das culturas e melhorar a funcionalidade do solo. Este estudo avaliou a eficácia de um consórcio fúngico-bacteriano (Trichoderma asperellum, Pseudomonas fluorescens e Bacillus subtilis) na promoção do crescimento de soja e milho sob condições edafoclimáticas distintas no Brasil. Ensaios de campo foram realizados em cinco locais, Rio Grande do Sul, Santa Catarina, São Paulo e Minas Gerais. Os tratamentos incluíram o consórcio (200 g ha⁻¹, no sulco de plantio) combinado com 50% ou 100% da dose recomendada de nitrogênio. Avaliou-se biomassa seca da parte aérea, concentrações foliares de N e P, rendimento de grãos e atividade microbiana do solo (hidrólise de diacetato de fluoresceína). O consórcio melhorou significativamente todas as variáveis em ambas as culturas. Na soja, a biomassa aumentou 10,7-13,4% e o rendimento de grãos 9,2-9,9%; N e P foliares aumentaram 10,2-12,4%, e a atividade enzimática do solo até 11,0%. No milho, a biomassa cresceu 10,4-11,8% e o rendimento de grãos 13,1-13,9%; N e P foliares aumentaram 9,5-14,1% e a atividade enzimática do solo até 12,5%. Respostas positivas foram mantidas com 50% da fertilização nitrogenada. Estes resultados demonstram que o consórcio fúngico-bacteriano otimiza a aquisição de nutrientes e a atividade microbiana do solo, melhorando o desempenho das culturas sob condições ambientais variáveis com menor aporte de N, apoiando seu potencial como ferramenta biológica para o manejo sustentável de nutrientes em sistemas de soja e milho.
Termos para indexação:
Eficiência agronômica; condicionador biológico do solo; cultura do milho; cultura da soja; microrganismos promotores de crescimento vegetal (MPCV).
Introduction
Brazil, a major global producer of soybeans and corn, faces the structural challenge of high and volatile fertilizer costs, stemming from its heavy reliance on imports (especially of phosphorus and nitrogen), the volatility of international prices, geopolitical shocks, and logistical bottlenecks. Inorganic phosphorus consumption in Brazil is estimated to be approximately 2.2 Tg per year, with projections of an increase to 4.6 Tg by 2050 (Withers et al., 2018), and phosphorus flow analysis revealed that, in 2013, the country had a total inflow of 1.7 × 10³ Gg P/year, mostly in the form of phosphate fertilizers (Sipert & Cohim, 2020). In the case of nitrogen, Brazil will continue to be a major importer of urea, as the expansion of domestic production faces economic barriers related to the cost of natural gas (Santos & Szklo, 2016). Fertilizers account for a significant portion of the production cost of these crops: for corn, phosphorus plays a significant role (Prado & Fernandes, 2001), while nitrogen is the nutrient that most burdens production costs (Duete et al., 2009). A key mitigation strategy is the use of microbial inoculants formulated with plant growth-promoting microorganisms (Zambrano Gavilanes et al., 2023).
Plant growth-promoting microorganisms (PGPMs) play a central role in plant development and the sustainability of modern agriculture, offering biotechnological solutions that enhance crop productivity and plant health in an environmentally responsible manner. These microorganisms - including beneficial bacteria and fungi - colonize the rhizosphere, plant tissues, and phyllosphere, contributing to nutrient uptake, resistance to biotic and abiotic stresses, and pathogen suppression (Harman et al., 2004; Lugtenberg & Kamilova, 2009). PGPMs are capable of fixing atmospheric N, solubilizing insoluble soil phosphates, and producing phytohormones and other bioactive substances that stimulate plant growth, thereby reducing the need for synthetic fertilizers and pesticides. This sustainable approach has gained increasing attention due to its potential to boost agricultural productivity while minimizing the environmental impacts associated with excessive agrochemical use.
Among the most promising PGPMs are Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis, whose multiple modes of action have been extensively documented. These microorganisms enhance plant nutrition through nutrient solubilization, production of phytohormones such as indole-3-acetic acid (IAA), and secretion of siderophores that increase iron availability in soils with low micronutrient bioavailability (Ahmed & Holmström, 2014; Chen et al., 2006; Orozco-Mosqueda, Santoyo & Glick, 2023; Peix et al., 2015).
Beyond their role in plant nutrition, PGPMs also contribute to biological control. T. asperellum produces hydrolytic enzymes and antimicrobial compounds capable of degrading the cell walls of phytopathogens such as Fusarium oxysporum and Rhizoctonia solani, while also stimulating the production of defense-related secondary metabolites in plants (Benítez et al., 2004; Harman et al., 2004). P. fluorescens and B. subtilis produce natural antibiotics such as phenazines, pyoluteorin, and lipopeptides, which inhibit the growth of a wide range of plant pathogens (Ongena & Jacques, 2008; Raaijmakers et al., 2010).
A further relevant contribution of PGPMs is the activation of systemic acquired resistance (SAR), which primes plants to respond more effectively to future infections and stress events. This defense mechanism, triggered by elicitor compounds produced by P. fluorescens and B. subtilis, is analogous to an immune response in animals and reduces the reliance on chemical pesticides (Van Loon, Bakker, & Pieterse, 1998). PGPMs also help mitigate abiotic stresses such as drought and salinity. Many strains produce the enzyme ACC deaminase, which breaks down 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a stress-related plant hormone, thereby promoting healthy plant growth under adverse conditions (Wang et al., 2020). Studies with Bacillus spp. further demonstrate their ability to enhance water and nutrient use efficiency and to regulate hormonal balance in stressed plants (Radhakrishnan, Hasem, & Abd Allah, 2017; Sukkasem et al., 2018).
In addition to promoting plant growth, PGPMs contribute to soil fertility and structure. The formation of biofilms by P. fluorescens and B. subtilis creates protective microhabitats for beneficial microorganisms, stabilizes the soil microbiota, and fosters synergistic interactions among microbial species. These effects result in increased functional biodiversity and long-term soil health (Radhakrishnan, Hasem, & Abd_Allah, 2017). Moreover, PGPMs assist in the recovery of soils degraded by intensive agricultural practices, improving water retention, soil structure, and beneficial microbial activity.
In this context, the fungal-bacterial consortium of T. asperellum, P. fluorescens, and B. subtilis, represents a promising strategy for sustainable agriculture. This study aimed to evaluate the ability of these microorganisms to promote growth and increase productivity in soybean and corn crops.
Material and Methods
General description
Field trials were conducted to assess the efficacy and agronomic viability of a fungal-bacterial consortium composed of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis. The product is commercially named BIOSSER TS and is hereafter referred to as BIOSSER throughout the study. The study adhered to the regulatory protocols established by the Brazilian Ministry of Agriculture, Livestock, and Supply (MAPA) for the registration of plant growth-promoting microorganisms and soil biological conditioners, in accordance with Normative Instructions SDA No. 13/2011 (Brasil, 2011), and No. 53/2013 (Brasil, 2013).
To evaluate the agronomic performance of the biological product under diverse environmental conditions, trials were carried out during the 2023/2024 growing season (October 2023 to June 2024) across five representative regions: Rio Pardo (RS), Xaxim (SC), Mafra (SC), Inúbia Paulista (SP), and Cristais (MG). These locations were selected for their contrasting soil characteristics, temperature ranges, and rainfall patterns, ensuring broad representativeness and applicability of the results (Tables 1 and 2).
The experimental design followed a factorial scheme combining two nitrogen topdressing levels (50% and 100% of the recommended dose) with six treatments: two negative controls (uninoculated with 50% and 100% N), two positive controls (Biofree inoculation at 300 mL/ha with 50% and 100% N), and two fungal-bacterial consortium treatments (200 g/ha applied in-furrow with 50% and 100% N) (Table 3 and 4). The Biofree inoculant used as positive control is formulated with Azospirillum brasilense and Pseudomonas fluorescens, minimum concentration of 1 x 108 CFU mL⁻¹. The fungal-bacterial consortium also exhibited a minimum concentration of 1 x 10⁹ CFU mL⁻¹. Each site consisted 36 plots (6 treatments x 6 replicates). Each plot measured 24 m² (4 m × 6 m), with a 10 m² net area designated for data collection, excluding border rows.
Prior to planting, soil samples were analyzed for chemical and physical characteristics (Table 5), which informed site-specific nitrogen-phosphorus-potassium (N-P-K) basal fertilization tailored to local conditions. Nitrogen rates were adjusted according to each treatment to evaluate the interaction between inoculation with the fungal-bacterial consortium and nitrogen availability. Fertilization and liming practices were defined based on soil analysis and expected yield, following official regional guidelines: the Manual of Liming and Fertilization for Rio Grande do Sul and Santa Catarina (Sociedade Brasileira de Ciência do Solo - SBCS, 2016) for the experiments conducted in Rio Pardo (RS), Xaxim, and Mafra (SC); the Boletim Técnico 100 - Recommendations for Fertilization and Liming for the State of São Paulo (Cantarella et al., 2022) for the experiment carried out in Inúbia Paulista (SP); and the Recommendations for the Use of Correctives and Fertilizers in Minas Gerais - 5th Approximation (Comissão de Fertilidade do Solo do Estado de Minas Gerais - CFSEMG, 1999) for the experiment conducted in Cristais (MG).
Data collection
To assess the effects of treatments, data were collected on critical agronomic and biological variables: dry shoot biomass, grain yield, plant nutrient status, soil microbial activity, and nodulation (number and mass). For biomass assessment, 50 plants were sampled from the central working area at the end of the vegetative stage. Samples were oven-dried at 60 °C to constant weight, and values extrapolated based on plant density per hectare.
Grain yield was determined from the same 50 plants, with moisture adjusted to 13%, and results expressed in kg/ha. Soybean was harvested at full maturity (R8) (Neumaier et al., 2000), and corn at the dough (R4) to dent (R5) stages (Magalhães & Durães, 2006), in alignment with standard phenological guidelines.
Soil microbial activity was assessed by fluorescein diacetate (FDA) hydrolysis, using soil samples collected from each plot. Microbial enzymatic activity was quantified by spectrophotometry as an indicator of overall metabolic function (Alef & Nannipieri, 1995).
Determination of N and P Levels in Plant Tissue
Collected leaf tissue samples were oven-dried at 60 °C to constant weight, ground, and digested with sulfuric acid for macronutrient analysis (N and P). The P was quantified via colorimetric analysis, and N content was determined using the micro-Kjeldahl method, according to the procedure outlined by Tedesco et al. (1995).
Determination of Fluorescein Diacetate (FDA) hydrolysis
Soil microbial activity was evaluated through fluorescein diacetate (FDA) hydrolysis, a method recognized for its high sensitivity in detecting microbiological activity, surpassing the responsiveness of conventional chemical and physical analyses. For this, 1.0 g of soil was incubated at 25 °C for 2 hours with 0.1 mL of a 4.8 mmol L⁻¹ FDA solution and 19.9 mL of phosphate buffer (60 mmol L⁻¹ sodium phosphate, pH 7.6). The reaction was stopped by adding 20 mL of acetone, followed by centrifugation at 4,000 rpm for 5 minutes. The resulting supernatant was filtered, and absorbance was read at 490 nm using a spectrophotometer, following Alef and Nannipieri (1995).
Statistical analysis
Data was initially assessed for normality and homogeneity of variances using Bartlett’s and Shapiro-Wilk tests, followed by analysis of variance (ANOVA). When significant effects were observed, Scott-Knott test was employed for post-hoc multiple comparisons. All statistical procedures were performed at a 10% significance level using R software (version 4.3.1; R Core Team, 2023), with the AgroR package (Shimizu et al., 2024).
Results and Discussion
The soybean and corn crops responded strongly to inoculation with the fungal-bacterial consortium of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis (BIOSSER). This responsiveness was evident in nearly all parameters evaluated across the five experimental sites - Rio Pardo (RS), Xaxim (SC), Mafra (SC), Inúbia Paulista (SP), and Cristais (MG). Below are the mean values for the treatments without inoculation (NI), BIOSSER (200 g/ha-1), and the positive control (Biofree), applied to soybean and corn plants fertilized with 50% and 100% of the recommended nitrogen dose. The evaluated variables include shoot dry biomass (SDB), grain yield, foliar nitrogen and phosphorus concentrations, and soil enzymatic activity measured by fluorescein diacetate (FDA) hydrolysis, along with the corresponding average percentage increases relative to the control treatments N50%-NI and N100%-NI.
Soybean crop
Shoot dry biomass production
Soybean plants exhibited uniform establishment across all experimental sites during the early growth stages, with no visible signs of nutrient deficiency (Table 6). Growth remained consistent throughout the cycle until harvest, which was performed at full maturity (R8 stage), immediately following physiological maturity. The consistent plant vigor observed across locations indicates favorable crop establishment and the absence of abiotic or biotic constraints during the vegetative phase.
The efficacy of the microorganisms present in BIOSSER has been previously indicated in different crops and edaphoclimatic conditions. For instance, T. asperellum has shown substantial increases in biomass and enhanced resistance to soilborne pathogens such as Phytophthora capsici in tomato and pepper crops (Benítez et al., 2004). Similarly, P. fluorescens and B. subtilis are well-documented for their synergistic effects on nutrient acquisition, growth promotion, and pathogen suppression.
Across all locations, BIOSSER treatments outperformed the uninoculated control with 50% of the recommended nitrogen dose (N50-NI) and indicated comparable, and in some cases superior, performance to the positive control (Biofree). In many instances, BIOSSER also exceeded biomass production levels recorded in the uninoculated control with full nitrogen fertilization (N100-NI), highlighting its effectiveness even under reduced nitrogen input.
The mean shoot dry biomass (SDM) values from the five sites were 4,729 t for the N50-NI control, 5,236 t for BIOSSER (200 g/ha-1), and 5,312 t for the positive control. Under full nitrogen fertilization, the averages were 4,801 t for N100-NI, 5,444 t for BIOSSER, and 5,390 t for the positive control. The average increase in SDM with BIOSSER relative to N50-NI was 10.7%, ranging from 7.2% to 13.8% across sites. Under N100 fertilization, the mean increase was 13.4%, with variations from 9.0% to 16.2%. Positive responses to BIOSSER inoculation were consistent across all sites, with significant biomass gains compared to uninoculated controls. The effect of nitrogen fertilization rate and the interaction between inoculation and N dose were statistically non-significant in most locations, indicating that the biomass response was primarily driven by microbial inoculation rather than by N rate adjustments.
The positive impact of BIOSSER on biomass production can be attributed to the complementary mechanisms of its microbial components. T. asperellum enhances biomass accumulation by solubilizing nutrients and releasing bioactive compounds that promote plant growth and suppress soilborne pathogens (Seņkovs et al., 2021). P. fluorescens produces siderophores that increase iron bioavailability - essential for plant energy metabolism - while also synthesizing antimicrobial compounds that protect roots (Ge et al., 2015). B. subtilis promotes growth through the production of indole-3-acetic acid (IAA), a phytohormone that stimulates root development and nutrient uptake even in low-fertility soils (Yendyo & Pandey, 2018).
Grain yield
Grain yield is a key indicator for evaluating the efficiency of biological products, whether as inoculants or soil conditioners. Across the five evaluated locations, the mean soybean grain yield for the uninoculated negative control (NI) was 4,204 kg ha⁻¹, compared to 4,592 kg ha⁻¹ with BIOSSER inoculation (200 g ha⁻¹) and 4,567 kg ha⁻¹ for the positive control (Biofree) under 50% of the recommended nitrogen dose. Under full nitrogen fertilization (100% N), yields averaged 4,407 kg ha⁻¹ for the NI treatment, 4,752 kg ha⁻¹ for BIOSSER, and 4,823 kg ha⁻¹ for the positive control (Figure 1).
Effect of the application of the fungal-bacterial consortium, based on the PGPMs Trichoderma asperellum, Pseudomonas fluorescens and Bacillus subtilis, under ntioxida nitrogen topdressing rates on grain yield (kg há-¹) in soybean crop. Lowercase letters indicate significant ntioxidan (Scott-Knott; p≤0.1) among inoculation treatments within each nitrogen dose, while uppercase letters indicate ntioxidan between nitrogen doses within each inoculation treatment.
Inoculation with BIOSSER resulted in mean yield increases (averaged across sites) of 9.2% (ranging from 7.2% to 13.8%) over NI under 50% N, and 9.9% (ranging from 5.6% to 15.1%) under 100% N (Table 6). These gains were consistently positive across all locations where the trials were conducted. Inoculation effects were statistically significant in all sites, whereas nitrogen fertilization effects were significant in only three out of the five sites (Xaxim - SC, Mafra - SC, Inúbia Paulista - SP). The interaction between inoculation and nitrogen dose was significant in just one site (Mafra - SC). Treatments with BIOSSER consistently achieved the highest mean yields when compared to uninoculated controls.
The yield improvements observed with BIOSSER are likely due to the complementary mechanisms of its microbial consortium - Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis. T. asperellum enhances nutrient solubilization and root health, while producing bioactive compounds that suppress soil-borne pathogens. P. fluorescens increases iron bioavailability via siderophore production and synthesizes antimicrobial metabolites that protect the rhizosphere. B. subtilis, known for its multifaceted benefits, promotes root growth through phytohormone (indole-3-acetic acid) production and has been shown to improve yields in wheat and rice (Iqbal et al., 2022).
By enhancing nutrient use efficiency, improving plant health, and supporting root system development, the microbial blend in BIOSSER offers a promising biotechnological approach to boost soybean productivity - even under reduced nitrogen fertilization - contributing to more sustainable and resource-efficient agricultural practices.
Plant nutritional status
Leaf nitrogen concentration is a key parameter for evaluating plant nutritional status and the efficiency of nitrogen uptake. Across the five experimental sites, the mean leaf N concentration under 50% N fertilization was 53.7 g kg⁻¹ for the uninoculated control (NI), 60.3 g kg⁻¹ for BIOSSER 200 g ha⁻¹, and 58.9 g kg⁻¹ for the positive control. Under 100% N fertilization, the mean values were 55.5 g kg⁻¹ (NI), 61.9 g kg⁻¹ (BIOSSER), and 61.6 g kg⁻¹ (positive control) (Table 6). Compared to the uninoculated treatment with 50% N (N50-NI), BIOSSER increased leaf N content by 12.4%. Under 100% N (N100-NI), the increase was 11.5%. These gains were consistent across sites, indicating the capacity of BIOSSER to improve nitrogen assimilation even with reduced fertilization rates.
Analysis of variance showed that inoculation significantly influenced leaf N concentration (p < 0.10) at all sites. Nitrogen fertilization effects were significant in Rio Pardo - RS, Xaxim - SC, Inúbia Paulista - SP, and Cristais - MG, but not in Mafra - SC. The interaction between inoculation and nitrogen dose was not significant in any of the evaluated sites, indicating that the benefits of inoculation were consistent regardless of nitrogen level.
Leaf phosphorus concentration followed a similar pattern. Under 50% N fertilization, mean P content was 2.9 g kg⁻¹ for NI, 3.2 g kg⁻¹ for BIOSSER, and 3.2 g kg⁻¹ for the positive control. At 100% N, the values were 2.9 g kg⁻¹ (NI), 3.3 g kg⁻¹ (BIOSSER), and 3.2 g kg⁻¹ (positive control) (Table 6). Relative to N50-NI, BIOSSER increased mean leaf P content by 10.2%, while for N100-NI, the increase reached 12.4%. These increases highlight the ability of BIOSSER to enhance P uptake, an essential nutrient for plant energy metabolism and grain production.
Inoculation effects on leaf P concentration were statistically significant (p < 0.10) at all sites. Nitrogen fertilization effects were significant only in Rio Pardo - RS and Inúbia Paulista - SP. Significant interactions between inoculation and nitrogen dose were detected in Xaxim - SC and Inúbia Paulista - SP, indicating that, in these environments, the response to inoculation was influenced by nitrogen availability.
Across both nutrients, BIOSSER consistently produced the highest mean N and P concentrations, surpassing the uninoculated control and, in several cases, the positive control. These effects are attributed to the combined action of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis, which enhance nutrient availability through mechanisms such as nutrient solubilization, siderophore production, pathogen suppression, and phytohormone synthesis. This synergistic action supports more efficient nutrient uptake, reducing dependence on synthetic fertilizers and promoting soil health.
The fungal-bacterial consortium increased nutrient availability through complementary mechanisms such as phosphate solubilization — via the secretion of organic acids and phosphatases (Kapri & Tewari, 2010; Saxena et al., 2019) — production of siderophores like pyoverdine and bacillibactin, which chelate iron and indirectly support N and P assimilation (Nagata, Oobo & Aozasa, 2013; Zhao et al., 2014; Trapet et al., 2016), pathogen suppression through mycoparasitism, antibiosis, and lipopeptide production (Wang et al., 2019; Nagaraju et al., 2023), and synthesis of phytohormones such as indole-3-acetic acid (IAA), which stimulates root development and increases the nutrient absorption surface area (Qi & Zhao, 2012; Saxena et al., 2019; Poveda & Eugui, 2022). Additionally, Pseudomonas and Bacillus strains contribute to nitrogen availability through biological fixation and organic matter mineralization (Zeng et al., 2022; Zambrano Gavilanes et al., 2023). This synergistic action supports more efficient nutrient uptake, reducing dependence on synthetic fertilizers and promoting soil health (Khatoon et al., 2020; Sharma et al., 2023).
Soil microbial activity
Soil enzymatic activity, assessed through fluorescein diacetate (FDA) hydrolysis, is a sensitive indicator of microbial metabolic activity and overall soil health. Across the five evaluated sites, mean FDA hydrolysis rates under 50% of the recommended nitrogen dose were 70.1 μg g⁻¹ dry soil h⁻¹ for the uninoculated control (NI), 77.8 μg g⁻¹ dry soil h⁻¹ for BIOSSER, and 76.6 μg g⁻¹ dry soil h⁻¹ for the positive control. Under 100% N fertilization, the means were 74.1 μg g⁻¹ dry soil h⁻¹ (NI), 81.1 μg g⁻¹ dry soil h⁻¹ (BIOSSER), and 80.5 μg g⁻¹ dry soil h⁻¹ (positive control) (Table 6).
When compared to the uninoculated controls, BIOSSER increased mean FDA hydrolysis rates by 11.0% (ranging from 8.6% to 15.4%) under 50% N and 9.5% (ranging from 7.7% to 10.9%) under 100% N. These increases were consistent across all sites, indicating that inoculation with BIOSSER enhanced soil microbial activity regardless of nitrogen fertilization level. Notably, the relative gain was slightly higher under the reduced nitrogen dose, suggesting that BIOSSER can help sustain or even boost microbial activity while lowering nitrogen inputs - an important consideration for sustainable agricultural practices.
Statistical analysis revealed that inoculation effects were significant at all sites, while nitrogen fertilization effects were significant at all locations except Cristais - MG. The interaction between inoculation and nitrogen dose was not significant in any of the evaluated sites, suggesting that the positive effects of BIOSSER on soil enzymatic activity were independent of nitrogen application rate.
The consistently higher FDA hydrolysis observed in BIOSSER treatments can be attributed to the synergistic action of T. asperellum, P. fluorescens, and B. subtilis, which are known to stimulate soil microbial biomass and enzymatic processes through mechanisms such as organic matter decomposition, nutrient solubilization, and pathogen suppression. Enhanced enzymatic activity not only reflects improved microbial functioning but also supports nutrient cycling and soil health, thereby contributing to better plant growth and productivity.
These synergistic interactions originate from the complementary mechanisms employed by each microorganism, including: (i) production of extracellular hydrolytic enzymes by Trichoderma spp., such as chitinases, glucanases, and cellulases, which contribute to the decomposition of organic matter and pathogen suppression (Poveda & Eugui, 2022); (ii) production of antibiotics (e.g., 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin) and siderophores (e.g., pyoverdine and pyrochelin) by P. fluorescens, which suppress pathogens and chelate iron, making it unavailable to competitors (Poveda & Eugui, 2022; Cheng et al., 2023); and (iii) formation of robust endospores and biofilms by B. subtilis, which provide long-term survival in the rhizosphere and stable root colonization (Poveda & Eugui, 2022; Cheng et al., 2023). Added to this, one of the main drivers of increased FDA hydrolysis in soils inoculated with BIOSSER can be attributed to enhanced organic matter decomposition: T. asperellum is recognized for its potent lignocellulolytic enzyme system, including cellulases, hemicellulases, and lignin-modifying enzymes (Poveda & Eugui, 2022), which breaks down plant-derived polymers into simple sugars and organic acids, feeding the entire microbial community; subsequently, P. fluorescens and B. subtilis mineralize these degradation products, releasing CO₂ and nutrients (N, P, S) into bioavailable forms (Chaudhary et al., 2023; Wu et al., 2023). The increased enzymatic activity, evidenced by greater FDA hydrolysis, not only reflects enhanced microbial functioning but also sustains nutrient cycling and soil health, thereby contributing to improved plant growth and productivity.
Number and mass of nodules
Nodule formation in soybean is a critical indicator of symbiotic nitrogen fixation efficiency, directly influencing plant nitrogen nutrition. Across the five evaluated sites, the mean nodule number per plant under 50% N topdressing was 24.0 for BIOSSER, 23.9 for the positive control, and 23.8 for the uninoculated control (NI), with no statistically significant differences among treatments. Under 100% N topdressing, the means were 23.9 (BIOSSER), 23.6 (positive control), and 23.6 (NI), again without significant differences (Table 6).
Similarly, nodule dry mass showed no significant variation among treatments. Under 50% N, mean dry nodule mass was 51.5 mg plant⁻¹ for BIOSSER, 53.0 mg plant⁻¹ for the positive control, and 52.1 mg plant⁻¹ for the NI control. Under 100% N, mean values were 52.7 mg plant⁻¹ for both BIOSSER and the positive control, and 52.1 mg plant⁻¹ for NI.
Statistical analysis suggests that neither inoculation nor nitrogen topdressing rate significantly influenced nodule number or mass in the evaluated sites. The lack of treatment effects may be attributed to the presence of well-adapted native Bradyrhizobium populations in Brazilian soils, which often dominate nodule occupancy and limit the measurable benefits of additional inoculation - particularly in fields with a history of soybean cultivation. This competitive advantage of native strains has been widely reported (Hungria et al., 2010), and may explain the uniform nodulation responses across treatments.
Despite the absence of significant differences in nodulation parameters, the yield and nutrient uptake improvements observed in BIOSSER treatments suggest that its beneficial effects on soybean growth may be more related to other plant growth-promoting mechanisms - such as enhanced nutrient solubilization, phytohormone production, and improved soil enzymatic activity - than to increased biological nitrogen fixation alone.
Corn crop
Shoot dry biomass production
The increases in shoot dry biomass (SDB) observed with BIOSSER treatments highlight the strong growth-promoting potential of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis in maize. Across the five evaluated sites, mean SDB under reduced nitrogen fertilization (50% N) was 6.52 t ha⁻¹ for the uninoculated control (NI), 7.20 t ha⁻¹ for BIOSSER at 200 g ha⁻¹, and 7.32 t ha⁻¹ for the positive control (Table 7). Under 50% N, BIOSSER produced an average SDB increase of 10.4% compared with NI, ranging from 8.3% to 17.2% across sites. With full nitrogen fertilization (100% of the recommended dose), mean SDB values were 7.49 t ha⁻¹ (NI), 8.38 t ha⁻¹ (BIOSSER), and 8.28 t ha⁻¹ (positive control), with BIOSSER delivering an average increase of 11.8% over NI, ranging from 8.4% to 14.9%.
Across all sites, BIOSSER inoculation resulted in positive SDB responses regardless of nitrogen rate. The effects of inoculation and nitrogen fertilization were statistically significant at all sites, whereas the interaction between inoculation and nitrogen rate was significant only at Rio Pardo (p ≤ 0.01) and Cristais (p ≤ 0.05). In every case, BIOSSER recorded the highest SDB means relative to uninoculated controls.
These results suggest the capacity of the BIOSSER microbial consortium to enhance maize biomass production. This effect likely stems from the complementary mechanisms of action of microorganism’s components (T. asperellum, P. fluorescens, and B. subtilis). As also observed in soybean trials, the synergistic use of these microorganisms consistently enhances plant biomass by improving root health and nutrient use efficiency, representing a promising and sustainable alternative to boost agricultural productivity while reducing reliance on synthetic fertilizers.
Poveda and Eugui (2022) demonstrated that co-inoculation of Trichoderma with Bacillus and Pseudomonas results in greater growth promotion and disease control than single inoculations, as these microorganisms occupy different ecological niches, exhibit compatibility, and employ complementary mechanisms that collectively enhance plant performance. After colonizing the root epidermis, T. asperellum releases bioactive molecules that induce resistance and produce auxin-like compounds, stimulating root branching and increasing shoot biomass (Harman, 2006; Contreras-Cornejo et al., 2016). P. fluorescens acts in plant nutrition, hormonal modulation, pathogen inhibition, and phosphate solubilization (Vacheron et al., 2016). Moreover, B. subtilis — recognized for its ability to form resistant endospores, a characteristic that makes it particularly suitable for commercial biofertilizer formulation — demonstrates consistent tolerance to water stress in maize and beans, improving physiological parameters such as relative water content, chlorophyll content, and antioxidant enzyme activity (Lima et al., 2019). The combined use of these three microorganisms produces synergistic effects that exceed the sum of individual contributions, as demonstrated by the results obtained in this study.
Grain yield
Inoculation with BIOSSER consistently increased maize grain yield across the five evaluated sites, both under reduced (50% N) and full (100% N) nitrogen fertilization (Table 7). Under 50% N, mean grain yield rose from 8,088 kg há⁻¹ in the uninoculated control (NI) to 9,213 kg há⁻¹ with BIOSSER at 200 g há⁻¹ - há average gain of 13.9% (ranging from 10.2% to 20.8%) - and 9,051 kg há⁻¹ for the positive control. Under 100% N, yields increased from 9,288 kg há⁻¹ (NI) to 10,506 kg há⁻¹ with BIOSSER - há average gain of 13.1% (ranging from 8.7% to 15.7%) -while the positive control yielded 10.21 t há⁻¹ (Figure 2).
Effect of the application of the fungal-bacterial consortium, based on the PGPMs Trichoderma asperellum, Pseudomonas fluorescens and Bacillus subtilis, under different nitrogen topdressing rates on grain yield (kg ha-¹) in corn crop. Lowercase letters indicate significant differences (Scott-Knott; p≤0.1) among inoculation treatments within each nitrogen dose, while uppercase letters indicate differences between nitrogen doses within each inoculation treatment.
Yield gains with BIOSSER were positive at all sites regardless of nitrogen rate. Both inoculation and nitrogen fertilization effects were statistically significant across all locations, while the interaction between inoculation and nitrogen rate was significant in three of the five sites (Xaxim - SC, Mafra - SC, and Inúbia Paulista - SP). In all cases, BIOSSER recorded the highest mean yields compared to uninoculated controls.
A direct economic interpretation of these results highlights the practical impact of the technology. Under 50% N fertilization, BIOSSER increased maize grain yield by an average of 1,125 kg ha⁻¹ (equivalent to 18.7 bags of 60 kg) compared to NI. Under 100% N fertilization, the mean gain was 1,218.7 kg ha⁻¹ (20.3 bags of 60 kg) over NI. Such yield increments demonstrate the potential of BIOSSER to enhance crop productivity even under reduced nitrogen fertilization.
Plant nutritional status
Nutrient concentrations in plant tissues are influenced by a combination of environmental, edaphic, and management factors. Across the five evaluated sites, maize leaf nitrogen (N) and phosphorus (P) concentrations showed consistent increases with BIOSSER inoculation under both reduced and full nitrogen fertilization regimes. Under 50% N fertilization, mean leaf N and P contents were 23.5 g kg⁻¹ (N) and 2.5 g kg⁻¹ (P) for the uninoculated control (NI), 26.4 g kg⁻¹ (N) and 2.9 g kg⁻¹ (P) for BIOSSER at 200 g ha⁻¹, and 25.4 g kg⁻¹ (N) and 2.9 g kg⁻¹ (P) for the positive control. Under 100% N fertilization, mean values were 27.4 g kg⁻¹ (N) and 2.8 g kg⁻¹ (P) for NI, 30.4 g kg⁻¹ (N) and 3.0 g kg⁻¹ (P) for BIOSSER, and 29.4 g kg⁻¹ (N) and 3.0 g kg⁻¹ (P) for the positive control (Table 7).
When compared to the uninoculated 50% N treatment (N50-NI), BIOSSER increased leaf N and P concentrations by an average of 12.0% and 14.1%, respectively. Under 100% N fertilization, the gains were 10.9% for N and 9.5% for P. These increments were more pronounced under reduced nitrogen conditions, highlighting the potential of BIOSSER to optimize nutrient uptake even with lower fertilizer inputs.
The improvement in N and P status reflects the activity of the plant growth-promoting microorganisms (PGPM) Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis, which enhance nutrient acquisition through multiple mechanisms, including nitrogen fixation, phosphorus solubilization, siderophore production, and stimulation of root growth. This aligns with previous studies showing that co-inoculation with PGPM can increase N and P uptake, root biomass, and grain yield even under water stress or reduced chemical fertilization (Castro-Restrepo et al., 2022; Yendyo & Pandey, 2018).
Soil microbial activity
The ability of Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis to enhance nutrient release - through increased enzymatic and biological activity in the soil - helps explain the higher maize productivity observed with BIOSSER compared to uninoculated controls (NI). Because these microorganisms act by conditioning the soil, improving nutrient cycling, and stimulating root development, their benefits are not crop-specific and may extend to other species of agronomic interest beyond soybean and maize.
Across the five evaluated sites, mean hydrolysis of fluorescein diacetate (FDA) was 62.3 µg g⁻¹ dry soil h⁻¹ for NI, 68.6 for BIOSSER, and 68.2 for the positive control under 50% of the recommended N dose. Under 100% N, the averages were 63.4 (NI), 71.3 (BIOSSER), and 68.9 (positive control). Inoculation with BIOSSER increased FDA hydrolysis by an average of 10.2% (range: 7.5-13.2%) under 50% N and 12.5% (range: 10.1-16.3%) under 100% N (Table 7). These increases were positive at all sites, regardless of nitrogen rate.
The effects of inoculation and nitrogen fertilization were statistically significant at all locations except for nitrogen fertilization in Mafra - SC, while the inoculation × nitrogen rate interaction was not significant in any site. BIOSSER consistently produced the highest FDA hydrolysis values for maize.
The observed increases in soil microbial activity can be attributed to the synergistic action of the PGPM in BIOSSER. T. asperellum is known to stimulate soil enzymatic activity and enhance phosphorus availability; P. fluorescens produces siderophores that improve micronutrient uptake and contributes to root system health; and B. subtilis increases nutrient absorption efficiency through the production of phytohormones such as indole-3-acetic acid (IAA). Together, these microorganisms promote nutrient mineralization, phosphorus solubilization, pathogen suppression, and abiotic stress mitigation, creating a more favorable rhizosphere environment. These findings, in line with previous studies (Fernandes et al., 2020; Moreno-Lora et al., 2022; Sarkar et al., 2021; Sivasakthi et al., 2013), reinforce the potential of BIOSSER to improve soil health, nutrient availability, and crop productivity while reducing dependence on synthetic fertilizers.
The incorporation of the BIOSSER consortium (Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis) into conventional agricultural systems requires considering interferences caused by seed treatments, insecticides, and fungicides. Systemic fungicides (metalaxyl, fludioxonil, trifloxystrobin) restrict root colonization by beneficial fungi and reduce phosphorus uptake, while contact fungicides show minimal effects (Jin et al., 2013). The insecticide Imidacloprid (1000 ppm) inhibits Trichoderma spp. growth by 3.7-16.2%, and Thiamethoxam by 7.5-10% (Prasanna et al., 2022). Root colonization by Bacillus subtilis is compromised by multiple agrochemical mixtures (e.g., Captan + Lorsban + Allegiance) when the inoculant is applied at a reduced dosage (Jensen, Percich & Graham, 2002). Foliar fungicides (strobilurins, triazoles) affect fungal communities more than bacterial ones, altering the structure of the edaphic microbiota (Santísima-Trinidad et al., 2018). Neonicotinoid insecticides (thiamethoxam, dinotefuran) genetically alter bacterial communities and their carbon utilization profiles (Yu et al., 2020), while fipronil inhibits nitrogen cycling functions (Sim et al., 2022). However, the results demonstrate that, under the conditions of this study, the use of chemical seed treatments did not influence the efficacy of the BIOSSER consortium, suggesting that the evaluated strains exhibit compatible tolerance with the agrochemicals employed. Nevertheless, it is still recommended to perform prior compatibility tests between the consortium strains and the products adopted on each property, as well as to stagger applications to minimize risks of direct and simultaneous exposure.
Conclusions
This field study, conducted in various environments, indicates the agronomic effectiveness of BIOSSER (Trichoderma asperellum, Pseudomonas fluorescens, and Bacillus subtilis) in soybean and corn under contrasting edaphoclimatic conditions in Brazil. The formulation increased biomass, grain yield, foliar concentrations of N and P, and soil microbial enzymatic mechanisms, improving nutrient acquisition and rhizosphere functionality. The product shows potential to improve nutrient use efficiency, reduce synthetic N inputs, and support sustainable agricultural production systems.
Acknowledgements
We would like to thank all the farmers that made their lands available for the conduction of the 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
