Performance of biological control agents in the control of soybean pathogens

Desempenho de agentes de controle biológico no controle de patógenos da cultura da soja

Abstract

Soybean (Glycine max [L.] Merrill) is the main oilseed crop produced worldwide. The oil and protein extracted from its grains serve as the basis for human and animal nutrition and as raw materials for biofuel production. Brazil holds the position of the largest producer and exporter of this commodity. However, some factors limit high yields, such as diseases, which are commonly managed with chemical pesticides that lead to ecosystem contamination and selection of resistant organisms. Given the demand for more sustainably produced food, biological inputs emerge as an alternative. In this context, the present study aimed to evaluate the effect of isolates of B. velezensis, B. megaterium, and T. longibrachiatum in the biocontrol of pathogens affecting soybean crops, as well as its potential for phosphorus and potassium solubilization, and its ability to induce resistance in plants. The experiments were conducted at the Federal University of Technology - Paraná, Dois Vizinhos Campus. In vitro confrontations were carried out using four biocontrol agent isolates against the pathogens C. truncatum, C. kikuchii, S. glycines, S. sclerotiorum, M. phaseolina, R. solani, D. longicolla, and F. tucumaniae. The experimental design was completely randomized with four replications. The data obtained were subjected to the Shapiro-Wilk normality test, ANOVA, followed by regression analysis using the Rbio software. The results indicate that the isolates of. B. velezensis, B megaterium, and T. longibrachiatum exhibit in vitro antagonistic effects against the tested pathogens.

Keywords:
antagonism; biological inputs; phytopathogen control; resistance induction; solubilization; phosphorus; potassium

Resumo

A soja (Glycine max [L.] Merrill) é a principal oleaginosa produzida mundialmente. O óleo e a proteína extraída dos seus grãos, servem como base para a alimentação humana e animal e matéria prima para produção de biocombustíveis. O Brasil, ocupa o posto de maior produtor e exportador da commodity. Contudo, alguns fatores causam limitações nos altos rendimentos, ao exemplo das doenças, comumente manejadas com defensivos químicos os quais geram a contaminação dos ecossistemas e seleção de organismos resistentes.Visto a demanda por alimentos produzidos de maneira mais sustentável, os bioinsumos surgem como uma alternativa. Neste sentido, o presente trabalho teve como objetivo, avaliar o efeito de isolados de B. velezensis, B. megaterium e T. longibrachiatum no biocontrole de patógenos que acometem a cultura da soja, bem como seu potencial para solubilização de fósforo e potássio, e capacitade de induzir resistência em plantas. Os experimentos foram realizados na Universidade Tecnológica Federal do Paraná, Campus Dois Vizinhos. Com os quatro isolados de agentes de bicontrole, realizou-se o confronto in vitro, dos patógenos C. truncatum, C. kikuchii, S. glycines, S. sclerotiorum, M. phaseolina, R. solani, D. longicolla e F. tucumaniae. O delineamento adotado foi o inteiramente casualizado com quatro repetições. Os dados obtidos foram submetidos ao teste de normalidade de Shapiro-Wilk, a ANOVA seguido da análise de regressão no software Rbio. Os resultados indicam que os isolados de B. velezensis, B. megaterium e T. longibrachiatum apresentam in vitro efeito antagonista contra os patógenos confrontados.

Palavras-chave:
antagonismo; bioinsumos; controle de fitopatógenos; indução de resistência; solubilização; fósforo; potássio

1. Introduction

Brazil is the leading producer and exporter of soybean (Glycine max [L.] Merrill). Its grains provide oils and proteins rich in human and animal nutrition, and are also an exclusive raw material for various industrial products and biofuel production (Hartman et al., 2011; Abdulkhani et al., 2017). The growing demand for healthier food has highlighted the need for safer and more sustainable management alternatives that promote a progressive replacement or combination with the chemical pesticides currently used on a large scale in farming practices (Rouphael and Colla, 2018; Rezende et al., 2021).

Sustainable and economically viable alternatives have been increasingly promoted. Among them, the use of bioinputs based on microorganisms effective in pathogen biocontrol and plant growth promotion has emerged as one of the safest and most environmentally sustainable strategies (Soumare et al., 2019; Macik et al., 2020; Meyer et al., 2022). Such approaches mitigate impacts such as ecosystem contamination, human and animal health risks, and pathogen resistance when compared to fungicides (Albuquerque et al., 2016).

Despite advances in strategies and technologies applied to crop management, there are still constraints to achieving high grain yield in the field. Significant losses are caused by a wide range of pathogens, including fungi, bacteria, nematodes, and viruses (Bandara et al., 2020; Galeano et al., 2024). Lin et al. (2022) emphasized that yield losses and disease severity are influenced by multiple factors, such as sowing region, cultivar used, cropping year, pathogen strain virulence, and disease management practices.

Soybean is affected by numerous diseases (Henning et al., 2014). Root diseases are particularly damaging, such as charcoal rot caused by Macrophomina phaseolina (Lodha and Mawar, 2020); white mold caused by Sclerotinia sclerotiorum (Smolińska and Kowalska, 2018); seedling damping-off caused by certain Fusarium species; sudden death syndrome (SDS) or red root rot caused by Fusarium spp. (Rosati et al., 2018; Lin et al., 2022); Phomopsis seed decay and root rot caused by Diaporthe longicolla (Santos et al., 2011; Ghissi et al., 2014); and damping-off caused by Rhizoctonia solani (Palacioğlu et al., 2024).

In addition to root diseases, foliar diseases also cause significant losses, such as Asian soybean rust caused by Phakopsora pachyrhizi (Wrather et al., 2010); anthracnose caused by Colletotrichum truncatum (Medeiros et al., 2022); Septoria brown spot caused by Septoria glycines (Allen et al., 2017); and Cercospora leaf blight and purple seed stain caused by Cercospora kikuchii (Soares et al., 2015).

Among the most relevant biocontrol agents are the genera Trichoderma and Bacillus. Trichoderma spp. are saprophytic fungi, cosmopolitan inhabiting soils, belonging to the Ascomycota. They exhibit antagonistic potential against different pathogens (Blaszczyk et al., 2014). Bacillus spp., in turn, are Gram-positive, rod-shaped, motile, spore-forming bacteria (Villarreal-Delgado et al., 2018; Beskrovnaya et al., 2021), categorized as rhizobacteria (Sansinenea, 2019).

These microorganisms colonize plant roots and the rhizosphere, where plant–microorganism interactions occur. They display multiple plant-beneficial modes of action, including antibiosis through the production of metabolites toxic to phytopathogens; degradation of chitin, a component of fungal cell walls; competition for nutrients and space; induction of systemic resistance in plants; and plant growth, by producing phytohormones, siderophores, and phosphorus-solubilizing compounds (Segarra et al., 2010; Borges Chagas et al., 2015; Grover et al., 2021; Tyśkiewicz et al., 2022; Meyer et al., 2022; Nunes et al., 2024).

In this context, exploring new biocontrol agents may support crop management, leading to greater profitability and more sustainable farming systems. Therefore, the present study evaluated the effect of Bacillus spp. and Trichoderma spp. isolates in controlling the main diseases affecting soybean cultivation.

2. Material and Methods

2.1. Characterization of the experimental area

The experiments were conducted in the Biological Control and Plant Health laboratories of the Federal University of Technology – Paraná, Dois Vizinhos Campus (UTFPR-DV), located in the Southwest region of Paraná State (25°41’35” S; 53°05’37” W).

2.2. Isolation and cultivation of microorganisms

The biocontrol agents used in this study were the isolates Bacillus velezensis (BC 004), Bacillus velezensis (BC 005), Bacillus megaterium (BC 003), and Trichoderma longibrachiatum (TC 002), bioprospected and identified by DNA sequencing, obtained from SRM Brazil Technology.

The phytopathogens included Cercospora kikuchii, Colletotrichum truncatum, Septoria glycines, and root disease-causing fungi such as Diaporthe longicolla, Fusarium tucumaniae, Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. These were obtained from the microorganism collection of the Plant Health Laboratory at the Federal University of Technology – Paraná, Dois Vizinhos Campus, originally sourced from EMBRAPA Soybean (Londrina, Brazil).

Both the biocontrol agents and the phytopathogens were subcultured in a laminar flow hood and grown on Petri® dishes containing potato dextrose agar (PDA). Subsequently, they were incubated in a BOD chamber at 25 °C, with a 12-hour photoperiod, for 7 days.

2.3. In vitro dual culture antagonism assay

To evaluate direct antagonism through in vitro pairing, the methodology described by Dennis and Webster (1971) was followed. Each experimental unit consisted of Petri® dishes (9 cm in diameter) containing 20 mL of PDA medium, previously prepared and autoclaved. The challenging phytopathogens included foliar disease-causing fungi (Cercospora kikuchii, Colletotrichum truncatum, Septoria glycines) and root disease-causing fungi (Diaporthe longicolla, Fusarium tucumaniae, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum).

In a laminar flow hood, a sterile platinum loop was used to transfer a 7 mm mycelial disc of each pathogen to one side of the Petri® dish, positioned 1 cm from the edge. On the opposite side, also 1 cm from the edge, was placed a 7 mm disc of each of the four studied isolates (Bacillus velezensis BC 004, Bacillus velezensis BC 005, Bacillus megaterium BC 003, and Trichoderma longibrachiatum TC 002), cultivated as described in section 2.2. The control treatment consisted only of pathogen discs without biocontrol agents.

The Petri dishes were sealed with PVC film, and two lines were drawn on the bottom of each plate with a millimeter ruler, one horizontal and one vertical, to monitor the mycelial growth of the colonies. Subsequently, the plates were incubated in a BOD chamber at 25 °C with a 12-hour photoperiod. The mycelial growth of the phytopathogens was assessed daily, every 24 h, until the control fully covered the plate. For this evaluation, measurements were taken with a millimeter ruler. For each plate, the two measurements were averaged to obtain the mean colony growth. The percentage of mycelial growth inhibition (PGI) was calculated using the Formula 1 proposed by Menten et al. (1976), as follows:

$$PGI=\left(C-T\right)*100C$$ (1)

where C = colony growth in the control, and T = colony growth in the treatment.

A completely randomized experimental design was adopted, with four replicates per treatment. Data were subjected to the Shapiro–Wilk normality test, followed by ANOVA (Scott–Knott at 5% probability) and regression analysis using the Rbio software (Bhering, 2017).

2.4. Solubilization of Phosphorus (P) and Potassium (K)

Phosphorus solubilization was evaluated in modified NBRIP medium (Nautiyal, 1999), and potassium solubilization in Pikovskaya Agar medium (Velázquez-Gurrola and Ramos-Alegría, 2015). After autoclaving, the media were poured into test tubes and inoculated with 4 mm mycelial discs of the isolates Bacillus velezensis (BC 004 and BC 005), Bacillus megaterium (BC 003), and Trichoderma longibrachiatum (TC 002). The tubes were sealed and incubated in a BOD chamber at 25 °C, with a 12 h photoperiod, for five days.

Evaluations were performed every 24 h, observing solubilization halos: in the case of phosphorus, by the color change of the medium, and in potassium, by the change from blue to yellow. The solubilization index (SI) was calculated as the ratio between the diameter of the solubilization zone and that of the colony (Chagas et al., 2017).

The experimental design was completely randomized, with four replicates per treatment. Data were analyzed by Shapiro–Wilk, ANOVA (Scott–Knott, 5%), and regression in Rbio software (Bhering, 2017).

2.5. Plant resistance induction responses

2.5.1. Plant cultivation in the greenhouse

The soybean cultivar Brasmax Zeus IPRO® (maturity group 5.5) was grown in 8 L pots with commercial substrate. The design was completely randomized, with five treatments: control and four isolates (Bacillus velezensis BC 004 and BC 005, Bacillus megaterium BC 003, and Trichoderma longibrachiatum TC 002), in four replicates, with two plants per pot. At the V3 stage, plants were sprayed with a suspension of the isolates (1×10¹⁰ spores mL⁻¹) and, after 24 h, inoculated with Erysiphe diffusa (1×10⁶ spores mL⁻¹). Samples for analysis were collected at five time points: 0, 48, 96, 144, and 192 hours after inoculation.

The E. diffusa was used solely as an elicitor to induce the expression of defense enzymes following the application of the biocontrol agents, considering the favorable environmental conditions and timing for disease development.

2.5.2. Preparation of the enzymatic extract

Enzymatic extracts were obtained according to Moerschbacher et al. (1988), as described by Guzzo and Martins (1996). Samples of 0.3 g of plant tissue were macerated with glass microbeads, Dowex resin, and PVPP in sodium borate buffer. After incubation for 5 min, the material was centrifuged (20,000 rpm, 4 ºC, 20 min), and the supernatant was collected for enzymatic analyses.

2.5.3. Determination of total proteins

Total protein content was determined according to Bradford (1976). For each replicate, 40 µL of the plant extract was diluted in 460 µL of distilled water and added to 1 mL of Bio-Rad reagent (Sigma). After vortex homogenization, the samples were read in a spectrophotometer at 595 nm.

2.5.4. Determination of chitinase

Chitinase activity was measured according to Moerschbacher et al. (1988), as described by Guzzo and Martins (1996). Plant extract (500 µL) was incubated with specific substrate (100 mg) in 0.2 M sodium phosphate buffer (400 µL) at 50 ºC for 40 min. After centrifugation (7,000 rpm, 10 min), the supernatant was analyzed in a spectrophotometer at 595 nm.

2.5.5. Determination of β-1,3-glucanase

β-1,3-Glucanase activity was determined according to Guzzo and Martins (1996). Plant extract (200 µL) was incubated with 400 µL of 50 mM sodium acetate buffer (pH 5.0) and 200 µL of Curdlan substrate (Sigma) at 40 ºC for 50 min. The reaction was stopped with 200 µL of 2N HCl, followed by cooling on ice and centrifugation (10,000 rpm, 5 min). Absorbance was measured at 600 nm.

2.5.6. Determination of phenylalanine ammonia-lyase (PAL)

PAL activity was determined according to Rodrigues et al. (2006). Samples of 0.5 g of plant tissue were macerated in TRIS-HCl buffer (pH 8.0), centrifuged (6,000 rpm, 4 ºC, 10 min), and the supernatant was used as the enzymatic extract. The extract was incubated with phenylalanine (40 ºC, 30 min), the reaction was stopped on ice, and absorbance was measured at 290 nm.

2.5.7. Determination of total phenolic compounds

Total phenolic compounds were quantified according to Bieleski and Turner (1966), with extraction in MCA solution (methanol:chloroform:water, 6:2.5:1.5) and centrifugation. The obtained supernatant was subjected to the Folin–Ciocalteu method (Jennings, 1981). After reaction with alkaline sodium carbonate solution, absorbance was determined at 760 nm.

2.5.8. Statistical analysis

The design was completely randomized, with four replicates per treatment. Data were subjected to the Shapiro–Wilk normality test, followed by ANOVA (Scott–Knott, 5%) and regression analysis using the Rbio software (Bhering, 2017).

3. Results and Discussion

3.1. Antagonistic effects on pathogens causing foliar and root diseases

For the control of foliar disease-causing pathogens C. kikuchii, C. truncatum, and S. glycines, in the in vitro dual culture antagonism assay with the three isolates B. megaterium (BC 003), B. velezensis (BC 004), and B. velezensis (BC 005), differences among treatments were observed, and the isolates exhibited antagonistic potential against the mycelial growth of the phytopathogens. As shown in Figures 1 and 2, and Tables 1 and 2, in the confrontation with C. kikuchii, isolates BC 003, BC 004, and BC 005 reduced the radial growth of the pathogen by 21% compared to the control. In the analysis of the confrontation assay against C. truncatum (Figure 1), a reduction in pathogen growth was observed: 41.1% for isolate BC 003, 37% for isolate BC 004, and 34.2% for isolate BC 005. Furthermore, reductions in the mycelial growth of S. glycines were also observed, with isolate BC 003 reducing growth by 38.9%, isolate BC 004 by 42.9%, and isolate BC 005 by 23.8%, compared with the control.

Figure 1
Antagonistic effect of the isolates T. longibrachiatum (TC 002), B. velezensis (BC 004), B. velezensis (BC 005), and B. megaterium (BC 003) on the mycelial growth (cm) of C. truncatum, S. glycines and C. kikuchii, respectively, over four days. Source: Author’s own work.

Figure 2
Plates at 7 days of evaluation: Relationship of the phytopathogen controls C. truncatum, S. glycines, and C. kikuchii, and confrontations performed with B. megaterium (BC 003), B. velezensis (BC 004), and B. velezensis (BC 005), respectively. Source: Author’s own work.

Table 1
Linear equation and adjusted R² values related to the mycelial growth of C. truncatum, S. glycines and C. kikuchii.

Colletotrichum truncatum	Equation	Adjusted R2
Control	y = 1.7208x + 0.2663	0.9924
BC 003	y = 0.8295x + 1.1625	0.9804
BC 004	y = 0.829x + 1.2925	0.9436
BC 005	y = 0.9778x + 1.1462	0.9586
Septoria glycines	Equation	Adjusted R2
Control	y = 1.4723x + 0.3033	0.9975
BC 003	y = 0.8983x + 1.45	0.9489
BC 004	y = 0.5817x + 1.7583	0.9666
BC 005	y = 0.4767x + 1.9833	0.8576
Cercospora kikuchii	Equation	Adjusted R2
Control	y = 1.555x + 0.65	0.9945
BC 003	y = 1.3017x + 0.7167	0.997
BC 004	y = 1.16x + 1.175	0.9818
BC 005	y = 1.16x + 1.175	0.9818

• Source: Author’s own work.

Table 2
Linear equation and adjusted R² related to the mycelial growth of D. longicolla, F. tucumaniae, M. phaseolina, R. solani, and S. sclerotiorum.

Diaporthe longicolla	Equation	Adjusted R2
Control	y = 1.5133x + 0.9933	0.9928
TC 002	y = 0.775x + 1.725	0.9481
BC 003	y = 0.8183x + 2.095	0.9557
BC 004	y = 0.817x + 2.141	0.9597
BC 005	y = 0.8667x + 2.0467	0.9717
Sclerotinia sclerotiorum	Equation	Adjusted R2
Control	y = 2.2867x - 1.3333	0.9947
TC 002	y = 0.4575x + 0.5625	0.8197
BC 003	y = 1.5667x - 0.6417	0.9432
BC 004	y = 1.5917x - 0.9167	0.9785
BC 005	y = 1.4x - 0.525	0.9109
Fusarium tucumaniae	Equation	Adjusted R2
Control	y = 2.194x - 0.23	0.9859
TC 002	y = 0.4973x + 2.0425	0.6
BC 003	y = 0.981x + 1.405	0.8757
BC 004	y = 0.81x + 1.75	0.7797
BC 005	y = 0.9402x + 1.241	0.8987
Rhizoctonia solani	Equation	Adjusted R2
Control	y = 1.5567x + 0.4733	0.9871
TC 002	y = 0.3033x + 1.1667	0.6037
BC 003	y = 0.925x + 0.7517	0.9143
BC 004	y = 1.025x + 0.8017	0.8717
BC 005	y = 0.8233x + 0.92	0.8703
Macrophomina phaseolina	Equation	Adjusted R2
Control	y = 1.86x + 1.6583	0.9289
TC 002	y = 0.19x + 2.7417	0.7905
BC 003	y = 1.4283x + 1.6583	0.9448
BC 004	y = 1.1283x + 2.0583	0.9919
BC 005	y = 1.3517x + 1.5333	0.963

The findings of this study are consistent with previous reports. Teixeira et al. (2024) investigated the biocontrol activity of B. velezensis against C. truncatum and C. kikuchii and found that the antagonist effectively inhibited the mycelial growth of the phytopathogens. Similarly, Mantecón (2008) demonstrated that the use of B. subtilis and B. pumilus controlled brown spot (Septoria glycines) in soybean under field conditions.

Other species of the genus Bacillus, such as B. amyloliquefaciens, have also shown in vitro inhibitory effects against C. truncatum, reducing mycelial growth by 84.3%. In addition, it protected alfalfa (Medicago sativa) plants from anthracnose under greenhouse conditions, with a biocontrol efficacy of 82.59% (Hu et al., 2021). The growing interest in biocontrol agents has directed substantial attention toward exploring the antagonistic potential of the genus Bacillus in the management of a wide range of phytopathogenic fungi (Fan et al., 2018; Yang et al., 2020; Torres et al., 2020; Avozani et al., 2022; Joly et al., 2021; El-Sersawy et al., 2021; Platel et al., 2022; Medhioub et al., 2022; Soliman et al., 2022).

For the confrontations carried out with root pathogens, it was observed that the isolate T. longibrachiatum (TC 002) differed from the other isolates, showing superior performance in reducing the mycelial growth of soilborne pathogens compared with the Bacillus isolates (Figure 3). Guzmán-Guzmán et al. (2023) reported the potential and benefits of species from the genus Trichoderma spp. for disease control, with T. longibrachiatum standing out due to its ability to parasitize, compete, produce secondary metabolites, antibiosis, and induce resistance.

Figure 3
Antagonistic effect of the isolates T. longibrachiatum (TC 002), B. velezensis (BC 004), B. velezensis (BC 005), and B. megaterium (BC 003) on the mycelial growth (cm) of D. longicolla, S. sclerotiorum, F. tucumaniae, R. solani, and M. phaseolina, respectively, over four days. Source: Author’s own work.

In this context, the graphs showing the confrontation assay (Figure 3) indicate that the mycelial growth of D. longicolla was reduced by 31.2% compared with the control when challenged with isolate TC 002. For Bacillus spp. isolates BC 003 and BC 004, reductions of 27.1% were observed, and for BC 005, a reduction of 21%. In the confrontation with F. tucumaniae, radial growth was reduced by 56.47% for TC 002, and by 43.53%, 47.05%, and 45.88% for isolates BC 003, BC 004, and BC 005, respectively, as also shown in Figure 4. Other authors, such as Akintayo et al. (2023), have highlighted the inhibitory efficiency of B. velezensis strains in the biocontrol of different Diaporthe spp. In more detail, strain ES1-02 reduced the mycelial growth of the fungus by up to 40%. These strains have proven to be effective biocontrol agents with promising yields of cyclic lipopeptides (CLPs).

Figure 4
Plates at 7 days of evaluation: Relationship of the phytopathogen controls D. longicolla, S. sclerotiorum, F. tucumaniae, R. solani, and M. phaseolina, and confrontations performed with T. longibrachiatum (TC 002), B. megaterium (BC 003), and B. velezensis (BC 004 and BC 005), respectively.

Ou et al. (2022) obtained promising results with B. megaterium (HGS7) in inhibiting the growth of the Fusarium spp. complex. Similarly, Higashi (2020) studied B. velezensis in phytopathogen control and concluded that it exerted strong antagonism against the Fusarium spp. complex and S. sclerotiorum. El-Morsy et al. (2023) observed that T. longibrachiatum exhibited antifungal activity against F. equiseti, inhibiting growth by up to 60%. Trichoderma spp. has a long history as a biocontrol agent against several pathogenic species of the genus Fusarium spp. (Kareem et al., 2016; Larran et al., 2020; Chen et al., 2021).

For M. phaseolina, the isolate TC 002 reduced the growth of the phytopathogen by 60% compared with the control, while BC 003, BC 004, and BC 005 reduced it by 17.64%, 23.5%, and 22.4%, respectively (Figures 3 and 4). Sridharan et al. (2021) reported that T. longibrachiatum reduced the growth of M. phaseolina by 58%, in addition to confirming its antibiosis effect. Ebtehag et al. (2009) also reported the biocontrol efficacy of B. megaterium against M. phaseolina in soybean seedlings. Pastrana et al. (2016) highlighted the commercial formulation based on B. megaterium and B. laterosporus (Fusbact®) for controlling charcoal rot in strawberry, caused by the fungus M. phaseolina. Hong et al. (2022) reported that B. velezensis inhibited the growth of M. phaseolina and F. oxysporum f. sp. fragariae by 64.7% and 55.2%, respectively. The authors observed that the phytopathogens displayed mycelial abnormalities such as degradation and deformation of the hyphal cell wall compared with the normal hyphal structure in the control group.

In the confrontation tests with R. solani (Figures 3 and 4), a reduction of 71.8% was observed with the T. longibrachiatum isolate (TC 002), and an average reduction of 43.5% to 47.1% in the phytopathogen growth was observed with the Bacillus spp. isolates. Furthermore, for S. sclerotiorum, the TC 002 isolate promoted a reduction of 75.3%, while Bacillus spp. reduced pathogen growth by an average of 22.4% to 24.7% compared with the control (Figures 3 and 4).

Rahimi Tamandegani et al. (2020) observed similar in vitro control effects against A. solani and R. solani, with biocontrol indices of 96.05% and 92.16%, respectively. The mycelial growth of the phytopathogens was mitigated, and the colony diameter was reduced in confrontation with isolates of T. asperellum and T. longibrachiatum.

Other promising results regarding B. velezensis in the control of R. solani have been reported in the literature. Strains SQR9 (Liu et al., 2022a), ES2-4 (Wang et al., 2023), Q-426 (Liu et al., 2022b), and C3-3 (Suárez-Bautista et al., 2024) demonstrated the ability to reduce the pathogen through the production of antimicrobial compounds that disrupt fungal cell walls and suppress pathogen growth. In addition, these strains promote plant growth through mechanisms such as root colonization and the production of secondary metabolites, including phytohormones and siderophores, which enhance nutrient uptake and modulate plant growth (Liu et al., 2022b; Wang et al., 2023).

In an in vitro study, the B. megaterium strain HGS7 inhibited 83.8% of the hyphal growth of S. sclerotiorum (Ou et al., 2022). Teixeira et al. (2021) reported that B. velezensis exhibited antifungal activity against S. sclerotiorum, M. phaseolina, B. cinerea, and R. solani, with 60% inhibition of mycelial growth, confirming the clear antagonistic effect of the microorganism under study. Torres et al. (2020) demonstrated the antifungal activity of B. velezensis, with spore germination inhibition and subsequent mycelium formation reduced by over 80% for Sclerotinia sclerotiorum and Fusarium oxysporum. Similar results were reported by Maral-Gül and Eltem (2024), where Bacillus sp. isolates exhibited percentage mycelial growth inhibition of 65.6% against B. cinerea, 42.2% against F. solani, and 55.6% against R. solani, all of which are transmitted phytopathogenic fungi.

Numerous studies have also reported the antagonistic effects and mycoparasitism of Trichoderma spp. against S. sclerotiorum. Khan et al. (2020), when studying different isolates of Trichoderma, observed reductions in S. sclerotiorum compared with the control. Haddad et al. (2017) studied 120 strains of Trichoderma spp., of which 66 showed the ability to reduce sclerotia germination by more than 50%, and 22 completely inhibited the germination of S. sclerotiorum.

3.2. Nutrient solubilization results for Phosphorus (P) and Potassium (K)

All evaluated isolates (Trichoderma longibrachiatum, Bacillus megaterium, and Bacillus velezensis BC 004 and BC 005) exhibited the ability to solubilize phosphorus in NBRIP medium (Table 3), with a progressive increase in halo size over time (Table 4 and Figure 5), similar to the reference microorganism T. koningiopsis. The reduction in medium pH (3.7–5.5) confirms the production of organic acids, a mechanism widely described as responsible for phosphorus availability (Prajapati et al., 2012).

Table 3
Regression equation and adjusted R² related to the defense response of soybean plants inoculated with T. longibrachiatum (TC 002), B. megaterium (BC 003), and B. velezensis (BC 004 and BC 005), in the expression of Chitinase and β-1,3-Glucanase.

Treatments	Equation	Adjusted R2
Chitinase
Control	y = -0.0013x2 + 0.0164x - 0.0068	0.733
TC 002	y = 0.0275x - 0.028	0.8564
BC 003	y = 0.0259x - 0.0261	0.8705
BC 004	y = -0.005x2 + 0.0315x - 0.0164	0.9189
BC 005	y = -0.0075x2 + 0.0556x - 0.0426	0.7315
β-1,3-Glucanase
Control	y = -0.0004x2 + 0.0033x - 0.0012	0.637
TC 002	y = 0.0028x - 0.0012	0.7058
BC 003	y = 0.0025x - 0.0014	0.8451
BC 004	y = -0.0019x2 + 0.0112x - 0.0075	0.8345
BC 005	y = -0.0016x2 + 0.01x - 0.007	0.5833

Table 4
Phosphorus and potassium solubilization index of the isolates T. longibrachiatum (TC 002), B. megaterium (BC 003), B. velezensis (BC 004), and B. velezensis (BC 005).

Time (h)	PHOSPHORUS					POTASSIUM
	Reference	TC 002	BC 004	BC 005	BC 003	Reference	TC 002
‘24	0.57 aA	0.60 aA	0.60 aA	0.47aA	0.53 aA	0.81 aA	0.77 aA
48	1.17 bA	1.23 bA	0.73 bA	0.90 bA	0.90 bA	1.70 bA	1.50 bA
72	1.77 cA	1.80 cA	1.27 cA	1.63 cA	1.57 cA	2.27 cA	2.07 cA
96	2.70 dA	2.60 dA	2.09 dA	2.60 dA	2.87 dA	3.01 dA	2.67 dA

• Lowercase letters compare treatments within rows, whereas uppercase letters compare treatments across columns. Means followed by the same letter do not differ statistically according to the Scott–Knott clustering test at the 5% probability level.

Figure 5
Phosphorus solubilization gradient of the isolates T. longibrachiatum (TC 002), B. megaterium (BC 003), B. velezensis (BC 004), and B. velezensis (BC 005), on the fourth day of evaluation. Source: Author’s own work.

In the case of potassium, only T. longibrachiatum (TC 002) showed significant solubilization (Figure 6), corroborating studies reporting the efficiency of Trichoderma species in potassium availability and promoting plant growth (Chen et al., 2021; Yu et al., 2023; Chungopast et al., 2023). The Bacillus isolates did not exhibit potassium solubilization activity under the conditions evaluated, although other authors describe this capacity in different species of the genus (Singh et al., 2015; Verma et al., 2015; Ali et al., 2021).

Figure 6
Potassium solubilization gradient of the isolates T. longibrachiatum (TC 002), B. megaterium (BC 003), and B. velezensis (BC 004 and BC 005) on the fourth day of evaluation.

3.3. Plant resistance induction responses

The isolates T. longibrachiatum (TC 002), B. megaterium (BC 003), and B. velezensis (BC 004 and BC 005) induced defense responses in soybean, particularly activating the enzymes chitinase and β-1,3-glucanase, while total proteins, phenolic compounds, and PAL did not show significant differences among treatments (Figure 7). The peak enzymatic activity occurred at 72 h for BC 004 and BC 005, and at 96 h for TC 002 and BC 003.

Figure 7
Defense response of soybean plants inoculated with the isolates T. longibrachiatum (TC 002), B. megaterium (BC 003), and B. velezensis (BC 004 and BC 005) in the expression of Chitinase, β-1,3-Glucanase, phenolic compounds, proteins, and PAL, respectively. CVs% = 23.32; 29.29; 14.69; 17.90; and 18.63, respectively.

Similar results have been reported in other studies, where B. velezensis and Trichoderma species demonstrated the ability to induce chitinases and β-1,3-glucanases associated with the biocontrol of phytopathogens (Nifakos et al., 2021; Hong et al., 2022; Silva et al., 2011). These effects are related to the release of metabolites and elicitors that stimulate systemic plant resistance (Zhong et al., 2024; Shen et al., 2019).

Thus, the results confirm the potential of the evaluated isolates as resistance inducers in soybean, highlighting especially B. velezensis and T. longibrachiatum, which align with the known role of these microorganisms as growth-promoting and biocontrol agents.

4. Conclusion

The isolates B. megaterium (BC 003) and B. velezensis (BC 004 and BC 005) exhibited in vitro antagonistic effects against the pathogens C. truncatum, S. glycines, and C. kikuchii. Furthermore, isolates BC 003, BC 004, and BC 005, as well as T. longibrachiatum (TC 002), demonstrated in vitro control of D. longicolla, S. sclerotiorum, F. tucumaniae, R. solani, and M. phaseolina. The observed modes of action of these isolates included antibiosis, competition for space and nutrients, and nduction of resistance in treated soybean plants, activating the chitinase and glucanase enzyme pathways, in addition to the solubilization of phosphorus and potassium.

Acknowledgements

The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq), CAPES, and the Federal University of Technology – Paraná (UTFPR), Dois Vizinhos Campus.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

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

History

• Received
  20 Sept 2025
• Accepted
  14 Jan 2026